An ER-resident lipid scramblase is crucial for biogenesis and function of apicomplexan parasite secretory organelles

Domain: biology
Published: 2025-12-13
Editorial outcome: major_revision

Abstract
Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, contain specialized secretory organelles such as micronemes, rhoptries, and dense granules, which are essential for parasite motility, host cell invasion, development, and egress. DedA superfamily proteins are implicated in lipid mobilization, which is a key requirement for organelle biogenesis. Herein, we identify and investigate the vacuole membrane protein 1 (VMP1), a DedA superfamily member, of P. falciparum (PfVMP1) and T. gondii (TgVMP1). PfVMP1 and TgVMP1 are ER-localized lipid scramblases. TgVMP1 depletion adversely affects parasite development, motility, host cell invasion, and egress. These phenotypes are consistent with impaired rhoptry and dense granule biogenesis, and decreased secretion of micronemes and rhoptries in TgVMP1-depleted parasites, indicating a crucial role for TgVMP1 in the biogenesis and function of these organelles. TgVMP1 depletion impairs lipid droplet homeostasis and ER organization, and causes loss of intravacuolar network in the parasitophorous vacuole, which are key for parasite development. Restoration of the ER-localized lipid scramblase by complementing TgVMP1-depleted parasites with PfVMP1 or a homolog as distant as human VMP1 rescues the depleted parasites, indicating their functional conservation and a crucial role for ER-resident lipid scramblase activity in the biogenesis and function of secretory organelles.

Introduction
Protozoans of the phylum Apicomplexa are intracellular obligate parasites, which cause diseases in humans, livestock, and wild animals1. Plasmodium and Toxoplasma are the most studied apicomplexans, causing malaria and toxoplasmosis, respectively. The central feature of apicomplexans is the apical complex, consisting of the apical polar ring at the apex of the cell and specialized secretory organelles called rhoptries and micronemes2. Another specialized secretory organelle of apicomplexans is dense granules, which are present throughout the cytoplasm. Microneme and rhoptry secretions are essential for parasite motility, invasion, formation of the parasitophorous vacuole (PV) around the parasite, and egress2–9. Dense granule secretion contributes to the formation of the intravacuolar network (IVN) within the PV, host cell remodeling, nutrient import, and export of various substances across the parasitophorous vacuole membrane (PVM)6,9,10. As these organelles are unique and essential, proteins present in their secretions are being pursued as drug and vaccine targets. The biogenesis of micronemes, rhoptries, and dense granules is far from clear; nonetheless, the ER and Golgi have been shown to be the sites of biogenesis of the vesicles and proteins destined to these organelles11. Lipid scramblases, which move lipids between the membrane leaflets, have been shown to be crucial for various cellular processes, including membrane/organelle biogenesis, lipid distribution, and regulation of ER-organelle contacts12. DedA (downstream of hisT E. coli DNA gene A) superfamily proteins are implicated in lipid distribution and organelle biogenesis, and have members in all the life forms13,14. The vacuole membrane protein 1 (VMP1) is the best studied DedA superfamily member, and DedA superfamily proteins have yet to be reported in parasitic protozoans. This prompted us to identify and investigate the DedA superfamily proteins in apicomplexan parasites.

VMP1 was first identified as an overexpressed gene in the rat model of experimental acute pancreatitis15. All the characterized VMP1 homologs are ER-localized multi-pass transmembrane proteins, and share a stretch of amino acids that is called the DedA domain, a characteristic feature of DedA superfamily13,16. VMP1 has roles in a variety of cellular processes, including the formation of autophagosomes, regulation of the ER-organelle contact sites, lipid transport and distribution, secretion of lipoproteins, and formation of viral replication organelles17–27.

A major function of VMP1 is in the formation of autophagosomes at an early stage. Mammalian VMP1 has been shown to recruit the PI3K complex (VPS34/Beclin1/Vps15/Atg14L) at the phagophore assembly site via interaction with Beclin1, which then generates PI3P to initiate phagophore formation28,29. The cellular abnormalities observed due to the loss of VMP1 in mammalian cells are also mirrored in VMP1 knock-out unicellular eukaryotes Dictyostelium discoideum and Chlamydomonas reinhardtii, which exhibited fragmented ER, distorted Golgi, impaired endocytosis and exocytosis, damaged mitochondria, and accumulation of ubiquitinated-protein aggregates and lipid droplets (LDs)30–32. VMP1 has been shown to activate the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), which transports cytosolic Ca2+ into ER33, thereby, inducing dissociation of ER-membrane contacts. Consistently, depletion of Homo sapiens VMP1 (HsVMP1) increased the number of ER contacts with autophagosomes, mitochondria, LDs, and endolysosomes33,34.

Mammalian VMP1 and the transmembrane protein 41B (TMEM41B), a DedA superfamily protein as well, have recently been shown to function as lipid scramblases and are implicated in maintaining lipid asymmetry across the ER leaflets and at the ER contact sites with the membranes of other organelles23,35. VMP1 and TMEM41B have been shown to be required for the formation of virus-induced replication organelles in contact with ER, and this function has been attributed to their interaction with viral proteins and mobilization/distribution of lipids at the ER-virus replication organelle site26,27,36,37. VMP1 has been shown to be key for the formation of LDs and secretion of lipoproteins, which likely depend on its scramblase activity24,34. The pleiotropic functions of VMP1 are plausible based on its scramblase activity and involvement in several ER-associated processes, as ER is the hub of lipid synthesis, organelle biogenesis, and formation of dynamic physical contacts with various organelles and structures38. In this study, we identified a VMP1 homolog in T. gondii (TgVMP1) and P. falciparum (PfVMP1), and investigated whether it has a role in the biogenesis and function of micronemes, rhoptries, and dense granules. We show that TgVMP1 and PfVMP1 are ER-localized lipid scramblases. T. gondii VMP1 is crucial for the biogenesis and function of secretory organelles, maintenance of ER organization, formation of IVN, and LD homeostasis.

Results
Apicomplexan parasites contain DedA superfamily members
The phylum Apicomplexa contains obligate intracellular parasites, including Plasmodium, Toxoplasma, Cryptosporidium, Babesia, Eimeria, Theileria, and Neospora, which cause diseases in humans, livestock and wild animals. To identify the apicomplexan DedA superfamily members, we searched the genome databases of P. falciparum and T. gondii (https://veupathdb.org/veupathdb/app) by BLAST using the amino acid sequences of DedA superfamily proteins of human (HsVMP1), S. cerevisiae (ScTVP38), and Escherichia coli (YohD, YdjZ, YqaA) as queries. The BLAST searches predicted multiple DedA domain-containing proteins in T. gondii (TGME49_244370, TGME49_279370, TGME49_248870, and TGME49_301350) and P. falciparum (PF3D7_1474600, PF3D7_1365700, and PF3D7_0611000). Of these predicted proteins, TGME49_244370 and PF3D7_1474600 showed higher sequence identity (29.3-30.3%) with HsVMP1 than the remaining proteins (10.2–20.1%), and we termed TGME49_244370 and PF3D7_1474600 as the T. gondii VMP1 (TgVMP1) and P. falciparum VMP1 (PfVMP1), respectively. Since VMP1 homologs have crucial roles in several processes, including organelle biogenesis, we focussed on TgVMP1 and PfVMP1. TgVMP1 and PfVMP1 homologs are also present in several apicomplexans, which share 28.1-32.6% sequence identity with HsVMP1, and 34.5-83.3% sequence identity among themselves (Supplementary Table 1). Apicomplexan VMP1 homologs, including TgVMP1 and PfVMP1, contain the characteristic multi-transmembrane domain architecture and DedA domain of the DedA superfamily (Supplementary Fig. 1A). The DedA domain is characterized by [F/Y]XXX[R/K] and GXXX[V/I/L/M]XXXX[F/Y] sequence motifs, and two predicted re-entrant loops (Supplementary Fig. 1B)13. These motifs are YXXXY and GXXXMXXXXF in PfVMP1 and TgVMP1. The Gly residue in the GXXX[V/I/L/M]XXXX[F/Y] motif has been shown to be essential for the function of D. discoideum VMP134, and is also conserved in TgVMP1 and PfVMP1. The AlphaFold structures of PfVMP1 and TgVMP1 also overlapped with that of HsVMP1 (RMSD of 3.736 Å between HsVMP1 and PfVMP1, and 3.362 Å between HsVMP1 and TgVMP1) (Supplementary Fig. 1C)39,40. The conserved DedA superfamily domain architecture and structural similarity of PfVMP1 and TgVMP1 with HsVMP1 indicate that these belong to the DedA superfamily.

PfVMP1 and TgVMP1 localize to ER
Towards characterization of apicomplexan VMP1 proteins, the endogenous PfVMP1 and TgVMP1 coding sequences in P. falciparum D10 and T. gondii RH TIR1 strains were tagged in-frame at their 3’-ends with the coding sequences of GFP and AIDHA, respectively. Cloned recombinant P. falciparum (PfVMP1GFPKI) and T. gondii (RHTIR1-VMP1AIDHA) parasites showed desired modification of the respective target locus (Supplementary Fig. 2). PfVMP1GFPKI parasites showed expression of PfVMP1GFP throughout the asexual erythrocytic stages (Fig. 1A, B), which co-localized with ER-Tracker (Fig. 1C), indicating that PfVMP1 is an ER-resident protein.

RHTIR1-VMP1AID parasites would express the AID receptor TIR1 and TgVMP1 with C-terminal mini-AID-3×HA (TgVMP1AIDHA). mAID or mAID-fusion protein undergoes proteasomal degradation in the presence, but not in the absence, of auxin/indole-3-acetic acid (IAA) in the cells expressing TIR141. RHTIR1-VMP1AID tachyzoites expressed TgVMP1AIDHA (Fig. 1D). To check the subcellular localization of TgVMP1, we designed a GFP-based ER-reporter (ER-GFP) that contained BiP signal sequence at the beginning and ER-retention signal (HDEL) at the end of GFP. We also used the P. falciparum Golgi reassembly stacking protein (GRASP)-GFP as a Golgi-reporter (GRASP-GFP)42,43. We episomally expressed ER-GFP or GRASP-GFP in RHTIR1-VMP1AID parasites, which showed substantial co-localization of ER-GFP with TgVMP1AIDHA (Fig. 1E, F), indicating that TgVMP1 is present in the ER. On the other hand, only a minor proportion of TgVMP1AIDHA co-localized with GRASP-GFP in the region proximal to the ER.

TgVMP1 is essential for parasite development
We used two knockdown approaches for PfVMP1. In the first approach, the endogenous PfVMP1 coding region was tagged in-frame at the 3’-end with cDD-glmS ribozyme coding sequence in P. falciparum D10 strain to obtain a trimethoprim- and glucosamine-inducible conditional knockdown line (PfVMP1dKD), which would express PfVMP1cDDHA fusion protein (Supplementary Fig. 3A). In the second approach, the endogenous PfVMP1 coding region was tagged in-frame with mAIDHA-PcDT5U/TIR1-Flag-T2A/HD/PcDT3’U sequence in P. falciparum 3D7 strain to generate an auxin-inducible knockdown line (PfVMP1AID), which would express PfVMP1AIDHA and TIR1-Flag-human DHFR proteins (Supplementary Fig. 3E). PfVMP1dKD and PfVMP1AID parasites showed desired modification of the target loci and expressed PfVMP1cDDHA and PfVMP1AIDHA (Supplementary Fig. 3B, C, F, G), respectively. However, both the parasites neither showed any noticeable depletion of the fusion protein nor any growth defect under knockdown conditions (Supplementary Fig. 3C, D, G, H). PfVMP1AID parasites expressed TIR1 (Supplementary Fig. 3I), which ruled out failure of auxin-inducible knockdown due to its absence. It is possible that the intrinsic nature and ER membrane localization of PfVMP1 rendered it refractory to degradation in case of cDD and glmS systems, as has also been reported for other proteins44,45. The auxin-inducible knockdown was quite effective for TgVMP1 in T. gondii but not for PfVMP1 in P. falciparum, which might be due to differences in the specificity of E3 ligases of P. falciparum and T. gondii for TIR1. Similar to our observation, previous studies showed limited success of the auxin-inducible system in P. falciparum46. Hence, we could not investigate PfVMP1 further in P. falciparum.

On the other hand, RHTIR1-VMP1AID parasites showed efficient depletion of TgVMP1AIDHA in the presence of IAA (+IAA), with an near completed depletion in 3 h, whereas the levels of control proteins (SAG1 and ROP1) remained unchanged (Fig. 2A), confirming IAA-inducible depletion of TgVMP1AIDHA. Consistently, the TgVMP1AIDHA signal, but not the SAG1 signal, was almost absent in the IFA images of +IAA RHTIR1-VMP1AID parasites, whereas both the signals were prominent in -IAA parasites (Fig. 2B).

We next assessed the effect of TgVMP1AIDHA depletion on parasite development. T. gondii tachyzoites invade host cells, replicate every 6–8 h for about 2–3 days to produce 64-128 cells within the PV, and finally egress to infect the neighboring uninfected host cells47. Repeated rounds of lytic cycles cause lysis of infected host cells, leaving clear areas in between the uninfected cells, which are called plaques. RHTIR1-VMP1AID and parental RHTIR1 parasites were cultured in the presence (+IAA) or absence (-IAA) of IAA for 8 days, and assessed for plaques. +IAA or -IAA RHTIR1 parasites and -IAA RHTIR1-VMP1AID parasites formed plaques of similar size, indicating similar growth (Fig. 2C, D). On the other hand, +IAA RHTIR1-VMP1AID parasites did not form plaques (Fig. 2C, D), indicating that TgVMP1 is essential for overall parasite development. To assess intracellular development, RHTIR1 and RHTIR1-VMP1AID tachyzoites were cultured for 24 h with (+IAA) or without (-IAA) IAA, and the number of parasites/PV was counted. The majority of +IAA RHTIR1-VMP1AID parasites did not progress beyond 1 parasite/PV stage, whereas the majority of -IAA RHTIR1-VMP1AID parasites and ±IAA RHTIR1 parasites developed to 4–8 parasites/PV stage (Fig. 2E), indicating an essential role of TgVMP1 in intracellular tachyzoite development.

We next evaluated the effect of TgVMP1 depletion on in vivo development of RHTIR1-VMP1AID tachyzoites. BALB/c mice were infected with RHTIR1-VMP1AID tachyzoites, maintained with (+IAA) or without (-IAA) IAA, and monitored for 21 days. As a control, a group of naïve mice was also maintained with IAA (+IAA). All the infected -IAA mice succumbed to infection by day 10, whereas only 50% of the infected +IAA mice succumbed to infection by day 15 (Fig. 2F). This is in contrast to the complete block of parasite development upon TgVMP1 depletion in plaque assay, which might be due to differences in auxin bioavailability in in vitro and in vivo experimental settings. The peritoneal exudates of dead +IAA mice had few parasites as compared with several parasites in the peritoneal exudates of dead –IAA mice (Fig. 2G). The peritoneal exudates of infected +IAA mice that survived for 21 days did not show parasites. The +IAA naïve mice survived and did not exhibit any visible symptoms, indicating that IAA was not toxic at the concentration used. The delayed death and 50% survival of infected +IAA mice indicate that TgVMP1 is critical for tachyzoite development in vivo.

TgVMP1 is crucial for gliding motility, invasion, and egress
The motile and invasive stages of apicomplexan parasites, including P. falciparum and T. gondii, rely on gliding motility to migrate, invade the host cells, egress from the infected cell, and disperse48. Tachyzoites shed surface proteins like SAG1 during gliding, which can be used to track gliding trails. For assessing motility, RHTIR1-VMP1AID parasites were grown with (+IAA) or without (-IAA) IAA, purified, allowed to glide on FBS-coated coverslips, fixed, and stained for SAG1. 64.0% of the -IAA RHTIR1-VMP1AID tachyzoites exhibited gliding motility with an average trail length of 53.0 µm as compared with 21.8% of the +IAA RHTIR1-VMP1AID tachyzoites with an average trail length of 26.4 µm (Fig. 3A–C), indicating a significant defect in gliding motility upon TgVMP1 depletion. Gliding motility is powered by glideosome, an actomyosin motor between the parasite plasma membrane and the inner membrane complex. The plasma membrane protein SAG1 and inner membrane complex protein 1 (IMC1) showed similar localization in +IAA and –IAA RHTIR1-VMP1AID tachyzoites (Supplementary Fig. 4), indicating that these two cellular structures are unaffected in TgVMP1-depleted parasites and unlikely to be responsible for the gliding motility defect. Furthermore, the viability of purified +IAA (82.6%±6.8) and –IAA (87.7%±4.0) RHTIR1-VMP1AID tachyzoites was comparable, indicating that the decreased motility of +IAA parasites is not associated with parasite viability.

For assessing invasion, purified +IAA and –IAA RHTIR1 and RHTIR1-VMP1AID parasites were allowed to invade HFF cells, fixed, and labeled for extracellular and invaded parasites. The +IAA RHTIR1-VMP1AID parasites showed significantly reduced invasion efficiency as compared with –IAA RHTIR1-VMP1AID parasites and ±IAA RHTIR1 parasites (Fig. 3D, E), indicating that TgVMP1 is crucial for host cell invasion.

For assessing egress, +IAA and –IAA intracellular RHTIR1-VMP1AID and RHTIR1 tachyzoites were treated with the calcium ionophore ionomycin, which has been used to induce rapid egress of intracellular parasites49, and processed for IFA using antibodies to the PV protein GRA1. RHTIR1 tachyzoites showed dispersal irrespective of the IAA treatment. The majority of -IAA RHTIR1-VMP1AID parasites were dispersed away from the PV, whereas +IAA RHTIR1-VMP1AID parasites remained as clumps near the PV (Fig. 3F, G), indicating a key role of TgVMP1 in egress.

TgVMP1 depletion did not affect conoid extrusion
Conoid extrusion, gliding motility, and secretions of micronemes and rhoptries are key sequential events for host cell invasion by T. gondii. Conoid is a hollow cone-like structure of tubulin fibers in the apical complex of T. gondii and certain other apicomplexans50. Conoid extrusion regulates motility, invasion, and egress by activating microneme secretion and actomyosin motor3. Since TgVMP1 depletion decreased gliding motility, invasion, and egress, we compared purified –IAA and +IAA RHTIR1-VMP1AID tachyzoites for conoid extrusion by treating them with the calcium ionophore A23187, which induces conoid extrusion51. The number of parasites showing conoid extrusion was similar in +IAA and –IAA parasites (Supplementary Fig. 5A, B), ruling out a role of TgVMP1 in this key event.

