VPS26A retromer complex and SNX27 mediate stress-induced Golgi bypass of membrane proteins

Domain: chemistry
Published: 2026-04-06
Editorial outcome: minor_revision

Abstract
Many proteins can reach the cell surface through a Golgi-independent unconventional protein secretion (UPS) pathway, particularly under cellular stress conditions. However, the molecular mechanisms that mediate UPS remain largely elusive. In this study, VPS26A-containing retromer complex, along with the sorting nexin SNX27, is identified as a regulator of UPS of transmembrane proteins, including the trafficking-deficient ∆F508 mutant CFTR, which causes cystic fibrosis, and the SARS-CoV-2 spike protein, associated with COVID-19. A targeted CRISPR knockout screen identified VPS26A as a key contributor in the UPS of ∆F508-CFTR. Subsequent molecular analyses revealed that SNX27 recruits ∆F508-CFTR to the VNPS26A-VPS35-VPS29 retromer complex, facilitating its transport to the cell surface under UPS-inducing conditions. Additionally, VPS26A and SNX27 are necessary for UPS of the spike protein, enabling the formation of intact SARS-CoV-2 virions. These findings suggest that the retromer complex and SNX27, known for their roles in recycling endosomes, mediate previously unrecognized functions in the UPS of transmembrane proteins.

Introduction
Cellular proteins are transported to appropriate sites after synthesis to perform their specific functions within subcellular microdomains. It is well established that most secretory and transmembrane proteins are transported via the endoplasmic reticulum (ER)-to-Golgi pathway1,2. However, increasing evidence suggests that proteins can move through alternative pathways in addition to the typical protein transport pathway3,4. While much remains to be uncovered about these alternative pathways, researchers have identified a number of cytosolic and membrane proteins that reach the plasma membrane via Golgi-independent routes5,6, collectively known as unconventional protein secretion (UPS) pathways. UPS can be categorized in to three or four subtypes, one of which involves the Golgi bypass of transmembrane proteins. A well-known example of this is the Golgi-independent transport of the cystic fibrosis transmembrane conductance regulator (CFTR)7.

CFTR is an anion channel that facilitates the transport of chloride and bicarbonate anions in epithelial cells