TgVMP1 is crucial for microneme secretion
Decreased motility, host cell invasion, egress, and intracellular development of TgVMP1-depleted parasites prompted us to look into the effect of TgVMP1 depletion on micronemes, rhoptries, and dense granules, which are crucial for these processes in Toxoplasma and Plasmodium2,52. Micronemes are situated at the apical end, and their secretion is required for motility, invasion, and egress3. We compared the localization and secretion of selected microneme proteins in +IAA and –IAA RHTIR1-VMP1AID tachyzoites. For microneme organization, +IAA and -IAA intracellular parasites were processed for IFA and compared for localization of selected microneme proteins (apical membrane antigen 1 (AMA1), microneme protein 2 (MIC2), and microneme protein 3 (MIC3)). All these proteins showed similar predominant apical localization in +IAA and –IAA parasites, ruling out any adverse effect of TgVMP1 depletion on microneme organization (Fig. 4A–D; Supplementary Fig. 6A, B). Transmission electron microscopy (TEM) images confirmed the presence of both apical and lateral micronemes in +IAA and –IAA parasites (Supplementary Fig. 6C), which was also corroborated by MIC2 labeling in immunoelectron microscopy (IEM) images (Supplementary Fig. 7A). Furthermore, the TEM micrographs showed comparable number of apical micronemes in –IAA (9.9±5.9/apical region, n=22 parasite sections) and +IAA (9.0±4.4/apical region, n=22 parasite sections) parasites, which together with the IFA data indicate that TgVMP1 depletion did not affect microneme organization. For microneme secretion, purified +IAA and –IAA RHTIR1-VMP1AID parasites were treated with ethanol, which induces microneme secretion that is known as extracellular secreted antigens (ESA)53. We used MIC2 as an ESA marker, which is proteolytically processed by the microneme protein proteases MPP1 and MPP2, and is predominantly present in the shorter form in ESA54. The level of larger MIC2 form was higher in the pellet fraction of +IAA parasites than that of –IAA parasites (Fig. 4E, F). The ESA fraction of +IAA parasites had less of the processed form of MIC2, along with the larger MIC2 form, as compared with the presence of only the processed form of MIC2 in –IAA parasites (Fig. 4E, G), which indicated both impaired microneme secretion and processing of MIC2 upon TgVMP1 depletion.

TgVMP1 is crucial for rhoptry biogenesis and secretion
Rhoptries are situated at the apical end, and their secretion is essential for host cell invasion, formation of PV around the parasite inside the host cell, and modulation of host cell responses4,5. We compared intracellular +IAA and –IAA RHTIR1-VMP1AID parasites for rhoptry organization and secretion using ROP1 as a marker. For rhoptry organization, intracellular +IAA and –IAA tachyzoites were processed for IFA, TEM, and IEM. IFA images showed ROP1 localization to characteristic rhoptry-like elongated structures with a bulbous base in the majority (85.3%) of -IAA parasites as compared with only a small number (11.2%) of +IAA parasites (Fig. 5A, B). 59.4% of +IAA parasites also showed ROP1 signal in the PV compared to 10.9% of -IAA parasites (Fig. 5C, D). Similar to the intracellular parasites, purified -IAA parasites also exhibited ROP1 localization to rhoptry-like elongated structures, which was not the case with +IAA parasites (Fig. 5E). In agreement with the IFA data, TEM images showed apical rhoptries in 52.2% of -IAA parasites (n=23 parasites), whereas only 18.2% of +IAA parasites (n=22 parasites) had apical rhoptries (Fig. 6A). ROP1 labeling of the similar structures in IEM micrographs indicated ROP1 targeting to these structures (Supplementary Fig. 7B). However, compared to the IFA data, IEM micrographs did not show a clear ROP1 signal in the PV of –IAA and +IAA parasites, which might be due to differences in the two experimental procedures. The loss of rhoptries in TgVMP1-depleted parasites indicates a crucial role for TgVMP1 in rhoptry formation.

For rhoptry secretion, we checked for ROP1-labeled e-vacuoles, which are discharged into the host cell during invasion. E-vacuole discharge can be monitored by treating the parasites with cytochalasin D (CytD) that prevents invasion without affecting e-vacuole secretion. The CytD-treated purified +IAA and –IAA parasites were allowed to invade HFF cells, the cells were processed for IFA using anti-ROP1 antibodies, and scored for ROP1 positive e-vacuoles. ~16% of the +IAA parasites secreted e-vacuoles into the host cell as compared with ~34% of the –IAA parasites (Fig. 6B, C), indicating a crucial role for TgVMP1 in rhoptry secretion.

TgVMP1 is required for dense granule biogenesis and IVN formation
Dense granules are secretory organelles throughout the parasite cytoplasm, which secrete an arsenal of secretory factors, collectively called GRA proteins, into the PV lumen immediately after invasion and during parasite development6,9,10. Proteins secreted by dense granules associate with and contribute to the formation of IVN, which has been suggested to act as a scaffold to position and organize the replicating parasites within the PV9,10. Some GRA proteins have also been shown to be transported to the PVM and host cell to mediate transport across the PVM and modulate host processes, respectively6,52. We evaluated intracellular +IAA and –IAA RHTIR1-VMP1AID parasites for dense granules by IFA using GRA1 and GRA2 as markers, and by TEM. The IFA images showed GRA1 and GRA2 localization to distinct structures in the PV of -IAA parasites, whereas these proteins had diffuse localization in the PV of +IAA parasites (Fig. 7A; Supplementary Fig. 8A). The number of parasites with diffuse GRA1 or GRA2 localization was significantly higher in +IAA parasites (83.6% for GRA1, 79.0% for GRA2) than that in –IAA parasites (9.6% for GRA1, 14.6% for GRA2) (Fig. 7B; Supplementary Fig. 8B). TEM images showed the absence of tubulo-vesicular IVN in the PV of 57.7% +IAA parasites (n=123 PVs) as compared with 7.8% –IAA parasites (n=64 PVs) (Fig. 7C), indicating a crucial role for TgVMP1 in IVN formation.

For dense granule biogenesis, purified +IAA and –IAA tachyzoites were processed for IFA and compared for the levels of intracellular GRA1 and GRA2. +IAA parasites showed higher levels of these proteins than –IAA parasites (Fig. 7D; Supplementary Fig. 8C). -IAA RHTIR1-VMP1AID parasites showed distinct GRA1- or GRA2-labeled dense granules in the cytoplasm (Fig. 7E; Supplementary Fig. 8D). On the other hand, in addition to the cytoplasmic GRA1 and GRA2-labeled dense granules, +IAA RHTIR1-VMP1AID parasites also showed perinuclear accumulation of GRA1 and GRA2. The perinuclear accumulation of GRA1 or GRA2 was significantly higher in +IAA parasites than that in –IAA parasites (Fig. 7F; Supplementary Fig. 8E), and both GRA1 and GRA2 co-localized with ER-GFP in +IAA parasites (Fig. 7G; Supplementary Fig. 8F), which indicated a key role of TgVMP1 in dense granule biogenesis. TEM micrographs of both –IAA and +IAA parasites showed dense granules (Figs. 6A and 7C), which was corroborated by IEM showing GRA2 labeling of dense granules in the parasite cytoplasm and IVN in the PV (Supplementary Fig. 7C). The TEM micrographs of +IAA parasites contained less number of cytoplasmic dense granules (1.5/parasite section, n=153) than those of –IAA parasites (3.2/parasite section, n=94), which together with the IFA data indicate a crucial role of TgVMP1 in dense granule biogenesis. We next compared dense granule secretion in the ESA of purified –IAA and +IAA parasites by IEM using antibodies to GRA2. The number of GRA2-gold particles was comparable (Supplementary Fig. 7D, E), suggesting that impaired dense granule biogenesis in TgVMP1-depleted parasites did not affect secretion. Nonetheless, the majority of the gold particles were associated with electron-dense material in –IAA parasites as opposed to +IAA parasites (Supplementary Fig. 7D), suggesting a defect in the post-secretion process that might be required for the formation of IVN.

TgVMP1 depletion altered LD homeostasis
The DedA superfamily proteins are proposed to be lipid scramblases; human VMP1 and TMEM41B have been shown to have roles in lipoprotein and lipid transport, LD biogenesis, and lipid distribution22–24. This prompted us to evaluate the effect of TgVMP1 depletion on lipid homeostasis using LDs as a marker. LDs contain neutral lipids such as triglycerides and cholesterol esters, are formed in the ER bilayer, and bud off into the cytoplasm55. We stained purified +IAA and –IAA RHTIR1-VMP1AID tachyzoites with Nile red to label LDs56, and observed the parasites by live-cell confocal microscopy. +IAA parasites had a significant reduction in the number and increase in the size of LDs as compared with -IAA parasites (Fig. 8A–C). TEM micrographs confirmed the presence of much larger-sized LDs in +IAA parasites than in -IAA parasites (Fig. 8D), indicating a key role for TgVMP1 in LD homeostasis. Additionally, LDs were closely associated with the ER membrane in +IAA parasites, which was not evident in –IAA parasites. Although we attempted to determine membrane lipid distribution in TgVMP1-depleted parasites using click lipids and fluorophores, we did not succeed due to widespread non-specific labeling of the parasite by click fluorophores. Further optimization of the use of click lipids or alternative approaches is required to determine if TgVMP1-depletion affects membrane lipid distribution.

TgVMP1 depletion disrupted ER organization
The microneme, rhoptry, and dense granule proteins have been shown to be synthesized in ER, and vesicles containing these proteins bud off the ER/Golgi regions and follow the endosomal vesicular trafficking pathway to reach/mature into these organelles11. Perinuclear clustering of dense granule proteins GRA1 and GRA2 with ER-GFP in TgVMP1-depleted parasites prompted us to investigate whether TgVMP1 depletion compromised the ER. We compared the localization of ER-GFP in intracellular –IAA and +IAA RHTIR-VMP1AID::BiP-GFP-HDEL parasites by IFA and TEM. –IAA parasites showed uniform ER-GFP localization around the nucleus, which was also extended toward the apical region (Fig. 8E, F). On the other hand, ER-GFP was prominently clustered around the nucleus in a significant number of +IAA parasites (Fig. 8E, F). TEM micrographs showed ER tubules stretched throughout the +IAA parasites, including in close contact with LDs, whereas these were mostly perinuclear in –IAA parasites (Fig. 8G). These data indicate impaired ER organization, which might contribute to various phenotypes observed in TgVMP1-depleted parasites. These data highlight a key role of TgVMP1 in the ER functioning, which could include the maintenance of ER structure, formation of functional subdomains, and communication with other organelles.

TgVMP1 interactome supports its roles in the secretory organelle function and biogenesis
To understand how TgVMP1 depletion might impair the function and biogenesis of secretory organelles, we determined and analyzed the TgVMP1 interactome (Supplementary Fig. 9; Supplementary Table 2). TgVMP1 interactome contained proteins that have been shown to localize to micronemes (apical cap protein AC5 and MIC7), rhoptries (RON3, ROP8, ROP26, and ROP37), and dense granules (phosphatidylserine decarboxylase, cathepsin CPC2, and GRA12). The interactome also contained the proteins associated with gliding motility (PhiL1, GAPM3, GAPM2B, GAPM1A, and GRA12), processing of the microneme and rhoptry proteins (V-type proton ATPase subunit a1), and vesicle transport (sortilin-like receptor, Rab5b, glutathione S-transferase 2, and Rab2). Additionally, the TgVMP1 interactome contained a large number of proteins related to protein biosynthesis, cytoskeleton, maintenance of the parasite shape, and virulence. Among these proteins, the sortilin-like receptor (TgSORTLR) and V-type proton ATPase are specifically relevant in the context of secretory organelles. TgSORTLR is required for trafficking of the microneme and rhoptries proteins, and TgSORTLR knockdown impairs the biogenesis of these organelles57. The V-type proton ATPase is localized to the plasma membrane and endosomal compartments, and is required for the processing of microneme and rhoptry proteins during their transport through the endosomal compartments58. Furthermore, the V-type proton ATPase is also required for activation of the proteases that process microneme and rhoptry proteins. The presence of proteins localized to/associated with micronemes, rhoptries, dense granules and the motor complex in the TgVMP1 interactome suggests a direct or indirect role of TgVMP1 in the functioning and biogenesis of these organelles, which is in agreement with the defects observed in these organelles upon TgVMP1 depletion. The presence of several motility-associated proteins in the TgVMP1 interactome suggests an alternative mechanism by which TgVMP1 could contribute to parasite motility; however, this requires further investigation.

TgVMP1 and PfVMP1 are lipid scramblases, and restoration of the ER-localized scramblase activity rescued TgVMP1-depleted parasites
TgVMP1-depleted parasites showed dense granule accumulation in the ER, impaired LD homeostasis, and disrupted ER organization, suggesting a role for TgVMP1 in ER and ER-organelle contact sites. The ER-localized DedA superfamily proteins HsVMP1 and HsTMEM41B have been shown to have lipid scramblase activity, which has been proposed to be required for lipoprotein and lipid transport, LD biogenesis, lipid distribution, and the formation of viral replication organelles22,23,26,35. TgVMP1, PfVMP1, and HsVMP1 share similar domain architecture and AlphaFold structure, which prompted us to ask whether TgVMP1 and PfVMP1 function as lipid scramblases and restoration of the ER-localized scramblase activity in RHTIR1-VMP1AID parasites could rescue the growth defects.

Lipid scramblases translocate lipids between two membrane leaflets, which can be measured by monitoring the fluorescence signal intensity of fluorescent phospholipid (NBD-PS)-containing liposomes in the presence or absence of a membrane impermeable quencher (dithionite). The fluorescence signal of NBD-PS on the outer liposome leaflet would be quenched by dithionite, causing ~50% decrease in fluorescence intensity (Fig. 9A). On the other hand, translocation of NBD-PS between the outer and inner leaflets of the liposome by a lipid scramblase would result in the »50% decrease in fluorescence intensity (Fig. 9A). Addition of Triton X-100 would permeabilize the liposomes, allowing dithionite to enter and quench the NBD-PS fluorescence completely (Fig. 9A). For lipid scramblase activity, PfVMP1AIDHA and TgVMP1AIDHA were purified from PfVMP1AID and RHTIR1-VMP1AID parasites, respectively (Supplementary Fig. 10A). Purified TgVMP1AIDHA or PfVMP1AIDHA was incorporated into NBD-PS-containing liposomes to obtain TgVMP1AIDHA-liposomes or PfVMP1AIDHA-liposomes. Only NBD-PS-containing liposomes (empty-liposomes) were used as a control. Indeed, TgVMP1-liposomes and PfVMP1-liposomes showed »50% decrease in fluorescence intensity upon addition of dithionite as compared with ~50% decrease in fluorescence intensity of empty-liposomes (Fig. 9B), indicating that TgVMP1 and PfVMP1 are lipid scramblases.

For restoration of the ER-localized scramblase activity in RHTIR1-VMP1AID parasites, we ectopically expressed Myc-tagged PfVMP1 (PfVMP1Myc) and HsVMP1 (HsVMP1Myc) proteins in RHTIR1-VMP1AID parasites to obtain complemented parasites (RHTIR1-VMP1AID/PfVMP1Myc and RHTIR1-VMP1AID/HsVMP1Myc, respectively). We next compared the complemented parasites with RHTIR1-VMP1AID parasites for overall growth and intracellular development in the presence (+IAA) or absence (-IAA) of IAA. The complemented parasites expressed PfVMP1Myc or HsVMP1Myc (Supplementary Fig. 10B), which co-localized with TgVMP1AIDHA (Supplementary Fig. 10C), indicating that these proteins retain ER localization, even in a heterologous system. As expected, all three parasites formed plaques under –IAA conditions, whereas only complemented parasites, but not RHTIR1-VMP1AID parasites, formed plaques under +IAA conditions (Supplementary Fig. 10D), which indicated restoration of the overall growth by PfVMP1 or HsVMP1 in the absence of TgVMP1. 90.7% of the +IAA RHTIR1-VMP1AID parasites were arrested at the single parasite/PV stage as compared to 27.5% (for HsVMP1) and 56.1% (for PfVMP1) of the +IAA complemented parasites (Fig. 9C), indicating restoration of intracellular development by PfVMP1 or HsVMP1 in the absence of TgVMP1. We next checked if PfVMP1 and HsVMP1 could reverse the perinuclear accumulation of the dense granule protein GRA2 in TgVMP1-depleted parasites. <5.7% of the +IAA complemented parasites showed perinuclear accumulation of GRA2 as compared with 44.7% of the +IAA RHTIR1-VMP1AID parasites (Fig. 9D, E), indicating rescue of the dense granule biogenesis defect.

The rescue of growth and dense granule biogenesis defects of TgVMP1-depleted parasites by PfVMP1 and HsVMP1 indicates their functional conservation. This provides a mechanistic proof for a critical role of the ER-localized scramblase activity in the biogenesis and function of apicomplexan secretory organelles.

Methods
Materials
All the routinely used biochemicals were of molecular biology or cell culture grade, and were purchased from Thermo Fisher Scientific, Sigma-Aldrich, MP Biomedicals or Serva unless otherwise stated. Cell culture media and supplements, blasticidin S-hydrochloride, gentamicin, penicillin-streptomycin, and Giemsa stain were from Lonza and Thermo Fisher Scientific. Lipids (POPC, POPS, and NBD-PS) were from Avanti Polar Lipids. Dithionite was from Sigma-Aldrich. All the plasticware for cell culture was procured from standard manufacturers, including Thermo Fisher Scientific, Corning Inc., Nalgene, TPP, and Tarsons. Restriction enzymes and DNA-modifying enzymes were from New England Biolabs and Thermo Fisher Scientific. DNA and plasmid isolation kits were from QIAGEN and MACHERY-NAGEL. PrimeScript 1st strand cDNA synthesis kit was from Takara Bio. ProLong Diamond Antifade Mountant, DAPI, Hoechst, ER-Tracker Red stain (BODIPY TR Glibenclamide), and SuperSignal Chemiluminescent substrates were from Thermo Fisher Scientific. Trimethoprim, N-acetylglucosamine, indole-3-acetic acid (IAA), thapsigargin, xanthine, mycophenolic acid, calcium ionophore A23187, formaldehyde and Nile red were purchased from Merck. Ionomycin was from Tocris Bioscience. 5Ph-IAA was from MedChemExpress. Pyrimethamine was from MP Biomedicals. The protease inhibitor cocktail was from Roche. Glutaraldehyde, formaldehyde, osmium tetroxide, and sodium cacodylate were from Electron Microscopy Sciences. Uranyl acetate was from Lobachemie, and epoxy resin components were from TED Pella. Antibodies were from Thermo Fisher Scientific, Cell Signalling Technology, Santa Cruz Biotechnology or Jackson ImmunoResearch. GFP-Trap and HA-Trap antibodies were from ChromoTek. Pierce protein A/G magnetic beads were from Thermo Fisher Scientific. Human Foreskin Fibroblasts (HFF) were from ATCC, P. falciparum 3D7 and D10 strains, T. gondii RH TIR1 strain, and antibodies to T. gondii proteins (AMA1, MIC2, MIC3, ROP1, GRA1, GRA2, and SAG1) were procured from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). WR99210 was a kind gift from David Jacobus (Jacobus Pharmaceutical, Princeton, U.S.A.). Rabbit anti-IMC1 and anti-ACP polyclonal antibodies were a kind gift from Abhijit S. Deshmukh (NIAB, India) and Dhanasekaran Shanmugam (NCL, India), respectively. Rabbit anti-TIR1 antibody was from MYBIOSOURCE. Gold-conjugated goat-anti-mouse IgG was from Electron Microscopy Sciences. The pTKO-HXGPRT plasmid was a kind gift from Dr Nishit Gupta (BITS, India). pTUB1:YFP-mAID-3HA-DHFR-TS:HXGPRT (Addgene #87259) and pSAG1::Cas9-U6::sgUPRT (Addgene #54467) plasmids were a kind gift from Dr. David Sibley. pCTG-Fluc (originally sourced from Dr. Shobhona Sharma, TIFR, India) and pARL1a-PfGRASP-GFP (originally sourced from Dr. Lilach Sheiner, University of Glasgow, UK) were kind gifts from Dr. Swati Patankar (IIT Bombay, India).

Human blood was collected from healthy volunteers after obtaining informed consent according to the approved protocol of the Institutional Ethics Committee (IEC-103/2023, IEC-38/2015, IEC-38-R1/2015, IEC-38-R2/2015 and IEC-38-R3/2015) of Centre for Cellular and Molecular Biology, India. P. falciparum and T. gondii cultures and related experiments were carried out according to the approved protocols of the Institutional Biosafety Committee of Centre for Cellular and Molecular Biology, India. Animals were housed in cabin-type isolators at room temperature (22-25°C, 40–70% humidity, and 12/12 h dark/light photoperiod), and all the experiments on animals were performed according to the approved protocols (IAEC 09/2023) of the Institutional Animal Ethics Committees (IAEC) of Center for Cellular and Molecular Biology, India. All the studies were carried out as per the Declaration of Helsinki principles.

Sequence analysis
The amino acid sequences of VMP1 proteins of model organisms (Homo sapiens, ID: Q96GC9; Drosophila melanogaster, ID: Q9W2S1; Caenorhabditis elegans, ID: Q9XWU8; Arabidopsis thaliana, ID: Q5XF36 (KSM1) and F4I8Q7 (KSM2); Chlamydomonas reinhardtii, ID: A0A2K3DNE8; Dictyostelium discoideum, ID: Q54NL4; Danio rerio, ID: Q6NYY9), S. cerevisiae TVP38 (ID: P36164), and selected E. coli DedA superfamily proteins (YohD, ID: P33366; YdjZ, ID: P76221; YqaA, ID: P0ADR0) were obtained from the Uniprot database (https://www.uniprot.org/). The amino acid sequences of HsVMP1, ScTVP38, and E. coli DedA superfamily proteins (YohD, YdjZ, YqaA) were used as queries in the BLAST searches of the eukaryotic pathogen database VEupathDB (https://veupathdb.org/) to identify homologs in selected reference apicomplexan parasites, including T. gondii and P. falciparum (Supplementary Table 1). The amino acid sequences of apicomplexan VMP1 proteins were analyzed for the presence of conserved domains using CD BLAST (ncbi.nlm.nih.gov) or MOTIF (genome.jp/tools/motif/), and sequences were aligned using Clustal Omega72.

Parasite culture
P. falciparum 3D7 and D10 strains were maintained in human RBCs (at 2% hematocrit) in RPMI-1640 medium (supplemented with 2g/l sodium bicarbonate, 2g/l glucose, 25mg/ml gentamicin, 300mg/l L-glutamine, 100mM hypoxanthine, 0.5% albumax II) under a mixed gas environment (5% CO2, 5% O2 and 90% N2) at 37°C73–75. The synchrony of P. falciparum culture was maintained by treating the cells with 5% sorbitol when the majority of parasites were at the ring stage76. Genomic DNA was isolated from late trophozoite/schizont stage parasites using the NucleoSpin Tissue kit as recommended by the manufacturer.

Human foreskin fibroblast (HFF) cells were maintained in D10 medium (DMEM supplemented with 10% FBS, 2mM glutamine, 3.7g/l sodium bicarbonate, 155mg/l sodium pyruvate, 1% pen-strep) at 37°C under 5% CO2 environment. T. gondii RH TIR1-3FLAG strain (RHTIR1)77 was grown in confluent HFF cells, as has been reported previously78. Fully confluent HFF cells infected with RHTIR1 tachyzoites were scraped, and the suspension was passed through a 3µm filter to remove host debris. The filtrate was centrifuged at 800×g for 8 min, washed in incomplete DMEM medium (DMEM without antibiotics and FBS), and the purified parasite pellet was resuspended in an appropriate buffer as has been described in the following sections.

Genomic DNA (gDNA) was isolated from purified T. gondii RHTIR1 tachyzoites using the NucleoSpin Tissue kit following the manufacturer’s instructions. For T. gondii cDNA preparation, total RNA was isolated from the purified RHTIR1 T. gondii tachyzoites using the NucleoSpin RNA isolation kit according to the manufacturer’s instructions. 5μg of the DNA-free total RNA was used to prepare cDNA using PrimeScript 1st strand cDNA synthesis kit as per the manufacturer’s instructions.

Generation of recombinant P. falciparum parasites
The primers and synthetic DNAs used for the generation of transfection constructs are listed in Supplementary Table 3. To knock-in GFP immediately downstream of the 3’-PfVMP1 coding sequence, the 3’-coding sequence of PfVMP1 corresponding to 249 amino acid residues was amplified from P. falciparum 3D7 genomic DNA using PfVMP1-F1/PfVMP1-R1 primers, and cloned into the pGEM-T vector to obtain pGEMT-PfVMP1c plasmid. The pGEMT-PfVMP1c plasmid was digested with ApaI/KpnI to excise the PfVMP1c fragment, which was subcloned into similarly digested pGT-GFP plasmid to obtain pGT-PfVMP1c-GFP. The pGT-PfVMP1c-GFP plasmid was digested with ApaI/XhoI to excise the PfVMP1c-GFP insert, which was subcloned into similarly digested pNC vector to obtain pNC-PfVMP1c-GFP transfection plasmid. pNC and pGT-GFP plasmids have been described earlier79,80.

We constructed a dual knockdown plasmid, which would cause glmS-mediated mRNA degradation and mutant E. coli DHFR (cDD)-mediated protein degradation as has been described previously81. The 3’-coding sequence corresponding to 249 amino acid residues (flank1) of the PfVMP1 coding sequence and 1087bp of PfVMP1 3’-UTR region (flank2) were amplified from P. falciparum 3D7 genomic DNA using PfVMP1-fl1F/PfVMP1-R1 and PfVMP1-Fl2F/PfVMP1-Fl2R primer pairs, respectively. The flank1 and flank2 PCR products were cloned into the HBPFA18/cDDHA/GlmSAc plasmid at NotI/KpnI and AvrII/KasI sites, respectively, to obtain HB-PfVMP1-cDDHA-GlmSAc transfection plasmid. HBPFA18/cDDHA/GlmSAc plasmid has been described earlier82.

We also employed an auxin-inducible knockdown strategy for PfVMP1. We purchased synthetic XTEN-mAID-2×HA (mAIDHA) and PcDT5U/Tir1-Flag-2A/HD/PcDT3’U (TIR1-HD) DNAs, which were cloned into the HBPFA18/cDDHA/GlmSAc at KpnI/XhoI and AgeI/AvrII sites, respectively, to obtain HB-mAIDHA/TIR1-HD plasmid. The mAID-2×HA and TIR1-Flag-2A/HD coding sequences are codon-optimized for P. falciparum, and the TIR1 used here is F74G mutant of OsTIR183, which is inducible with 5Ph-IAA. The above PfVMP1 flank1 and flank2 regions were cloned into the HB-mAIDHA/TIR1-HD plasmid at NotI/KpnI and AvrII/KasI sites, respectively, to obtain the HB-PfVMP1-mAIDHA/TIR1-HD transfection plasmid.

Ring stage P. falciparum D10 (for PfVMP1c-GFP and HB-PfVMP1-cDDHA-GlmSAc plasmids) or 3D7 (for HB-PfVMP1-mAIDHA/TIR1-HD plasmids) parasites were transfected with 100µg of plasmid DNA by electroporation, and transfected parasites were selected with blasticidin S hydrochloride (at 2µg/ml for pNC-PfVMP1c-GFP) or WR99210 (at 1nM for HB-PfVMP1-cDDHA-GlmSAc and HB-PfVMP1-mAIDHA/TIR1-HD) to obtain resistant parasites as has been previously described84,85. Blasticidin-resistant parasites were cloned by dilution cloning to obtain clonal populations. The genomic DNA of each of the clonal D10 parasite lines was isolated, assessed by PCR for plasmid integration using locus-specific primer sets (5’-integration: PfVMP1-5con/GFPSEQR1; 3’-integration: PcDT5U-R1/PfVMP1-3con; wild-type: PfVMP1-5con/PfVMP1-3con; positive control: PfA8fl2-F1/PfA8fl2-R1). WRR99210 resistant parasites were cloned by dilution cloning to obtain clonal lines, genomic DNA was isolated from clonal lines, and assessed for plasmid integration into the target locus by PCR using locus-specific primer sets (HB-PfVMP1-cDDHA-GlmSAc-transfected parasites (5’-integration: PfVMP1-5con/PvAc-Con-R; 3’-integration: Hrp2-SeqF/PfVMP1-3con2; wild type-specific: PfVMP1-5con/ PfVMP1-3con2; positive control: PfA8-F1/PfA8fl2-R1), HB-PfVMP1-mAIDHA/TIR1-HD-transfected parasites (PfVMP1-5con/PfVMP1-3con2; positive control: PfUCH-F/PfUCH-R). The cloned recombinant parasites with PfVMP1c-GFP (PfVMP1GFPKI), PfVMP1/cDDHA-GlmS (PfVMP1dKD) or PfVMP1-mAIDHA/TIR1-HD (PfVMP1AID) locus were used for various experiments.

Generation of recombinant T. gondii parasites
The transfection cassette containing TgVMP1-mAID-3×HA coding sequence and HXGPRT selection cassette (TgVMP1-mAID-3HA/DHFR-TS:HXGPRT) was amplified from the pTUB1:YFP-mAID-3HA, DHFR-TS:HXGPRT plasmid using TgVMP1-Fkd2/TgVMP1-RP primers86. The TgVMP1-Fkd2 primer contained 50bp homology sequence corresponding to the immediately upstream of the TgVMP1 stop codon and overlapped with the 5’-mAID-3HA sequence. The TgVMP1-RP primer contained 50bp homology sequence corresponding to 133bp downstream of the TgVMP1 stop codon. The primer (SgDNA1) corresponding to the 20-nucleotide protospacer sequence in the 3’ UTR of TgVMP1, along with the SgDNARP primer, were used to amplify pSAG1::Cas9-U6::sgUPRT plasmid87, which generated a PCR product with the target guide RNA sequence and Cas9 expression cassette (pSAG1::Cas9-U6::sgTgVMP1). The PCR product was transformed into DH5α cells and sequenced to ensure the correctness of the guide RNA coding sequence.

T. gondii RHTIR1 tachyzoites were transfected with a mixture of TgVMP1-mAID-3HA/DHFR-TS:HXGPRT transfection cassette and pSAG1::Cas9-U6::s-gTgVMP1 plasmid, as has been described previously41. In brief, fully confluent HFF cells infected with RHTIR1 tachyzoites were scraped, tachyzoites were purified using a 3µm filter to remove host debris, centrifuged at 800×g for 8 min, and washed in incomplete DMEM medium (DMEM without antibiotics and FBS). 5×106 tachyzoites were mixed with 15µg each of the transfection cassette and plasmid, the volume was adjusted to 400µl using incomplete medium, transferred to a 2mm cuvette, and electroporated (at 1500V, 10µF capacitance, ∞ resistance). The electroporated parasites were transferred to confluent HFF cells in T25 flasks, grown in drug-free complete medium for 24h, followed by in selection medium (complete medium with 25µg/ml mycophenolic acid and 50µg/ml xanthine) for 48h. The intracellular parasites were syringe lysed, and 500µl of the lysed suspension was added to fully confluent HFF cells in a T25 flask, and grown in the selection medium. Resistant parasites were diluted to achieve 0.5 parasite/well in a 96-well tissue culture plate containing HFF monolayer, and allowed to grow until parasites appeared. These parasites were screened by PCR for the presence of the integration locus using TgVMP1-5con/TgVMP1-3con primer set, along with the TgVP1-5con/TgVP1-3con primer set for the TgVP1 gene as a positive control. Parasites showing the absence of wild-type TgVMP1 and presence of the integration locus were checked for expression of the TgVMP1-mAID-3×HA fusion protein (TgVMP1AIDHA) by western blotting using anti-HA antibodies. Pure clonal recombinant parasites (RHTIR1-VMP1AID) were used for further studies.

Expression of ER and Golgi reporters in RHTIR1-VMP1AID parasites
Tubulin (Tub) promoter region was amplified from RHTIR1 gDNA using TubF/TubR primers by PCR, and cloned into pMyc3x.LIC-DHFR plasmid at PacI/EcoRV sites to obtain pTub-DHFR plasmid. The N-terminal signal sequence (MTAAKKLSLFSLAALFCLLSVATLRPVAASD) and C-terminal ER-retention signal (HDEL) of T. gondii chaperonin BiP (TGRH88_051090) were added to the N-terminal and C-terminal, respectively, of GFP by PCR to generate the ER reporter BiP-GFP-HDEL. In brief, the BiP N-terminal signal sequence was amplified from T. gondii gDNA using BipSig-F/BipSig-GFP-Rrec primers. The GFP coding sequence was amplified from the pTKO-HXGPRT plasmid using GFP-Bip-Frec/GFP-HDEL-R primers. The two PCR fragments were recombined by PCR using primers BipSig-F/GFP-HDEL-R to obtain BiP-GFP-HDEL (ER-GFP) coding sequence, which was cloned at EcoRV/AvrII sites in pTub-DHFR plasmid to obtain pTub-ER-GFP plasmid. 15µg of this plasmid was transfected into RHTIR1-VMP1AID parasites to obtain RHTIR1-VMP1AID::ER-GFP parasites as has been described above in the “Generation of recombinant T. gondii parasites” section.

For the expression of Golgi-reporter PfGRASP-GFP, which has been used as a Golgi-reporter previously42, 15µg of the pARL1a-PfGRASP-GFP plasmid was transfected into RHTIR1-VMP1AID tachyzoites to obtain RHTIR1-VMP1AID::GRASP-GFP parasites as has been described in the “Generation of recombinant T. gondii parasites” section.

VMP1 expression
Cloned PfVMP1GFPKI parasites, which would express PfVMP1GFP fusion protein, and wild-type P. falciparum D10 parasites were cultured and synchronized as described in the “Parasite culture” section. Synchronized PfVMP1GFPKI parasites were harvested at ring, early trophozoite, late trophozoite, and schizont stages, purified by saponin lysis, and parasite pellets were resuspended in 6× pellet volume of 10mM Tris (pH 7.4). The suspension was mixed with 1/3rd volume of 4× SDS-PAGE sample buffer (1× buffer has 50mM Tris-HCl, 10% glycerol (v/v), 2% SDS, 2.5% β-mercaptoethanol (v/v), 0.05% bromophenol blue (w/v), pH 6.8), heated at 99°C for 10min, centrifuged at 20,000×g for 20min, and supernatants were processed for western blotting. In brief, the lysates were run in a 12% SDS-PAGE gel, transferred to an Immobilon-P-PVDF membrane, incubated with blocking buffer (TBS-T (10mM Tris, 150mM NaCl, 0.1% Tween-20, pH 7.4) with 3% fat-free milk powder), probed using rabbit anti-GFP monoclonal antibodies, followed by goat anti-rabbit-HRP-conjugated secondary antibodies (at 1:20,000 dilution). The membrane was washed, and the signal was developed using SuperSignal chemiluminescent substrate. For loading control, the membrane was stripped off, blocked, incubated with mouse anti-β-actin-HRP-conjugated antibodies (at 1:30,000 dilution), and the signal was developed using SuperSignal chemiluminescent substrate.

RHTIR1 and RHTIR1-VMP1AID parasites were cultured in confluent HFF cells for 24h post-invasion, as has been described in the “Parasite culture” section. Infected HFF cells were syringe lysed to release parasites, the lysate was passed through a 3µm filter, and the filtrate containing parasites was centrifuged at 800×g. The parasite pellet was resuspended in 6× pellet volume of 10mM Tris (pH 7.4), the suspension was mixed with 1/3rd volume of 4× SDS-PAGE sample buffer, heated at 99°C for 10min, centrifuged at 20,000×g for 20min, and supernatants were processed for western blotting as has been described above for PfVMP1GFPKI parasites. The membranes were incubated with rabbit anti-HA monoclonal antibodies (for TgVMP1AIDHA, 1:1000 dilution), followed by goat anti-rabbit-HRP-conjugated secondary antibodies (at 1:20,000 dilution). For loading control, the blot was stripped, blocked, and incubated with mouse anti-ROP1 antibodies (at 1:1000 dilution) for 2h, followed by HRP-conjugated goat anti-mouse antibodies.

To assess for TgVMP1 depletion, fully confluent HFF cells in T25 flasks were infected with the RHTIR1-VMP1AID parasites and allowed to grow for 24h. Parasites were subjected to grow in the presence of 500μM auxin (+IAA) for 0, 15, 30, 60, 120 and 180 min, respectively. Following this, cells were harvested by scraping the parasite-infected HFF cells and subjecting them to centrifugation at 800×g. The obtained pellets were washed with 1× PBS and centrifuged again at 800×g. The resultant pellets were processed for western blotting as described above. TgVMP1AIDHA levels were determined by probing the blots using rabbit anti-HA antibodies, followed by goat anti-rabbit-HRP-conjugated secondary antibodies, as described above. Mouse anti-ROP1 and anti-SAG1 antibodies (at 1:1000 dilution), followed by anti-mouse-HRP antibodies (at 1:20,000 dilution) were used to probe ROP1 and SAG1 as the loading controls.

VMP1 localization
To determine the subcellular localization of PfVMP1GFP, mixed-stage PfVMP1GFPKI parasites were washed with PBS, stained with Hoechst (1µg/ml in PBS), washed with PBS, immobilized on a poly-L-lysine-coated slide, washed again to remove unattached cells, and covered with a glass coverslip. Live parasites were observed under the 100× objective of Zeiss Axioimager. Z2 fluorescence microscope, images were acquired using AxioVision software, and analyzed using ImageJ software. For co-localization, trophozoite stage PfVMP1GFPKI parasites were grown in complete medium containing the ER-Tracker Red stain (200nM) for 20min at 37°C, Hoechst (1µg/ml in complete medium) was added to the culture, and the culture was incubated for 10min. The cells were washed with PBS, immobilized on a poly-L-lysine-coated slide, washed again to remove unattached cells, and then covered with a glass coverslip. Images of live cells were captured using the 100× objective of the Olympus FV300 confocal laser scanning microscope. The images were analyzed for co-localization, and Pearson’s correlation coefficient was determined using the JACoP plugin in Fiji.

For localization of TgVMP1AIDHA, RHTIR1-VMP1AID parasites were grown in HFF cells on coverslips in 12-well plates for 38h post-invasion without indole-3-acetic acid (IAA), and processed for immunofluorescence assay (IFA). In brief, the culture medium was aspirated, cells were washed with PBS, fixed with formaldehyde (4% in PBS) for 30min, washed with PBS, permeabilized using 0.1% Triton-X100 for 30min, washed with PBS, and blocked with blocking buffer (3% BSA in PBS) for 1h. The cells were incubated with rabbit anti-HA antibodies (at 1:200 dilution) for 2h. The coverslips were washed with PBS, incubated with Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG (at 1:1000 dilution) for 1h at room temperature, incubated with DAPI (10µg/ml in PBS) for 10min, washed with PBS, air-dried, and mounted using the ProLongTM diamond antifade mountant. The cells were observed under the 100× objective, and images were acquired on a Zeiss LSM880 confocal laser scanning microscope. Airyscan mode was used in Zeiss LSM880 microscope to obtain super-resolution micrographs, images were exported using Zen Black software, and processed using ImageJ software. Maximum intensity projections of z-stack images were obtained using the z-projection of ImageJ software. Pearson’s correlation coefficient was determined using the JACoP plugin in Fiji.

For co-localization of TgVMP1AIDHA with ER-GFP, RHTIR1-VMP1AID::ER-GFP parasites were grown without IAA, and processed for IFA using mouse anti-HA antibodies (at 1:200 dilution) and rabbit anti-GFP antibodies (at 1:200 dilution), followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG and Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) as has been described above for TgVMP1. Images were acquired using a Zeiss LSM880 confocal laser scanning microscope. Airyscan mode was used in Zeiss LSM880 microscope to obtain super-resolution micrographs, images were exported using Zen Black software, and processed using ImageJ software. Pearson’s correlation coefficient was determined using the JACoP plugin in Fiji. For co-localization of TgVMP1AIDHA with GRASP-GFP, RHTIR1-VMP1AID::GRASP-GFP parasites were grown without IAA, and processed for IFA using mouse anti-HA (at 1:200 dilution) and rabbit anti-GFP antibodies (at 1:200 dilution), followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG and Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) as has been described above for TgVMP1. The cells were observed under the 100× objective of Olympus FV3000 confocal laser scanning microscope, images were captured using Fluoview FV31S-SW software, and processed as described above for TgVMP1. Pearson’s correlation coefficient was determined using the JACoP plugin in Fiji.

For TgVMP1 localization under IAA-induced depletion condition, RHTIR1-VMP1AID parasites were grown in HFF cells on coverslips in 12-well plates for 24h post-invasion, followed by growth for an additional 14h in the presence of 0.1% ethanol (-IAA) or 500µM (+IAA) indole-3-acetic acid, and processed for IFA as has been described for TgVMP1. Infected HFF cells were processed for IFA using rabbit anti-HA (at 1:200 dilution) and mouse anti-SAG1 (at 1:500 dilution) antibodies for 2h, followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 647-conjugated goat anti-mouse antibodies) as has been described above for TgVMP1. The cells were observed under the 100× objective, and images were acquired using Airyscan mode on a Zeiss LSM 880 confocal laser scanning microscope, exported using Zen Black software, and processed using ImageJ software. Maximum intensity projections of the z-stacks were obtained using the z-projection of ImageJ software.

To study the effect of TgVMP1 depletion on the inner membrane complex, RHTIR1-VMP1AID parasites were grown with (+IAA) or without (-IAA) indole-3-acetic acid, and processed for IFA using rabbit anti-IMC1 antibodies (at 1:500 dilution) and mouse anti-HA (at 1:200 dilution) antibodies, followed by appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG and Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG (1:1000 dilution)) as has been described above for TgVMP1. The cells were observed under the 100× objective of Zeiss LSM880 confocal laser scanning microscope, images were acquired using Airyscan mode, exported using Zen Black software, and processed using ImageJ software. Maximum intensity projections of the z-stacks were obtained using the z-projection option in ImageJ software.

To study the effect of TgVMP1 depletion on ER, the RHTIR1-VMP1AID::ER-GFP parasites were grown in HFF cells on coverslips in 12-well plates for 24h post-invasion, and then grown for an additional 14h with (+IAA) or without (-IAA) IAA. The intracellular or purified parasites were fixed in formaldehyde (4%) and processed for IFA using rabbit anti-GFP (at 1:200 dilution) and mouse anti-HA (at 1:200 dilution) antibodies, followed by appropriate secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor 647-conjugated goat anti-mouse antibodies) as has been described above for TgVMP1. The cells were observed under the 100× objective of Olympus FV3000 confocal laser scanning microscope, and images were acquired using Fluoview FV31S-SW software and processed using ImageJ software.

Effect of PfVMP1 depletion on parasite growth
Wild-type P. falciparum D10 and 3D7 strains, PfVMP1dKD, and PfVMP1AID parasites were cultured and synchronized as described in the “Parasite culture” section. PfVMP1dKD and PfVMP1AID parasites were assessed for PfVMP1 depletion and its effect on parasite growth. To check conditional PfVMP1 depletion, ring stage PfVMP1dKD parasites were grown at 8-10% parasitemia under control (+TMP-GlcN), single knockdown (-TMP-GlcN, +TMP+GlcN) or dual knockdown (-TMP+GlcN) conditions for two consecutive cycles (96h) as has been described in the “Parasite culture” section. Parasites (at 8-10% parasitemia) were harvested at 48 and 96h time points, and parasite lysates were processed for western blotting using rabbit anti-HA antibodies (1:1000 dilution), followed by HRP-conjugated goat anti-rabbit antibodies (1:20,000 dilution) as has been described in the “VMP1 expression” section. For loading control, the membrane was stripped off and probed with mouse HRP-conjugated anti-β-actin antibodies (at 1:30,000 dilution). To assess the effect of PfVMP1 deletion on parasite growth, synchronized ring stage PfVMP1dKD parasites were grown under control (+TMP-GlcN), single knockdown (-TMP-GlcN; +TMP+GlcN) or dual knockdown (-TMP+GlcN) conditions for 4 consecutive cycles (192h). The number of parasite-infected RBCs was determined by FACS using a BD Fortessa FACS analyzer, as has been described earlier88, data were presented as % parasitemia over 4 cycles using GraphPad Prism.

Likewise, PfVMP1AID parasites were assessed for 5Ph-IAA-inducible PfVMP1 depletion and the effect of depletion on parasite growth. Briefly, three different clonal lines of the ring stage PfVMP1AID parasites were cultured in the presence or absence of 1μM 5Ph-IAA for two consecutive erythrocytic cycles, as has been described in the “Parasite culture” section. Parasites (at 8-10% parasitemia) were harvested at 24, 48, 72, and 96h time points. The parasite lysates were processed for western blotting using rabbit anti-HA antibodies (1:1000 dilution), followed by HRP-conjugated goat anti-rabbit antibodies (1:20,000 dilution) as has been described in the “VMP1 expression” section. The membrane was stripped off and probed for β-actin as a loading control using mouse HRP-conjugated anti-β-actin antibodies (at 1:30,000 dilution). For assessing the effect of PfVMP1 depletion on parasite growth, three different clonal lines of the ring stage PfVMP1AID parasites were cultured in the presence (+A) or absence (-A) of 1μM 5Ph-IAA for three consecutive erythrocytic cycles as has been described in the “Parasite culture” section. Parasitemia was maintained at 3–4%, Giemsa-stained smears were made at the end of each cycle, observed under the 100× objective of a light microscope, and at least 1000 RBCs were counted to determine the percent parasitemia. Data were presented as % parasitemia over three cycles using the GraphPad Prism.

To check TIR1 expression in PfVMP1AID parasites, wild-type 3D7 and three clonal lines of PfVMP1AID parasites were harvested at mid-trophozoite/schizont stage at 8-10% parasitemia. Parasite lysates were processed for western blotting using rabbit anti-TIR1 antibodies (1:1000 dilution), followed by HRP-conjugated goat anti-rabbit antibodies (1:20,000 dilution) as has been described in the “VMP1 expression” section. The membrane was stripped off and probed for β-actin as a loading control using mouse HRP-conjugated anti-β-actin antibodies (at 1:30,000 dilution).

Effect of TgVMP1 depletion on parasite growth
To assess the effect of TgVMP1 depletion at the protein level, we compared RHTIR1 and RHTIR1-VMP1AID parasites for the overall development by plaque assay and intracellular development by replication assay, essentially as has been previously described89,90. For plaque assay, fully confluent HFF monolayers in 6-well plates were infected with 500 freshly egressed RHTIR1 or RHTIR1-VMP1AID parasites per well, grown in the presence of 0.1% ethanol (-IAA) or 500µM IAA (+IAA) for 8 days at 37°C. The cells were fixed with methanol for 5min at room temperature, stained with Giemsa for 10min, and washed with distilled water. The plates were air-dried, observed for plaques, and images were acquired using the ChemiDoc Imaging System (Bio-Rad). The area of each of the 100 plaques from 3 independent experiments was measured using ImageJ software, and presented as mean plaque area (mm2) using the GraphPad Prism software.

For the replication assay, approximately 106 freshly purified RHTIR1 or RHTIR1-VMP1AID parasites were added to fully confluent HFF cells grown on coverslips in 12-well plates. After 3h of invasion, extracellular parasites were removed by washing with PBS, and infected cells were grown for 24h in the presence of 0.1% ethanol (-IAA) or 500µM (+IAA) IAA. The culture media was carefully aspirated, cells were fixed with formaldehyde (4% in PBS), and processed for IFA using mouse anti-GRA1 antibodies (1:500 dilution), followed by Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG antibodies and DAPI (10µg/ml) staining as has been described for TgVMP1 in the “VMP1 localization” section. The number of parasites/PV was determined by observing at least 100 random PVs from 3 independent experiments, and presented as the number of PVs containing different numbers of parasites as a percentage of the total number of PVs.

Effect of TgVMP1 depletion on in vivo development
Female BALB/cJ mice were infected with RHTIR1-VMP1AID parasites to determine the effect of TgVMP1 depletion on in vivo development, as has been described previously91. RHTIR1-VMP1AID parasites were cultured without IAA for 48h post-infection, and purified as described in the “Parasite culture” section. 8-10 weeks old 20 female BALB/cJ mice were infected intraperitoneally with purified RHTIR1-VMP1AID tachyzoites (200 parasites/mouse), and five age-matched mice were used as uninfected naïve controls. The infected mice were divided into 2 groups (10 mice/group). Mice in one infected group were given 0.2ml water with 12.5mg/ml indole-3-acetic acid (+IAA) by oral gavage every day, and also provided drinking water with IAA (0.5mg/ml) and sucrose (5% (w/v)). Another infected group was given 0.2ml water only by oral gavage every day and also provided drinking water with (5% sucrose (w/v)). The naïve control group was given 0.2ml water with 12.5mg/ml IAA by oral gavage every day and also provided drinking water with IAA (0.5mg/ml) and sucrose (5% (w/v)). Drinking water was changed every 2–3 days, and mice were monitored for 21 days. Peritoneal exudates of mice were collected on the day of their death and examined for the presence of parasites using mouse anti-SAG1 antibodies (for SAG1) and rabbit anti-HA antibodies (for TgVMP1AIDHA) by IFA as has been described in the “VMP1 localization” section.

Effect of TgVMP1 depletion on gliding motility
To assess the effect of TgVMP1 depletion on gliding motility of tachyzoites, we compared the motility of RHTIR1-VMP1AID parasites cultured with or without IAA as has been previously described92. In brief, RHTIR1-VMP1AID parasites were added to confluent HFF cells in a T75 flask, allowed to invade the cells for 3h, and extracellular parasites were removed by washing the cells with D10 medium. The cells were grown for 24h, followed by an additional 14h in the presence of 0.1% ethanol (-IAA) or 500µM IAA (+IAA), and parasites were purified as has been described in the “Parasite culture” section. For assessing parasite viability, purified -IAA or +IAA parasites were incubated in PBS with propidium iodide (PI; 5μg/ml) for 15min at room temperature, washed with PBS, and immediately observed under the 100× objective of Zeiss Axioimager. Z2 fluorescence microscope. Images were acquired using AxioVision software, analyzed using ImageJ software, and the number of PI-negative parasites was counted and presented as a percentage of the total number of parasites observed using GraphPad Prism.

2×106 purified tachyzoites were resuspended in HBSS, allowed to glide on FBS-coated coverslips in a 12-well plate for 15min, fixed with formaldehyde (4% in PBS), and processed for IFA using mouse anti-SAG1 antibodies (at 1/500 dilution), followed by Alexa Fluor 488-conjugated F(ab’) donkey anti-mouse IgG antibodies as has been described for TgVMP1 in the “VMP1 localization” section. Parasites were imaged for gliding trails under 100× objective of the Axioimager. Z2 using AxioVision software, and analyzed using ImageJ software. At least 100 parasites were scored for the presence of gliding trails in each of the three independent experiments, and the number of parasites showing gliding trails was presented as a percentage of the total number of parasites observed. Motile parasites in each of the three experiments were observed for measuring the gliding trail length, and presented as mean trail length (μm) using GraphPad Prism.

Effect of TgVMP1 depletion on host cell invasion
The invasion efficiency of RHTIR1-VMP1AID parasites was determined according to the previously reported two-color invasion assay93. RHTIR1-VMP1AID parasites were cultured with or without IAA as has been described in the “Effect of TgVMP1 depletion on gliding motility” section. Purified tachyzoites were added to confluent HFF cells grown on coverslips in 12-well plates (MOI 2:1), and incubated for 3h with 0.1% ethanol (–IAA) or 500µM IAA (+IAA). The cells were washed with PBS, fixed with formaldehyde (4% in PBS), and processed for IFA as has been described for TgVMP1 in the “VMP1 localization” section. In brief, the cells were first stained for extracellular parasites with mouse anti-SAG1 antibodies, followed by Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG antibodies (at 1:1000 dilution). The cells were washed with PBS, permeabilized with 0.1% Triton-X100, washed with PBS, blocked, incubated again with mouse anti-SAG1 antibodies to stain intracellular parasites, followed by Alexa Fluor 488-conjugated F(ab’) donkey anti-mouse IgG antibodies. The cells were observed for red/green (failed to invade) and green only (successful invasion) parasites under the 100× objective of a Zeiss Axioimager Z2 fluorescence microscope, and images were acquired using AxioVision software. The number of red and green parasites was determined, and the number of green parasites was presented as a percentage of the total number of parasites (% Invasion) observed using GraphPad Prism. The experiment was repeated three times, and at least 100 parasites were observed in each experiment.

Effect of TgVMP1 depletion on egress
HFF cells were grown on coverslips in 12-well plates, infected with purified RHTIR1 or RHTIR1-VMP1AID parasites (at 1:1 MOI), and grown for 48h, followed by an additional 14h with 0.1% ethanol (-IAA) or 500µM IAA (+IAA). The cells were washed with PBS, treated with HBSS containing 2µM ionomycin, a calcium ionophore that induces rapid T. gondii egress94. HBSS was gently removed from the wells, the cells were fixed with 4% formaldehyde (in PBS), processed for IFA using mouse anti-GRA1 antibodies, followed by Alexa Fluor 647-F(ab’) donkey anti-mouse IgG and DAPI as described in the “VMP1 localization” section. The cells were observed under 100× objective of the Zeiss Axioimager. Z2 microscope, images were acquired using AxioVision software, and the total number of sites with well-dispersed parasites or clumps of parasites, which mark the PVs from which parasites egressed, was determined by counting at least 100 vacuoles in each of the three independent experiments. The number of PVs with parasite clumps was presented as a percentage of the total number of PVs observed using GraphPad Prism.

Conoid extrusion assay
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section, and evaluated for conoid extrusion using the calcium ionophore A23187 as has been previously described95. Briefly, ~2×106 purified tachyzoites were washed with PBS, resuspended in HBSS, and allowed to settle on coverslips in a 12-well plate for 10min. The parasites were treated with 2µM A23187 (in HBSS) for 10min, fixed with 4% formaldehyde (in PBS), observed under 100× objective of the Axioimager. Z2 microscope, and images were acquired using AxioVision software, and analyzed using ImageJ software. At least 100 parasites were observed for conoid extrusion in each of the three independent experiments, and the number of parasites showing conoid extrusion was presented as a percentage of the total number of parasites observed using GraphPad Prism.

Effect of TgVMP1 depletion on microneme organization and secretion
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section, and assessed for microneme organization and secretion using antibodies to microneme proteins. For microneme organization, parasite-infected cells were fixed with 4% formaldehyde (in PBS), and processed for IFA as has been described in “VMP1 localization” section. The cells were incubated with antibodies to microneme proteins (rabbit anti-AMA1 at 1:500 dilution; mouse anti-MIC2 at 1:500 dilution; mouse anti-MIC3 at 1:500 dilution) and rabbit or mouse anti-HA antibodies (for TgVMPAIDHA at 1:200 dilution), followed by appropriate secondary antibodies (Alexa 488- or Alexa 647-conjugated F(ab’) donkey anti-mouse or anti-rabbit IgG) and DAPI (10µg/ml). The cells were observed under 100× objective of the Zeiss LSM 880 confocal laser scanning microscope, and images were acquired in Airyscan mode using Zen Black software, and processed using ImageJ software. Maximum intensity projections of z-stack images were obtained using the z-projection option of ImageJ software.

Purified tachyzoites were assessed for microneme secretion according to the previously reported method96. Briefly, ~3×107 purified tachyzoites were resuspended in 100µl of DGH medium (DMEM with 2mM glutamine and 10mM HEPES), treated with ethanol (1% v/v) at 37°C for 2min to induce secretion, incubated on ice to block further secretion, and centrifuged at 1000×g for 8min. The supernatant containing the extracellular secreted antigens (ESA) and the pellet were separated, each fraction was mixed with an equal volume of SDS-PAGE sample buffer, and assessed for MIC2 level by western blotting using mouse anti-MIC2 (at 1:2000 dilution) and anti-ROP1 antibodies as has been described in the “VMP1 expression” section. The signal intensities of ROP1 and MIC2 in pellet fractions were measured using ImageJ, the signal intensity of MIC2 was normalized as times of the ROP1 signal intensity for the corresponding parasites, and plotted using GraphPad Prism. The signal intensities of MIC2 in ESA fractions were measured, normalized as times of the MIC2 signal intensity in the corresponding pellet fraction, and plotted using GraphPad Prism. The experiment was repeated four times.

Effect of TgVMP1 depletion on rhoptry organization
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section, and assessed for rhoptry organization by IFA using anti-ROP1 antibodies as has been described in the “VMP1 localization” section. The infected cells were incubated with mouse anti-ROP1 (at 1:500 dilution) and rabbit anti-HA antibodies (for TgVMPAIDHA, at 1:200 dilution), followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG, Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) and DAPI (10µg/ml). The cells were observed under the 100× objective of the Zeiss LSM 880 confocal laser scanning microscope, and images were acquired in Airyscan mode using Zen Black software and processed using ImageJ software. Maximum intensity projections of z-stack images were obtained using Z-projection of ImageJ software.

For co-localization of rhoptries with the ER-GFP, RHTIR1-VMP1AID::ER-GFP parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section, tachyzoites were purified, and processed for IFA as described in the “VMP1 localization” section using mouse anti-ROP1 (at 1:500 dilution) and rabbit anti-GFP antibodies (for ER-GFP, at 1:200 dilution), followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG, Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) and DAPI (10µg/ml). The cells were observed under the 100× objective of the Zeiss LSM 880 confocal laser scanning microscope, images were acquired in Airyscan mode using Zen Black software, and processed using ImageJ software.

Effect of TgVMP1 depletion on rhoptry secretion
Rhoptries discharge their contents as vacuoles (e-vacuoles) into the host cell upon attachment with the host cell during invasion, which can be monitored using ROP1 as a marker, as has been previously described97. Briefly, RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section. 107 purified tachyzoites were treated with 1µM cytochalasin D (CytD) for 10min, which prevents invasion, but not rhoptry discharge98. 5×106 CytD-treated parasites were added to confluent HFF cells grown on coverslips in 12-well plates, incubated at 37°C for 15min, washed with PBS, fixed with formaldehyde (4% in PBS), and processed for IFA using mouse anti-ROP1 antibodies, followed by incubation with Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG and DAPI (10µg/ml) as has been described in the “VMP1 localization” section. The cells were observed under 100× objective of the Zeiss Axioimager. Z2 microscope, and at least 100 parasites were observed for the presence of e-vacuoles. The experiment was repeated three times, and data was presented as the number of e-vacuole-positive parasites as a percentage of the total parasites using GraphPad Prism.

Effect of TgVMP1 depletion on dense granule biogenesis
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section. Intracellular parasites were evaluated for localization of dense granule proteins GRA1 and GRA2 in the PV by IFA, as has been described in the “VMP1 localization” section. In brief, intracellular parasites were incubated with mouse anti-GRA1 (at 1:500 dilution) or mouse anti-GRA2 (at 1:500 dilution) and rabbit anti-HA antibodies (for TgVMPAIDHA, at 1:200 dilution), followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG and Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) and DAPI (10µg/ml). The cells were observed for GRA1 and GRA2 localization patterns under 100× objective of the Zeiss LSM 880 confocal laser scanning microscope, images were acquired in Airyscan mode using Zen Black software, and processed using ImageJ software. Maximum intensity projections of z-stack images were obtained using z-projection of ImageJ software. ≥50 PVs were examined for the presence of GRA1 and GRA2 localization patterns, and the experiment was repeated three times. The number of PVs with IVN was presented as a percentage of the total number of PVs observed using GraphPad Prism.

To determine biogenesis of dense granules, purified -IAA and +IAA tachyzoites were added on poly-L-lysine-coated slides, and processed for IFA using mouse anti-GRA1 (at 1:500 dilution) or mouse anti-GRA2 (at 1:500 dilution) antibodies, followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-mouse IgG) and DAPI (10µg/ml). The cells were observed under the 100× objective of the Olympus FV3000 confocal laser scanning microscope, and images were captured using Fluoview FV31S-SW software. For super-resolution images, cells were observed under the 100× objective of the Zeiss LSM 880 confocal laser scanning microscope, and images were acquired in Airyscan mode using Zen Black software. Images were processed using ImageJ software. The fluorescence signal intensity of GRA1 or GRA2 in each tachyzoite was measured using the Multi Measure option of the ROI manager, and calculated using the formula “IntDen-(Area of interest×mean signal intensity of the background)”. The mean signal intensity of +IAA parasites was plotted as a percentage of the mean signal intensity of -IAA parasites (% Signal intensity) based on multiple -IAA (n=338) and +IAA (n=383) parasites from three independent experiments. The cells were also analyzed for intraparasitic distribution of GRA1 or GRA2 signal, and the number of parasites with perinuclear accumulation of GRA1 or GRA2 signal was presented as a percentage of the total number of parasites observed using GraphPad Prism.

For co-localization of dense granules with ER-GFP, RHTIR1-VMP1AID::ER-GFP parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section, tachyzoites were purified, and processed for IFA as described in the “VMP1 localization” section using mouse anti-GRA1 (at 1:500 dilution) or mouse anti-GRA2 (at 1:500 dilution) and rabbit anti-GFP antibodies (for ER-GFP, at 1:200 dilution), followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG and Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) and DAPI (10µg/ml). The cells were observed under a 100× objective of the Zeiss LSM 880 confocal laser scanning microscope, images were acquired in Airyscan mode using Zen Black software, and processed using ImageJ software.

Effect of TgVMP1 depletion on lipid droplets
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section, tachyzoites were purified, stained with Nile red (250ng/ml in complete DMEM) at 37 ⁰C for 30min, washed with PBS, and observed for fluorescence (λex=488 nm, λem=530 nm) under 100× objective of the Zeiss LSM 880 confocal laser scanning microscope, images were acquired using Zen Black software, and processed using ImageJ software. The number of lipid droplets (LDs) was determined and presented as the number of LDs/parasite using GraphPad Prism. We also measured the surface area (µm2) of each LD using the analyze particles option of ImageJ software, and presented it as mean LD size (μm2) using GraphPad Prism.

Immunoprecipitation and mass spectrometry
For immunoprecipitation of TgVMP1AIDHA, RHTIR1 and RHTIR1-VMP1AID parasites were cultured without IAA for 48h post-invasion, as has been described in the “Parasite culture” section. The purified tachyzoite pellets were resuspended in 300µl of lysis buffer (10mM Tris, 150mM NaCl, 5mM EDTA, 0.1% NP-40, 10% glycerol, 1× protease inhibitor cocktail, pH 7.5), incubated for 30min on ice with intermittent mixing, sonicated using Bioruptor for 5min (30s On/30s Off at high amplitude), and the lysates were incubated for 30min in ice. The lysates were centrifuged at 4°C for 15min at 20,000×g, and the supernatants were collected. The pellets were again extracted with 150µl of lysis buffer as described above, and supernatants were pooled with the respective first supernatant. The supernatant was incubated with 5µg of rabbit anti-HA antibodies for 12h at 4°C, mixed with protein A/G magnetic beads (10µl slurry/2mg of protein) for 2h at 4°C, and washed with lysis buffer. The beads were washed with lysis buffer, mixed with an equal volume of 2× SDS-PAGE sample buffer, incubated at 99°C for 10min, and the supernatant was collected as an eluate. Aliquots of input, flow through, wash, and eluate were processed for western blotting using mouse anti-HA antibodies (1:1000 dilution), followed by goat HRP-conjugated anti-mouse antibodies (1:20,000 dilution), as has been described in the “VMP1 expression” section. The remaining eluate was subjected to mass spectrometry analysis.

The immunoprecipitates were run on a 12% SDS-PAGE gel until the protein size ladder completely entered the gel. The protein-containing region was excised and processed for trypsin digestion and peptide extraction as has been described previously81,82. The peptide solution was run on Q Executive HF (Thermo Scientific) for HCD mode fragmentation and LC-MS/MS analysis. The raw data were imported into the Proteome Discoverer 2.2 software (Thermo Scientific), and proteins were identified by searching against the T. gondii RH-88 Uniprot databases using the SEQUEST HT algorithm. The analysis parameters included trypsin enzyme specificity, a maximum of two missed cleavages, variable modifications, such as carbamidomethylation of cysteine, oxidation of methionine, and deamidation of asparagine/glutamine. Precursor tolerance was set to 5 ppm, and fragmentation tolerance was set to 0.05 Da. Peptide spectral matches (PSM) and peptide identifications grouped into proteins were validated using the Percolator algorithm and filtered to a 1% false discovery rate (FDR). The peptide abundances in RHTIR1 and TgVMP1AIDHA immunoprecipitates from three biological repeats were compared for enrichment analysis using MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/) with a single-factor statistical analysis. Imputation was done by replacing missing values with 1/5th of the minimum positive value of each variable, and normalization was performed based on the sum of the abundances. The abundance value was transformed to Log10, and the auto-scaling option was used for data scaling. Proteins enriched ≥1.5-fold with a p-value<0.05 in TgVMP1AIDHA immunoprecipitate compared to the RH-TIR1 immunoprecipitate were considered significant interactors of TgVMP1.

Scramblase assay
TgVMP1AIDHA and PfVMP1AIDHA proteins were purified from RHTIR1-VMP1AID and PfVMP1AID parasites, respectively, as described in the “Immunoprecipitation and mass spectrometry” section. Briefly, the parasite lysate supernatants were incubated with HA-Trap magnetic agarose beads (ChromoTek; 25µl beads for 2mg/ml protein) for 12h at 4°C. The beads were washed (wash buffer: 20mM Tris, 500mM NaCl, 0.03% n-dodecyl β-D-maltoside (DDM), pH 7.4), and the bound proteins were eluted in 150µl elution buffer (25mM Tris, 150mM NaCl, pH 7.4) with HA peptide (0.64mg/ml). The eluate was diluted ~100 fold with storage buffer (20mM Tris, 150mM NaCl, 0.03% DDM, 5% glycerol and 1× protease inhibitor cocktail, pH 7.4), concentrated to ~50µl using Amicon Ultra centrifugal filter (10 kDa cut-off) at 4°C, flash frozen in liquid nitrogen, and stored at -80°C until further use. Aliquots of input, flow through, washes, and eluate fractions were checked for the presence of TgVMP1AIDHA or PfVMP1AIDHA proteins by western blotting using rabbit anti-HA antibodies (at 1:1000 dilution), followed by goat HRP-conjugated anti-rabbit antibodies (1:20,000 dilution) as has been described in the “VMP1 expression” section.

Liposomes were prepared as has been reported previously22,99. In brief, POPC, POPS, and NBD-PS were dissolved in chloroform. POPC and POPS were mixed at 9:1 molar ratio along with 0.4 mol% NBD-PS. The lipid mixture was dried in a glass bottle under a nitrogen stream to form a uniform layer, followed by vacuum drying at room temperature for 12h. The dried lipid layer was rehydrated using rehydration buffer (50mM HEPES, 100mM NaCl, pH 7.5) to obtain multilamellar vesicles (MLVs) at a final concentration of 10.5mM. MLVs were subjected to 10 rounds of freeze-thaw cycles and diluted in rehydration buffer to 5.25mM. The sample was passed 21 times through a 0.4µm filter, followed by 11 times through a 0.2µm filter using a mini extruder (Avanti Polar Lipids) to obtain uniform-sized liposomes. The liposomes were stored at 4°C until further use.

Scramblase assay was performed as has been described previously22,99. Liposomes (5.25mM) were destabilized by mixing with DDM (0.36% w/v final), and the mixture was incubated at room temperature for 3h at 10 rpm on a nutator. The destabilized liposomes were mixed with storage buffer (with 0.36% DDM) or purified proteins (TgVMP1AIDHA or PfVMP1AIDHA in storage buffer with 0.36% DDM) at a 2:1 ratio (v/v) to prepare empty-liposomes or proteoliposomes (TgVMP1-liposomes or PfVMP1-liposomes), respectively. The samples were incubated at room temperature for 1.5h to allow protein incorporation into the liposomes. DDM was removed by incubating the samples serially with 25mg Bio-Beads for 1h at room temperature, 25mg Bio-Beads for 2h at room temperature, and 50mg Bio-Beads for overnight at 4°C. The liquid phase containing liposomes was separated and diluted to 200µM using rehydration buffer. 50µl of the empty or proteoliposome sample was transferred to a 96-well flat-bottom white polystyrene plate, and assessed for fluorescence signal (λex=470 nm, λem=530 nm) using the SpectraMax iD3 multi-mode microplate reader (Molecular Devices). The fluorescence signal intensity was measured at every 26s interval for 208s, followed by dithionite (10mM final concentration) for 416s, and then with Triton X-100 (0.5% (w/v)) for 208s. Relative fluorescence intensities of empty and proteoliposomes were calculated for each time point, and plotted as (Ft - FTriton)/(F0 - FTriton), where Ft is the fluorescence intensity at the corresponding time point, F0 is the initial fluorescence intensity, and FTriton is the fluorescence intensity after addition of Triton X-100 (at the 208s time point). The relative fluorescence intensity was plotted on the y-axis over time on the x-axis using GraphPad Prism.

Expression of PfVMP1 and HsVMP1 in RHTIR1-VMP1AID parasites
A synthetic and mammalian-codon optimized PfVMP1 coding sequence (PfVMP1syn) was amplified from the pUC-PfVMP1syn plasmid using PfVMP1-Frec/PfVMP1-R primers. The T. gondii tubulin promoter region was amplified from pCTG-Fluc plasmid using TgTub-F/TgTub-PfVMP1-Rrec primers100. The PfVMP1-Frec and TgTub-PfVMP1-Rrec primers contain overlapping sequences, which allowed recombination of PfVMP1syn and tubulin promoter fragments by PCR using TgTub-F/PfVMP1-R primers. The recombined PCR product was digested with PacI/AvrII and cloned into similarly digested pMyc3×.LIC-DHFR plasmid to obtain pTub-PfVMP1syn-3×Myc-DHFR plasmid. A synthetic and T. gondii-codon-optimized HsVMP1 coding sequence (HsVMP1syn) was cloned into pJET1.2/Blunt vector, excised with EcoRV/AvrII, and subcloned into similarly digested pTub-PfVMP1syn-3×Myc-DHFR plasmid to obtain pTub-HsVMP1syn-3×Myc-DHFR plasmids. 15µg of pTub-PfVMP1syn-3×Myc-DHFR or pTub-HsVMP1syn-3×Myc-DHFR plasmid was transfected into RHTIR1-VMP1AID parasites as has been described in the “Generation of recombinant T. gondii parasites” section. The transfected parasites were selected with 1µM pyrimethamine without IAA until the resistant parasites emerged. The resistant parasites with pTub-PfVMP1syn-3×Myc-DHFR plasmid (RHTIR1-TgVMP1AID::PfVMP1Myc) would express PfVMP1Myc, and the resistant parasites with pTub-HsVMP1syn-3×Myc-DHFR plasmid would express HsVMP1Myc. These parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA). The parasite lysates were evaluated for expression of TgVMP1AIDHA (rabbit anti-HA antibodies), PfVMP1Myc or HsVMP1Myc (mouse monoclonal anti-Myc antibodies at 1:1000 dilution), and SAG1 (mouse anti-SAG1 antibodies) as a loading control by western blotting, as has been described in the “VMP1 expression” section. The RHTIR1-TgVMP1AID::PfVMP1Myc and RHTIR1-TgVMP1AID::HsVMP1Myc parasites were assessed for co-localization of TgVMP1AIDHA with PfVMP1Myc or HsVMP1Myc, respectively, by IFA as has been described in the “VMP1 localization” section. The parasites were incubated with mouse anti-Myc (at 1:200 dilution) and rabbit anti-HA antibodies (for TgVMP1, at 1:200 dilution), followed by appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG and Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) and DAPI (10µg/ml). The cells were observed under a 100× objective of the Zeiss LSM880 confocal laser scanning microscope, images were acquired in Airyscan mode using Zen Black software, and processed using ImageJ software. Pearson’s correlation coefficient for co-localization was determined using the JACoP plugin in Fiji.

To study whether PfVMP1Myc or HsVMP1Myc complement the growth of TgVMP1-depleted parasites, the parental RHTIR1-VMP1AID and complemented (RHTIR1-TgVMP1AID::PfVMP1Myc, RHTIR1-TgVMP1AID::HsVMP1Myc) parasites were grown without (–IAA) or with (+IAA) IAA, and compared for overall growth by plaque assay and intracellular development by replication assay as has been described in the “Effect of TgVMP1 depletion on parasite growth” section. Plaques were observed using the 1× objective at 40× digital zoom of AXIO Zoom.V16 microscope, and images were taken using the Zen Blue software.

To evaluate whether PfVMP1Myc or HsVMP1Myc can rescue the dense granule biogenesis defect, the parental RHTIR1-VMP1AID and complemented (RHTIR1-TgVMP1AID::PfVMP1Myc, RHTIR1-TgVMP1AID::HsVMP1Myc) parasites were grown without (–IAA) or with (+IAA) IAA, and compared for localization of dense granule protein GRA2 as has been described in the “Effect of TgVMP1 depletion on IVN formation, and dense granule secretion and biogenesis” section.

Intracellular calcium monitoring
RHTIR1-VMP1AID parasites were cultured without (-IAA) or with (+IAA) IAA for 14h, and tachyzoites were purified as described in the “Parasite culture” section. The parasites were evaluated for intracellular calcium levels using Fluo-4 AM as has been described previously101. The purified parasites were washed with Ringer’s solution (10mM HEPES, 2mM CaCl2, 1mM MgCl2, 4.5mM KCl, 155mM NaCl, 10mM D-glucose, pH 7.4), incubated with 250nM of Fluo-4 AM for 10min, washed with and resuspended in Ringer’s solution, transferred to poly-L-lysine-coated glass slides, and observed under the 100× objective of Olympus FV3000 confocal microscope. Images were acquired using Fluoview FV31S-SW software, the fluorescence signal intensity/tachyzoite was measured using the Multi Measure option of the ROI manager of ImageJ software, and calculated using the formula “IntDen-(Area of interest×mean signal intensity of the background)”. Multiple parasites from three independent experiments were observed, and the mean signal intensity/parasite of +IAA parasites was presented as a percentage of the mean signal intensity/parasite of -IAA parasites using GraphPad Prism.

Transmission electron microscopy
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section. Intracellular parasites were processed for transmission electron microscopy (TEM) as has been previously described102. Briefly, parasites were washed with 0.1M phosphate buffer (PB, pH 7.4) and fixed (PB with 2.5% glutaraldehyde and 2% paraformaldehyde) overnight at 4°C. The fixed cells were scraped off, washed with PB, incubated in 1% osmium tetroxide (in PB) for 1h at room temperature, washed twice with distilled water, stained en bloc with 2% aqueous uranyl acetate for 1h, and washed twice with distilled water. The sample was dehydrated using increasing percentages of ethanol (30%, 70%, 80%, 90%, and 95%) for 10min each, followed by three treatments with 100% ethanol for 15min each. The sample was infiltrated with epoxy and ethanol mixtures (1:3 for 2h, 1:1 for 12h, and 3:1 for 3h) at room temperature. This was followed by sequential incubations in 100% epoxy resin for 3h, 12h, and 3h. The cells were transferred to 100 % epoxy and polymerized at 62⁰C for 72h. Ultrathin sections (50–70nm thickness) were cut using an ultramicrotome (Ultracut UCT; Leica Microsystems) using a glass knife. These sections were transferred onto a 200 mesh copper grid, air-dried, and stained with uranyl acetate and lead citrate for 5min each. These sections were visualized using a Talos L120C transmission electron microscope operating at 120kV, and imaged using 4k×4k Ceta CMOS camera. Images were acquired and processed using Velox software.

Immunoelectron microscopy
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section. Immunoelectron microscopy (IEM) of intracellular parasites was performed essentially as previously described102. Briefly, cells were washed with 0.1M phosphate buffer (PB), fixed (PB with 4% paraformaldehyde and 0.1% glutaraldehyde) for 15min at room temperature, followed by fresh fixative for 2h at 4°C. The cells were scraped off, washed with PB, dehydrated sequentially using increasing percentage of ethanol (30%, 50%, 70%, 80%, 90%, and 95%) for 10min each, and then dehydrated twice with absolute ethanol for 15min each. The cells were incubated twice in ethanol-LR white mixture (1:1) for 1h each with a change of the mixture. The cells were incubated in 100% LR white for 8h at 4°C, followed by incubation in 100% LR white for 2h at room temperature, and incubated at 55°C for 72h to polymerize. Ultrathin sections (50–70nm) were cut using an ultramicrotome and transferred onto nickel-coated grids. The grids were quenched with 50mM NH₄Cl (in PB) for 10min and blocked (2% skimmed milk in PBT (PB with 0.05% Tween-20)) for 1h at room temperature. The grids were incubated with mouse anti-ROP1, anti-GRA2 or anti-MIC2 antibodies (1:20 dilution in PBT with 3% BSA) for overnight at 4°C without agitation, washed with PBT, incubated with 6nm gold-conjugated goat-anti-mouse IgG antibodies (1:20 dilution in PBT with 3% BSA) for 1h, and washed with PBT. The grids were post-fixed with 2.5% glutaraldehyde for 10min, washed with distilled water, stained with uranyl acetate, washed again with distilled water, and air-dried. The sections were imaged as described above in the “Transmission electron microscopy” section.

We evaluated GRA2 secretion by extracellular parasites with modification of a previously described method103. Briefly, RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section. 5×103 purified tachyzoites (in 10μl DGH medium) were placed onto a glow-discharged carbon-coated 200-mesh nickel grids, and allowed to glide for 15min at room temperature. Excess solution was gently wicked off, the grids were fixed with 1% paraformaldehyde for 10min, and probed with anti-mouse-GRA2 primary antibodies (1:20 dilution), followed by 6nm gold-conjugated anti-mouse IgG (1:20 dilution) as has been described above for ultrathin sections. Gold particles localized in the vicinity of each parasite were counted and presented as the number of particles/1µm² area of the micrograph.

Effect of TgVMP1 depletion on apicoplast
RHTIR1-VMP1AID parasites were cultured with 0.1% ethanol (-IAA) or 500µM IAA (+IAA) as has been described in the “Effect of TgVMP1 depletion on gliding motility” section, and assessed for the presence of apicoplast by IFA using antibodies to acyl carrier protein (ACP) as has been described in the “VMP1 localization” section. The infected cells were incubated with rabbit anti-ACP (at 1:500 dilution) and mouse anti-HA (at 1:200 dilution) antibodies, followed by incubation with appropriate secondary antibodies (Alexa Fluor 488-conjugated F(ab’) donkey anti-rabbit IgG, Alexa Fluor 647-conjugated F(ab’) donkey anti-mouse IgG) and DAPI (10µg/ml). The cells were observed under the 100× objective of the Olympus FV3000 confocal laser scanning microscope, and images were acquired using Fluoview FV31S-SW software and processed using ImageJ software. Maximum intensity projections of z-stack images were obtained using the Z-projection of the ImageJ software. At least 150 parasites were observed to determine apicoplast/nucleus ratio in each of the three independent experiments, and data were plotted using GraphPad Prism.

Statistical analysis
The significance of the difference between the two groups was determined by using a two-tailed Student’s t-test, and the p-values are mentioned in the respective figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper entitled « An ER-resident lipid scramblase is crucial for biogenesis and function of apicomplexan parasite secretory organelles » by G. Srinivas Reddy et al describes the important role of the vacuole membrane protein 1 (VMP1), a DedA superfamily member of T. gondii (TgVMP1). They showed that PfVMP1 and TgVMP1 are ER-localized lipid scramblases. While none of their attempts to knock down PfVMP1 worked in P. falciparum, TgVMP1 depletion adversely affected parasite development, motility, host cell invasion, and egress. These phenotypes were linked to impaired rhoptry and dense granule biogenesis, and decreased secretion of micronemes in TgVMP1-depleted parasites, indicating a crucial role forTgVMP1. TgVMP1 depletion impaired lipid droplet homeostasis and ER organization. They rescued the ER-localized lipid scramblase by complementing TgVMP1-depleted parasites with PfVMP1 or a homolog as distant as human VMP1 rescued the depleted parasites, indicating their functional conservation and a crucial role for ER- resident lipid scramblase activity in the biogenesis and function of secretory organelles.\nThe manuscript is well written and the experiments were correctly designed and well-conducted. Therefore, I will recommend its publication after a minor revision. The data generated are almost convincing, except the comments below.\nThe quality of this can be improved by answering the following concerns.\n1) The authors wrote at the end of the abstract, the following sentence : « The essentiality of TgVMP1 for parasite development and likely functional conservation of apicomplexan VMP1 proteins highlight their drug-target potential ». This is unnecessary because it not convincing at all that PfVPM1 and TgVPM1, which are so conserved with the distant human counterpart and this later can perfectly complement the TgVPMM1 mutant, can be used for a drug-target design. Otherwise, the authors should tell how this can happen. I will suggest to remove this kind sentence, which have no true added value.\n2) The authors do not discuss why all attempts in P. falciparum to knock down PfVPM1 do not work at all whereas there is no difficulty to genetically disrupt the function of TgVPM1 in T. gondii. What is the reason ? This is surprising since TgVPM1 and PfVPM1 are the same multilayer tarnsmembrane proteins in these both closely related apicomplexan parasites.\n3) They authors indicate several times that TgVPM1 is involved in the biogenesis of rhoptries and dense granules while it is not implicated in microneme biogenesis. The data provided to reach this conclusion are not convincing because only confocal microscopy and few antibodies have used to perform these experiments. An immuno-electron microscopy (IEM) using few antibodies specific to rhoptries, micronemes and dense granules should be used to see the subcellular distribution of gold-particules on these organelles. IEM will also demonstrate whether these proteins are properly located in the space destination after secretion.\n4) The authors indicate that the secretion of ROP proteins are disturbed while ROP1 protein appears nicely and more concentrated in the PVM in the mutants relative to the parental parasites when carefully looking at the images provided. It will be more convincing to perform serial ultrathin sections in only one chosen small vacuole containing few intracellular parasites to obtain several images that will be superimposed to reconstitute the whole bodies of the mutants and parental parasites. These experiments will allow to determine whether they are less rhoptries in the mutants versus the wild type parasites because these organelles will be counted. The number of rhoptries in mutants vers wild type parasites may impact the level of protein secretion. In addition, this experimental strategy will show if the mutant parasites still have distal and basal micronemes because a negative impact in their distributions in the apical end of the parasites may also explain why there is an effect on their secretion.", "reviewer_2": "Here, the authors described in Toxoplasma and Plasmodium a lipid scramblase, the vacuole membrane protein 1 (VMP1), that they nicely showed to be associated with ER in both parasites. They present a range biochemical, ultrastructural and experimental cell biological data, to interrogate the role of Toxoplasma VMP1. They showed that parasites have a strong defect in replication, motility and invasion, that is associated with a defect in the biogenesis of their secretory organelles dense granules and rhoptries. Moreover, TgVMP1 depletion impaired lipid droplet homeostasis and ER organization, a phenotype that can be rescued with PfVMP1 or human VMP1.\nThe characterization of DedA-like protein in apicomplexa is novel. In other organisms, the cellular abnormalities observed due to the loss of VMP1 are multiple: fragmented ER, ER-contact, distorted Golgi, impaired endocytosis and exocytosis, damaged mitochondria, and accumulation of ubiquitinated-protein aggregates and lipid droplets (LDs)… Here the authors focused only on the biogenesis of secretory organelles and lipid droplets accumulation. The characterization of the secretory organelles in the mutant is incomplete. A full analysis is needed to better assess the importance of this DedA-predicted protein in parasites. Overall structural details of the mutant are missing and the interpretation of some results is confusing.\n1. The authors performed TEM but the images shown do not allow drawing any conclusion because the poor preservation of the material : no intracellular membranes can be seen and therefore, ER and Golgi cannot be observed. This is particulary important for ER, since they claimed a change in the localisation of ER marker in the mutant (Figure S9). While they showed a strong defect in dense granules secretion, they do not analyze if dense granules are visible by TEM in the mutant ; they only focused on rhoptries. TEM should also resolves if micronemes are properly formed and present at the apex, including lateral micronemes, and sub-conoid micronemes. The analysis by IFA is not sufficient. Overall, the ultrastructure of the mutant by TEM is not conclusive and cannot support the claimed drawn by IFA, in particular, analysis of ER, dense granules, mitochondria.\n2. The authors overlook the fact that the parasite has a very serious replication defect. 75% of vacuoles contains a single parasite. This can be explained by the absence of dense granule proteins secretion, which was recently shown to be important for intracellular parasite development (Chelaghma et al. ELife 2024 ; Fu et al. PloS Pathogen 2023). However, the phenotype of replication is more drastic than in any of the mutants of annuli defective for secretion of dense granules (Chelaghma et al, eLife 2024 ; Fu et al., PlosPathogen 2023) or biogenesis of secretory organelles (Morlon-Guyot et al., Cell Mic 2015 ; Sloves et al. Cell Host microbes, 2012). This might suggest a role of lipid droplets homeostasis in growth, or potential other overlooked effects (point 1).\n3. Since DedA is important in autophagosomes formation and that PI3K, ATG8, ATG3… are linked to Apicoplast in Apicomplexa, the presence and inheritence of the apicoplast during division need to be assessed.\n4. The phenotype related to dense granules is parcelar. We would like to see further investigation of whether or not they are formed (TEM, see above). If they are visible, then to quantify secretion in more details (release in the media by quantification of GRAs in the ESA). The loss of IVM is suggested in TEM but the authors cannot quantify the association of GRA proteins with IVN by IFA (Fig 6B and S8B), we can just say that they are or not in vacuolar space. Association with IVN required specific fractionnation procedures.\n5. IFA figure 5A. The authors say that the rhoptries are fragmented. The resolution of IFA do not permit to see details of the morphologies of the rhoptries, even to see individual rhoptries. The ROP1 signal seems to be in the vacuolar space upon depletion of TgVMP1. First this should be confirmed by adding phase contrast. Second, the release of rhoptry proteins within the lumen is already observed upon depletion of proteins involved in rhoptry biogenesis (SORTILIN, VPS11, retromer complex…), and this is not discussed. Why there is still important signal in -IAA in panel 5A ?\n6. GRASP-GFP staining is weird ; it normally looks like a bar above the nucleus near the ER. I don’t think that they can use these images to claim colocalisation with Golgi.\n7. The authors observed a strong defect in motility, secretion of micronemes and rhoptries. Since most of the parasites are not able to divide, it might be difficult to distinguish between a potential defect in parasite gliding motility vs. parasites that are dead or dying and thereby are unable to exhibit active motility. Same for microneme and rhoptry secretion assays.\n8. Some of the results deserve more discussion.\n-Any explanation of why dense granules proteins but not rhoptry proteins accumulated at the ER in the mutant ?\n-VMP1 is an ER-resident protein so is expected to play a role in this compartment. Since the three secretory organelles, micronemes, rhoptry and dense granules, transit through the ER, why only dense granules and rhoptries are affected ? MICs proteins may follow, at some level, a different trafficking pathway ?\n-it is surprising to see that 50% of mice survived after infection with the mutant (+IAA) while there is no plaques at all in vitro. The authors should comment on this discrepancy between in vitro and in vivo.\n9. The interpretation of mass spectrometry data are questionable. The authors performed three independ IPs, but do not show any statistical significance of the enrichment, that will help for the conclusions, especially because almost all the proteins that they used to make their point are not enriched in the three sets of data. This is not clear how they connect an association of VMP1 with these proteins (MICs, GRAs, ROPs, IMC proteins) and biogenesis of secretory organelles.\n10. The authors referred to the absence of network when they look at IFA of dense granule proteins. The IVN is not visible by IFA.\n11. Fig. S4A. For the fusion of miniAID in Plasmodium, did the authors control by WB the expression of Tir1 in the transgenic line, which is a prerequisite for this conditional KD ?\n12. Section PfVMP1 and TgVMP1 localize to ER. ‘Towards characterization of apicomplexan VMP1 proteins, we replaced wild-type PfVMP1 and TgVMP1 coding sequences …’ This is not replacement of gene but endogenous tagging.\n13. Figure S2 : Molecular sizes of PCR products on schematic and gels are missing.\n14. Table : add accession number TGGT1"}, "author_response": "RESPONSES TO THE REVIEWER COMMENTS\nBelow are our responses to the reviewers’ comments in a point-wise manner. The comments\nare italicized and excerpts from the manuscript are highlighted with the page No. and line No.\ncorresponding to the start of excerpt in the file “Reddy, et al_Revised Manuscript”.\nREVIEWER #1\nComment 1. The authors wrote at the end of the abstract, the following sentence: The\nessentiality of TgVMP1 for parasite development and likely functional conservation of\napicomplexan VMP1 proteins highlight their drug-target potential. This is unnecessary…..I\nwill suggest to remove this kind sentence…...\nResponse. As suggested, we have removed the sentence “The essentiality of TgVMP1 for parasite development and likely functional conservation of apicomplexan VMP1 proteins highlight their drug-target potential” from the abstract on page 2.\nComment 2. The authors do not discuss why all attempts in P. falciparum to knock down\nPfVPM1 do not work at all whereas there is no difficulty to genetically disrupt the function of\nTgVPM1 in T. gondii. What is the reason ? This is surprising since TgVPM1 and PfVPM1 are\nthe same multilayer tarnsmembrane proteins in these both closely related apicomplexan\nparasites.\nResponse: We were also surprised with the failure of PfVMP1 knock-down in P. falciparum.\nWe have discussed this point in the text on page 8, line 191: “…..It is possible that intrinsic\nnature and ER membrane localization of PfVMP1 rendered it refractory to degradation\nin case of cDD and glmS systems, as has also been reported for other proteins 44,45. The\nauxin-inducible knock-down was quite effective for TgVMP1 in T. gondii but not for\nPfVMP1 in P. falciparum, which might be due to differences in the specificity of E3 ligases\nof P. falciparum and T. gondii for TIR1. Similar to our observation, previous studies\nshowed limited success of the auxin-inducible system in P. falciparum 45. ”.\nComment 3. The authors indicate several times that TgVPM1 is involved in the biogenesis of\nrhoptries and dense granules while it is not implicated in microneme biogenesis. The data\nprovided to reach this conclusion are not convincing because only confocal microscopy and\nfew antibodies have used to perform these experiments. An immuno-electron microscopy (IEM)\nusing few antibodies specific to rhoptries, micronemes and dense granules should be used to\nsee the subcellular distribution of gold-particules on these organelles. IEM will also\ndemonstrate whether these proteins are properly located in the space destination after\nsecretion.\nResponse: We thank the reviewer for this valuable suggestion, and agree that confocal\nmicroscopy alone would not provide a complete picture of the secretory organelles. We have\nperformed extensive TEM and IEM experiments to assess the effect of TgVMP1 depletion of\nmicronemes, rhoptries, and dense granules. The TEM data are in agreement with the IFA data,\nand IEM data showed labelling of organelles with the corresponding marker protein-specific\ngold particles. The new figures and related results have been incorporated in the text as\ndescribed below for each organelle:\nMicronemes. Page 14, line 349: “Transmission electron microscopy (TEM) images\nconfirmed the presence of both apical and lateral micronemes in +IAA and –IAA\nparasites (Supplementary Fig. 6C), which was also corroborated by MIC2 labelling in\nimmunoelectron microscopy (IEM) images (Supplementary Fig. 7A). Furthermore, the\nTEM micrographs showed comparable number of apical micronemes in –IAA\n(9.9±5.9/apical region, n=22 parasites) and +IAA (9.0±4.4/apical region, n=22 parasites)\nparasites, which together with the IFA data indicate that TgVMP1 depletion did not\naffect microneme organization.”.\nRhoptries. Page 17, line 396: “……For rhoptry organization, intracellular +IAA and –\nIAA tachyzoites were processed for IFA, TEM, and IEM. IFA images showed ROP1\nlocalization to characteristic rhoptry-like elongated structures with a bulbous base in the\nmajority (85.3%) of -IAA parasites as compared with only a small number (11.3%) of\n+IAA parasites (Fig. 5A, B). Similar to the intracellular parasites, purified -IAA parasites\nalso exhibited ROP1 localization to rhoptry-like elongated structures, which was not the\ncase with +IAA parasites (Fig. 5E). In agreement with the IFA data, TEM images showed\napical rhoptries in 52.2% of -IAA parasites (n=23 parasites), whereas only 18.2% of +IAA\nparasites had apical rhoptries (n=22 parasites) (Fig. 5F). ROP1 labelling of the similar\nstructures in IEM micrographs indicated ROP1 targeting to these structures\n(Supplementary Fig. 7B)”.\nDense granules. Page 20, line 483: “….TEM micrographs of both –IAA and +IAA\nparasites showed dense granules (Fig. 5F, 6C), which was corroborated by IEM showing\nGRA2 labelling of dense granules in the parasite cytoplasm and IVN in the PV\n(Supplementary Fig. 7C). The TEM micrographs of +IAA parasites contained less\nnumber of cytoplasmic dense granules (1.5/parasite section, n=153) than those of –IAA\nparasites (3.2/parasite section, n=94), which together with the IFA data indicates a crucial\nrole of TgVMP1 in dense granule biogenesis. We next compared dense granule secretion\nin the ESA of purified –IAA and +IAA parasites by IEM using GRA2. The number of\nGRA2-gold particles was comparable (Supplementary Fig. 7D, E), suggesting that\nimpaired dense granule biogenesis did not affect secretion. Nonetheless, majority of the\ngold particles were associated with electron dense material in –IAA parasites as opposed\nto +IAA parasites (Supplementary Fig. 7D), suggesting a defect in the post-secretion\nprocess that might be required for the formation of IVN.”.\nComment 4. The authors indicate that the secretion of ROP proteins are disturbed while ROP1\nprotein appears nicely and more concentrated in the PVM in the mutants relative to the\nparental parasites when carefully looking at the images provided. It will be more convincing\nto perform serial ultrathin sections in only one chosen small vacuole containing few\nintracellular parasites to obtain several images that will be superimposed to reconstitute the\nwhole bodies of the mutants and parental parasites. These experiments will allow to determine\nwhether they are less rhoptries in the mutants versus the wild type parasites because these\norganelles will be counted. The number of rhoptries in mutants vers wild type parasites may\nimpact the level of protein secretion. In addition, this experimental strategy will show if the\nmutant parasites still have distal and basal micronemes because a negative impact in their\ndistributions in the apical end of the parasites may also explain why there is an effect on their\nsecretion.\nResponse: The reviewer’s comment is valuable, and we have addressed it below in a), b), and\nc).\na). ROP1 protein appears nicely and more concentrated in the PVM in the mutants relative to\nthe parental parasites when carefully looking at the images provided.\nResponse. Indeed, ROP1 signal appears to be predominant in the PV of +IAA parasites as\ncompared to that of -IAA parasites, suggesting abnormal secretion. Nonetheless, in contrast to\nthe –IAA parasite, ROP1 signal does not localize to characteristic rhoptry-like elongated\nstructures in the +IAA parasites, indicating their absence. We have revised our analysis and\ndetermined the number of +IAA and –IAA parasites with typical rhoptry-like elongated\nstructures (Fig. 5A, B) or diffuse ROP1 signal in the PV (Fig. 5C, D). The results are described\non page 17, line 397 (“…IFA images showed ROP1 localization to characteristic rhoptry-\nlike elongated structures with a bulbous base in the majority (85.3%) of -IAA parasites\nas compared with only a small number (11.3%) of +IAA parasites (Fig. 5A, B). 59.4% of\n+IAA parasites also showed ROP1 signal in the PV compared to 10.9% of -IAA parasites\n(Fig. 5C, D).…”).\nb). It will be more convincing to perform serial ultrathin sections in only one chosen small\nvacuole containing few intracellular parasites to obtain several images that will be\nsuperimposed to reconstitute the whole bodies of the mutants and parental parasites. These\nexperiments will allow to determine whether they are less rhoptries in the mutants versus the\nwild type parasites because these organelles will be counted. The number of rhoptries in\nmutants vers wild type parasites may impact the level of protein secretion.\nResponse. We could not obtain serial ultrathin TEM sections corresponding to the same\nparasite to reconstruct a 3D image. Nonetheless, we carried out fresh TEM and IEM\nexperiments, and added new data in support of the defects in secretory organelles in TgVMP1-\ndepleted parasites, which is mentioned above in response to comment 3.\nWe also performed super-resolution Airyscan microscopy with 100 nm Z-slices,\nfollowed by 3D reconstruction of ROP1 labelling in –IAA and +IAA parasites. As evident from\nthe below figure, the mutant parasites (+IAA) do not have typical elongated rhoptries.\nAccessory Fig. 1A. 3D reconstruction of ROP1 localization. -IAA and +IAA RHTIR1-\nVMP1AID parasites were evaluated for rhotpry organization by IFA using anti-ROP1\nantibodies. Super-resolution Airyscan micrographs of the whole PVs were acquired using Zeiss\nLSM 880 confocal laser scanning microscope under the 100× objective with 100 nm optical\nsections. The representative 3D reconstructed Airyscan image shows rhoptries (magenta) and\nnucleus DAPI (cyan) in a PV with 4 parasites. Note that ROP1 signal is restricted to\ncharacteristic rhoptry-like elongated structures in -IAA parasites, as compared with\npredominantly disordered structures in +IAA parasites. Scale bar = 5 µm.\nWe could not count the number of rhoptries/parasite using the above 3D reconstruction\nimages, as IFA does not offer the resolution required to distinguish individual rhoptries.\nMoreover, the number of rhoptries is variable in Toxoplasma, and it would also vary from\nsection to section of the TEM images observed. Hence, we determined the number of parasites\nwith apical rhoptries in mutant and control parasites, and it is included on page 17, line 402\n(“……..In agreement with the IFA data, TEM images showed apical rhoptries in 50% of\n-IAA parasites (n=24 parasites), whereas only 13.6% of +IAA parasites had apical\nrhoptries (n=22 parasites) (Fig. 5F). ROP1 labelling of the similar structures in IEM\nmicrographs indicated ROP1 targeting to these structures (Supplementary Fig.\n7B)…...…..”).\nc). In addition, this experimental strategy will show if the mutant parasites still have distal and\nbasal micronemes because a negative impact in their distributions in the apical end of the\nparasites may also explain why there is an effect on their secretion.\nResponse. We performed super-resolution Airyscan microscopy with 100 nm optical sections,\nfollowed by 3D reconstruction of MIC2 labelling in –IAA and +IAA parasites. The image\nshown below indicates that both the parasites contain apical and lateral micronemes.\nAccessory Fig. 1B. 3D reconstruction of MIC2 labelling. -IAA and +IAA RHTIR1-\nVMP1AID parasites were evaluated for microneme organization by IFA using anti-MIC2\nantibodies. Super-resolution Airyscan micrographs of the whole PVs were acquired using Zeiss\nLSM 880 confocal laser scanning microscope under the 100× objective with 100 nm optical\nsections. The representative 3D reconstructed Airyscan image shows localization of MIC2 in\na PV with 2 parasites. Note MIC2 labelling in apical (A) and lateral (L) regions of both the\n+IAA and -IAA parasites, indicating the presence of apical and lateral micronemes. Scale bar\n= 2 µm.\nThe above 3D reconstruction data is consistent with the IFA (Fig. 4A-D), TEM (Supplementary\nFig. 6C), and IEM (Supplementary Fig. 7A) data, which show both apical and lateral\nmicronemes in mutant and control parasites (page 14, line 347: “……All these proteins\nshowed similar predominant apical localization in +IAA and –IAA parasites, ruling out\nany adverse effect of TgVMP1 in microneme organization (Fig. 4A-D; Supplementary\nFig. 6A, B). Transmission electron microscopy (TEM) images confirmed the presence of\nboth apical and lateral micronemes in +IAA and –IAA parasites (Supplementary Fig.\n6C), which was also corroborated by MIC2 labelling in immunoelectron microscopy\n(IEM) images (Supplementary Fig. 7A). Furthermore, the TEM micrographs showed\ncomparable number of apical micronemes in –IAA (9.9±5.9/apical region, n=22 parasites)\nand +IAA (9.0±4.4/apical region, n=22 parasites) parasites, which together with the IFA\ndata indicate that TgVMP1 depletion did not affect microneme organization.”).\nWe have not included the Accessory Fig. 1A and B in the text, as the included TEM\nmicrographs offer better appreciation of rhoptries and micronemes in mutant vs control\nparasite. However, if desired by the reviewer, we will be glad to include these figures along\nwith videos (uploaded on the portal) in the main text.\nReviewer #2\nComment 1. The authors performed TEM but the images shown do not allow drawing any\nconclusion because the poor preservation of the material : no intracellular membranes can\nbe seen and therefore, ER and Golgi cannot be observed. This is particulary important for\nER, since they claimed a change in the localisation of ER marker in the mutant (Figure S9).\nWhile they showed a strong defect in dense granules secretion, they do not analyze if dense\ngranules are visible by TEM in the mutant ; they only focused on rhoptries. TEM should also\nresolves if micronemes are properly formed and present at the apex, including lateral\nmicronemes, and sub-conoid micronemes. The analysis by IFA is not sufficient. Overall, the\nultrastructure of the mutant by TEM is not conclusive and cannot support the claimed drawn\nby IFA, in particular, analysis of ER, dense granules, mitochondria.\nResponse: We greatly appreciate reviewer’s suggestions. As suggested, we performed fresh\nexperiments for TEM and IEM, and scanned several micrographs to evaluate the effects of\nTgVMP1 depletion quantitatively on secretory organelles. We have included brand new TEM\nand IEM micrographs and related results in the text as mentioned below.\nMicronemes: Page 14, line 349: “Transmission electron microscopy (TEM) images\nconfirmed the presence of both apical and lateral micronemes in +IAA and –IAA\nparasites (Supplementary Fig. 6C), which was also corroborated by MIC2 labelling in\nimmunoelectron microscopy (IEM) images (Supplementary Fig. 7A). Furthermore, the\nTEM micrographs showed comparable number of apical micronemes in –IAA\n(9.9±5.9/apical region, n=22 parasites) and +IAA (9.0±4.4/apical region, n=22 parasites)\nparasites, which together with the IFA data indicate that TgVMP1 depletion did not\naffect microneme organization.”.\nRhoptries. Page 17, line 396: “……For rhoptry organization, intracellular +IAA and –\nIAA tachyzoites were processed for IFA, TEM, and IEM. IFA images showed ROP1\nlocalization to characteristic rhoptry-like elongated structures with a bulbous base in the\nmajority (85.3%) of -IAA parasites as compared with only a small number (11.3%) of\n+IAA parasites (Fig. 5A, B). Similar to the intracellular parasites, purified -IAA parasites\nalso exhibited ROP1 localization to rhoptry-like elongated structures, which was not the\ncase with +IAA parasites (Fig. 5E). In agreement with the IFA data, TEM images showed\napical rhoptries in 52.2% of -IAA parasites (n=23 parasites), whereas only 18.2% of +IAA\nparasites had apical rhoptries (n=22 parasites) (Fig. 5F). ROP1 labelling of the similar\nstructures in IEM micrographs indicated ROP1 targeting to these structures\n(Supplementary Fig. 7B)”.\nDense granules. Page 20, line 483: “….TEM micrographs of both –IAA and +IAA\nparasites showed dense granules (Fig. 5F, 6C), which was corroborated by IEM showing\nGRA2 labelling of dense granules in the parasite cytoplasm and IVN in the PV\n(Supplementary Fig. 7C). The TEM micrographs of +IAA parasites contained less\nnumber of cytoplasmic dense granules (1.5/parasite section, n=153) than those of –IAA\nparasites (3.2/parasite section, n=94), which together with the IFA data indicates a crucial\nrole of TgVMP1 in dense granule biogenesis.”.\nLD. Page 22, line 536: “…TEM micrographs confirmed the presence of much larger size\nLDs in +IAA parasites than in -IAA parasites (Fig. 7D), indicating a key role for TgVMP1\nin LD homeostasis. Additionally, LDs were closely associated with ER membrane in +IAA\nparasite, which was not evident in –IAA parasites.”.\nER. Page 24, line 585: “..ER-GFP was prominently clustered around the nucleus in a\nsignificant number of +IAA parasites (Fig. 7E, F). TEM micrographs showed ER tubules\nstretched throughout the +IAA parasites, including in close contact with LDs, whereas\nthese were mostly perinuclear in –IAA parasites. These data indicate impaired ER\norganization..”.\nThe method sections related to TEM and IEM have been revised as well (page 57-59).\nComment 2. The authors overlook the fact that the parasite has a very serious replication\ndefect. 75% of vacuoles contains a single parasite. This can be explained by the absence of\ndense granule proteins secretion, which was recently shown to be important for intracellular\nparasite development (Chelaghma et al. ELife 2024 ; Fu et al. PloS Pathogen 2023). However,\nthe phenotype of replication is more drastic than in any of the mutants of annuli defective for\nsecretion of dense granules (Chelaghma et al, eLife 2024 ; Fu et al., PlosPathogen 2023) or\nbiogenesis of secretory organelles (Morlon-Guyot et al., Cell Mic 2015 ; Sloves et al. Cell Host\nmicrobes, 2012). This might suggest a role of lipid droplets homeostasis in growth, or potential\nother overlooked effects (point 1).\nResponse: We thank the reviewer for bringing up this point to our notice. We agree that the\nreplication defect upon TgVMP1 depletion is more severe than that has been reported in case\nof dense granule defect (Chelaghma, et al., 2024 (reference No. 66 in the text); Fu, et al., 2023\n(reference No. 67 in the text)) or micronemes and rhoptries (Sloves, et al. 2012 (reference No.\n56 in the text; Stasic, et al. 2029 (reference No. 57 in the text)). We acknowledge reviewer’s\npoint that this severe replication defect upon TgVMP1 depletion could be a combined result of\ndefects in secretory organelles and LD homeostasis. We have discussed this point on page 32,\nline 808 (“…The accumulation of enlarged LDs in close association with ER membrane\nin TgVMP1-depleted parasites indicates impaired LD homeostasis, which together with\nimpaired secretory organelle biogenesis could have contributed to the overall block of\nparasite growth..…”).\nThe other potential reasons for severe parasite growth defect are discussed on page 32,\nline 790 (“…Additionally, as IVN formation requires lipids from the host as a primary\nsource and parasite as a secondary source 9,65, impaired lipid homeostasis in TgVMP1-\ndepleted parasites could have limited lipid supply to the PV, thereby, adversely affecting\nIVN formation and parasite growth…”), page 32, line 806 (“…..ER-resident lipid\nscramblase activity is crucial for parasite development, which likely functions via\ntranslocation of lipids in the ER and the precursor vesicles destined to the secretory\norganelles………..This further indicates that the loss of ER-localized lipid scramblase\nactivity is the major reason for impaired secretory organelle biogenesis, ER organization,\nLD homeostasis, and IVN formation in TgVMP1-depleted parasites, consequently\nblocking the parasite development. ”),\nComment 3. Since DedA is important in autophagosomes formation and that PI3K, ATG8,\nATG3… are linked to Apicoplast in Apicomplexa, the presence and inheritence of the\napicoplast during division need to be assessed.\nResponse: Indeed, human VMP1 has been shown to be required for autophagy during early\nstages (references 28 and 29). As suggested, we addressed this point by comparing -IAA and\n+IAA parasites for the presence of apicoplast using antibodies to the apicoplast marker protein\nACP. We did not observe any noticeable difference in the presence of apicoplast across the\nnumber of –IAA and +IAA parasites observed, which has been mentioned on page 33, line\n822: “….HsVMP1 recruits the PI3K complex (VPS34/Beclin 1/Vps15) at the phagophore\nassembly site via interaction with Beclin 1, which is crucial for autophagosome formation\n28,29. A striking consequence of the ablation of autophagy pathway in T. gondii and P.\nfalciparum has been shown to be the loss of apicoplast, a relict plastid that produces fatty\nacid precursors 70. We compared TgVMP1-depleted and control parasites for the\npresence of apicoplast as a marker to assess whether TgVMP1 depletion affected\nautophagy. The proportion of apicoplast-containing TgVMP1-depleted and control\nparasites was comparable (Supplementary Fig. 12), suggesting that the observed\nphenotypes of TgVMP1-depleted parasites are not associated with autophagy-related\ndefects.”.\nComment 4. The phenotype related to dense granules is parcelar. We would like to see further\ninvestigation of whether or not they are formed (TEM, see above). If they are visible, then to\nquantify secretion in more details (release in the media by quantification of GRAs in the ESA).\nThe loss of IVM is suggested in TEM but the authors cannot quantify the association of GRA\nproteins with IVN by IFA (Fig 6B and S8B), we can just say that they are or not in vacuolar\nspace. Association with IVN required specific fractionnation procedures.\nResponse: This is a valid point, and we thank the reviewer for prompting us to further address\nthe effect of TgVMP1 depletion on dense granule biogenesis, secretion, and IVN formation.\nAs has been described in response to comment 1, we have included brand new TEM and IEM\ndata related to the effect of TgVMP1 depletion on dense granule biogenesis and IVN formation.\nThe related excerpts from result and discussion sections are mentioned below.\nPage 20, line 483: “….TEM micrographs of both –IAA and +IAA\nparasites showed dense granules (Fig. 5F, 6C), which was corroborated by IEM showing\nGRA2 labelling of dense granules in the parasite cytoplasm and IVN in the PV\n(Supplementary Fig. 7C). The TEM micrographs of +IAA parasites contained less\nnumber of cytoplasmic dense granules (1.5/parasite section, n=153) than those of –IAA\nparasites (3.2/parasite section, n=94), which together with the IFA data indicate a crucial\nrole of TgVMP1 in dense granule biogenesis.. …”.\nWe agree with the reviewer that IFA images showing diffuse GRA signal do not\nindicate loss of IVN. It was a misinterpretation on our part, and we have revised the concerned\nresult section on page 19, line 467 (“….The number of parasites with diffuse GRA1 or\nGRA2 localization was significantly higher in +IAA parasites (83.6% for GRA1, 79.0%\nfor GRA2) than that in –IAA parasites (9.6% for GRA1, 14.6% for GRA2) (Fig. 6B;\nSupplementary Fig. 8B). TEM images showed the absence of tubulo-vesicular IVN in the\nPV of 57.7% +IAA parasites (n=123 PVs) as compared with 7.8% –IAA parasites (n=64\nPVs) (Fig. 6C), indicating a crucial role for TgVMP1 in IVN formation.”).\nWe quantified GRA2-gold particles as a measure of dense granule secretion in ESA\nby IEM. The results are mentioned on page 20, line 488 (“…..We next compared dense\ngranule secretion in the ESA of purified –IAA and +IAA parasites by IEM using GRA2.\nThe number of GRA2-gold particles was comparable (Supplementary Fig. 7D, E),\nsuggesting that impaired dense granule biogenesis in TgVMP1-depleted parasites did not\naffect secretion. Nonetheless, majority of the gold particles were associated with electron\ndense material in –IAA parasites as opposed to +IAA parasites (Supplementary Fig. 7D),\nsuggesting a defect in the post-secretion process that might be required for the formation\nof IVN.”).\nOur IFA, TEM, and IEM data are consistent with each other, and show defects in dense\ngranule biogenesis, post-secretion assembly of dense granule-secreted proteins, and IVN\nformation, which are discussed on page 31, line 775 (“…..TgVMP1-depleted parasites\ncontained less number of dense granules and also showed accumulation of dense granule\nproteins in the ER. The impaired ER organization in TgVMP1-depleted parasites might\nhave affected the transport and assembly of dense granule proteins during transport\nthrough the ER/Golgi endo-membrane system, thereby, causing their accumulation in\nER. Although fewer, the cytoplasmic dense granules in TgVMP1-depleted parasites\nshowed normal secretion……………Both control and TgVMP1-depleted parasites\nsecreted comparable GRA2 in the ESA. However, in contrast to TgVMP1-depleted\nparasites, majority of the GRA2 was associated with electron dense material in the ESA\nof control parasites………….This suggests a defect in the form of secreted-GRA2 or post-\nsecretion assembly of GRA2 that might be necessary for IVN formation,\n…………………as IVN formation requires lipids from the host as a primary source and\nparasite as a secondary source 9,67, impaired lipid homeostasis in TgVMP1-depleted\nparasites could have limited lipid supply to the PV, thereby, adversely affecting\nIVN formation and parasite growth. .…..”).\nComment 5. IFA figure 5A. The authors say that the rhoptries are fragmented. The resolution\nof IFA do not permit to see details of the morphologies of the rhoptries, even to see individual\nrhoptries. The ROP1 signal seems to be in the vacuolar space upon depletion of TgVMP1.\nFirst this should be confirmed by adding phase contrast. Second, the release of rhoptry proteins\nwithin the lumen is already observed upon depletion of proteins involved in rhoptry biogenesis\n(SORTILIN, VPS11, retromer complex…), and this is not discussed. Why there is still important\nsignal in -IAA in panel 5A ?\nResponse: Once again, thank you for bringing up these valid points to our notice, which we\ninadvertently missed or misinterpreted. Certainly, IFA doesn’t offer the resolution required to\ncount individual rhoptries and assess rhoptry morphology. Accordingly, we have corrected the\ny-axis label of Fig. 5B, which now reads as: “%Parasites with normal rhoptries”. We have\nalso rephrased the concerned sentence on page 17, line 400: “..Similar to the intracellular\nparasites, purified -IAA parasites also exhibited ROP1 localization to rhoptry-like\nelongated structures, which was not the case with +IAA parasites (Fig. 5E)…..”).\nWe have added Fig. 5C with phase contrast to show ROP1 signal in the PV and\ndetermined the numbers of PVs with ROP1 signal (Fig. 5D), as mentioned on page 17, line 399\n(“….59.4% of +IAA parasites also showed ROP1 signal in the PV compared to 10.9% of\n-IAA parasites (Fig. 5C, D)…..”). We have discussed this result on page 31, line 766 (“…the\nloss of apical rhoptries and secretion of ROP1 in the PV of TgVMP1-depleted parasites\nare similar to those observed in parasites with ablation of TgSORTLR, V-type proton\nATPase or Vps11 56,57,65. It is possible that TgVMP1 contributes to rhoptry biogenesis via\ninteraction with TgSORTLR and V-type proton ATPase, and loss of this interaction in\nTgVMP1-depleted parasites could have adversely affected rhoptry biogenesis...”).\nThe faint signal in the “VMP1” panel of +IAA parasites in the previous Figure 5A could\nhave been due to incomplete TgVMP1 depletion. However, it doesn’t impact the result, as we\nhave evaluated at least 100 PVs from each of the 3 independent experiments to quantitate the\neffect of TgVMP1 on rhoptries (Fig. 5B). We have replaced this panel in the revised manuscript\nwith a better representative image.\nComment 6. GRASP-GFP staining is weird ; it normally looks like a bar above the nucleus\nnear the ER. I don’t think that they can use these images to claim colocalisation with Golgi.\nResponse: We agree with the reviewer that GRASP-GFP localizes to a bar-like structure above\nthe nucleus (Bisio, et al., 2020; Palletier, et al. 2002). We repeated the experiment to check\nwhether TgVMP1 colocalizes GRASP-GFP. Indeed, we also observed that GRASP-GFP\nlocalization is predominantly bar-like close to the nucleus. We have revised Fig.1F, and\nmentioned the related result in the text on page 6, line 146 (“….We episomally expressed ER-\nGFP or GRASP-GFP in RHTIR1-VMP1AID parasites, which showed substantial co-\nlocalization of ER-GFP with TgVMP1AID (Fig. 1E, F), indicating that TgVMP1 is\nHA\npresent in ER. On the other hand, only a minor proportion of TgVMP1 co-localized with\nGRASP-GFP in the region proximal to ER…….”).\nComment 7. The authors observed a strong defect in motility, secretion of micronemes and\nrhoptries. Since most of the parasites are not able to divide, it might be difficult to distinguish\nbetween a potential defect in parasite gliding motility vs. parasites that are dead or dying and\nthereby are unable to exhibit active motility. Same for microneme and rhoptry secretion assays.\nResponse: This is a valid and important comment. To address whether the observed defects\ncould be due to parasite death, we set up the experiment exactly as been described in the method\nsection “Effect of TgVMP1 depletion on gliding motility” on page 46, and performed\npropidium iodide (PI) staining of purified parasites as an indicator of cell death (Accessory Fig.\n2A). As shown in the below Accessory Fig. 2B, the -IAA and +IAA parasites showed similar\nviability, indicating that the observed defects in motility, invasion, and organelle secretion are\nnot due to the presence of dead parasites in the sample used in related experiments.\nAccessory Fig. 2. Viability of parasites. RHTIR1-VMP1AID parasites were cultured with\n0.1% ethanol (-IAA) or 500 µM indole-3-acetic acid (+IAA) as has been described in the\n“Effect of TgVMP1 depletion on gliding motility” section. Purified -IAA and +IAA parasites\nwere incubated with PI (5 μg/ml in PBS) for 15 min at room temperature, washed with PBS,\nimmediately observed under the 100 objective of Zeiss Axioimager.Z2 fluorescence\nmicroscope, images were acquired using AxioVision software, and analysed using ImageJ\nsoftware. A. The representative micrographs show PI staining (PI), DIC, and merge of the two\npanels (Merge). Scale bar = 10 µm. PI staining indicates cell death. B. The number of PI-\nnegative -IAA and +IAA parasites in “A” was counted and presented as a percentage of the\ntotal number of parasites observed (% Viable parasites) on y-axis for the indicated parasites on\nx-axis. Each data is from 2 independent experiments (-IAA, n=324 cells; +IAA, 452 cells). The\nsignificance of difference between two data sets is indicated (ns: not significant).\nWe have not included this figure in the text, as there are already 8 main figures and 13\nsupplementary figures. Nonetheless, we have included the result on page 13, line 283\n(“………the viability of purified +IAA (82.6% ± 6.8) and –IAA (87.7% ± 4.0) RHTIR1-\nVMP1AID tachyzoites was comparable, indicating that the decreased motility of +IAA\nparasites is not associated with parasite viability.”).\nFurthermore, the purified –IAA and +IAA parasites used for conoid extrusion, which\nwere obtained using the similar procedure, did not show any significant difference in conoid\nextrusion (Supplementary Fig. 5). This data also ruled out differences in viability of purified –\nIAA and +IAA parasites.\nComment 8. Some of the results deserve more discussion.\n-Any explanation of why dense granules proteins but not rhoptry proteins accumulated at the\nER in the mutant ?\n-VMP1 is an ER-resident protein so is expected to play a role in this compartment. Since the\nthree secretory organelles, micronemes, rhoptry and dense granules, transit through the ER,\nwhy only dense granules and rhoptries are affected ? MICs proteins may follow, at some\nlevel, a different trafficking pathway ?\n-it is surprising to see that 50% of mice survived after infection with the mutant (+IAA) while\nthere is no plaques at all in vitro. The authors should comment on this discrepancy between\nin vitro and in vivo.\nResponse: We agree with the reviewer’s points. This comments has 2 points, as suggested by\nthe reviewer, we have responded to these points below in a) and b).\na). “Any explanation of why dense granules proteins but not rhoptry proteins accumulated at\nthe ER………MICs proteins may follow, at some level, a different trafficking pathway ?”.\nResponse. We have discussed this point on page 32, line 794: “Different effects of TgVMP1\ndepletion on micronemes, rhoptries, and dense granules, could be due to differences in\nthe pathways leading to their biogenesis and secretion 3,4,6,49,67,68. It is possible that\nTgVMP1 directly/indirectly regulates the proteins in ER/Golgi that are involved at\ndifferent stages of transport/maturation of the proteins destined to these organelles,\nwhich is also supported by the presence of several proteins in TgVMP1 interactome that\nregulate biogenesis of secretory organelles at different stages. Similar observations have\nbeen reported earlier for TgSORTLR and V-type proton ATPase, which are required for\ntrafficking of microneme and rhoptry proteins 56,57, but not for dense granule proteins.\nSimilarly, depletion of apical annuli-associated proteins have been shown to have\ndifferent effects on dense granule secretion and plasma membrane proteins……..”.\nb). it is surprising to see that 50% of mice survived after infection with the mutant (+IAA) while\nthere is no plaques at all in vitro. The authors should comment on this discrepancy between\nin vitro and in vivo.\nResponse. We have discussed the difference in the effects of TgVMP1 depletion on in vitro\nand in vivo parasite viability on page 9, line 224 (“This is in contrast to the complete block\nof parasite development upon TgVMP1 depletion in plaque assay, which might be due to\ndifferences in auxin bioavailability in in vitro and in vivo experimental settings.”).\nComment 9. The interpretation of mass spectrometry data are questionable. The authors\nperformed three independ IPs, but do not show any statistical significance of the enrichment,\nthat will help for the conclusions, especially because almost all the proteins that they used to\nmake their point are not enriched in the three sets of data. This is not clear how they connect\nan association of VMP1 with these proteins (MICs, GRAs, ROPs, IMC proteins) and biogenesis\nof secretory organelles.\nResponse: We thank the reviewer for bringing up this point to our notice. We reanalysed our\nmass spectrometry data for statistically significant peptide enrichment in the TgVMP1AID\nHA\nimmunoprecipitate compared to the control as mentioned in the Method section on page 53,\nline 1463 (“….The peptide abundances in RHTIR1 and TgVMP1AID\nHA\nimmunoprecipitates from 3 biological repeats were compared for enrichment analysis\nusing MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/) with a single factor statistical\nanalysis. ……….Proteins enriched ≥1.5-fold with a P-value <0.05 in\nTgVMP1AID immunoprecipitate compared to the RH-TIR1 immunoprecipitate were\nHA\nconsidered significant interactors of TgVMP1.”).\nThe revised supplementary Table 2 only contains proteins that are ≥ 1.5-fold enriched\nwith p-value ≤0.05 in TgVMP1 immunoprecipitate. The revised data contains multiple proteins\nassociated with micronemes, rhoptries, and dense granules (page 25, line 597: “….TgVMP1\ninteractome contained proteins that have been shown to localize to micronemes (apical\ncap protein AC5 and MIC7), rhoptries (RON3, ROP8, ROP26, and ROP37), and dense\ngranules (phosphatidylserine decarboxylase, cathepsin CPC2, and\nGRA12)…………gliding motility (PhiL1, GAPM3, GAPM2B, GAPM1A, and GRA12),\nprocessing of the microneme and rhoptry proteins (V-type proton ATPase subunit a1),\nand vesicle transport (sortilin-like receptor, Rab5b, glutathione S-transferase 2, and\nRab2)…...”).\nSince the revised data also contains significantly enriched multiple organelle or\norganelle-associated proteins, it is logical to propose that TgVMP1 may contribute to the\nfunctioning and biogenesis of the secretory organelles via these proteins.\nComment 10. The authors referred to the absence of network when they look at IFA of dense\ngranule proteins. The IVN is not visible by IFA.\nResponse: We agree with the reviewer, and apologise for misinterpretation of the IFA data\n(Fig. 6B, Supplementary Figure 8B). Indeed, IFA images showing diffuse GRA signal does\nnot indicate loss of IVN. We have revised the concerned result section and determined the\nnumber of –IAA and +IAA PVs with IVN based on TEM micrographs on page 19, line 467\n(“….The number of parasites with diffuse GRA1 or GRA2 localization was significantly\nhigher in +IAA parasites (83.6% for GRA1, 79.0% for GRA2) than that in –IAA parasites\n(9.6% for GRA1, 14.6% for GRA2) (Fig. 6B; Supplementary Fig. 8B). TEM images\nshowed the absence of tubulo-vesicular IVN in the PV of 57.7% +IAA parasites (n=123\nPVs) as compared with 7.8% –IAA parasites (n=64 PVs) (Fig. 6C), indicating a crucial\nrole for TgVMP1 in IVN formation.”).\nComment 11. Fig. S4A. For the fusion of miniAID in Plasmodium, did the authors control by\nWB the expression of Tir1 in the transgenic line, which is a prerequisite for this conditional\nKD ?\nResponse: This is a valid point. As suggested, we performed western blot of PfVMP1AID\nparasites to check TIR1 expression using anti-TIR1 antibodies. We have discussed this point\nin the text (page 8, line 189: “….. PfVMP1AID parasites expressed TIR1 (Supplementary\nFig. 3I), which ruled out failure of auxin-inducible knock-down due to its absence.”.\nComment 12. Section PfVMP1 and TgVMP1 localize to ER. ‘Towards characterization of\napicomplexan VMP1 proteins, we replaced wild-type PfVMP1 and TgVMP1 coding sequences\n…’ This is not replacement of gene but endogenous tagging.\nResponse: We have rephrased this and other similar sentences on page 6, line 132 (“….the\nendogenous PfVMP1 and TgVMP1 coding sequences in P. falciparum D10 and T. gondii\nRHTIR1 strains were tagged in-frame at their 3’-ends with the coding sequences of GFP\nand AID , respectively.”), page 8, line 178 (“…the endogenous PfVMP1 coding region\nHA\nwas tagged in-frame at the 3’-end with cDDHA-glmS ribozyme coding sequence in P.\nfalciparum D10 strain to obtain a trimethoprim- and glucosamine-inducible conditional\nknock-down line (PfVMP1dKD)…”), and page 8, line 182 (“..the endogenous PfVMP1\ncoding region was tagged in-frame with mAID -PcDT5U/TIR1-Flag-T2A/HD/PcDT3’U\nHA\nsequence in P. falciparum 3D7 strain to generate an auxin-inducible knock-down line\n(PfVMP1AID)…”).\nComment 13. Figure S2 : Molecular sizes of PCR products on schematic and gels are missing.\nResponse: We thank the reviewer for such a detailed review and bringing up this point to our\nnotice. We have included molecular size markers and the sizes of expected PCR products in\nSupplementary Fig. 3 and 4.\nComment 14. Table : add accession number TGGT1\nResponse: As suggested, we have included both Uniprot and ToxoDB IDs for each protein in\nSupplementary Table 2.\nReferences\nBisio, et al. The ZIP code of vesicle trafficking in Apicomplexa: SEC1/Munc18 and SNARE\nproteins. MBio. 2020, 11(5), 10-1128.\nPelletier, L. et al. Golgi biogenesis in Toxoplasma gondii. Nature. 2002, 418, 548-552."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have answered all questions raised by this reviewer. They provide additional data that are very convincing and the manuscript have been considerably improved. Their finding is of a high significance ot the field. No other additional experiments are needed. The methodology sound very well and the conclusions are supported by the results obtained. Therefore, I will suggest to publish this work in Nature Communications."}, "author_response": ""}]

Decision letter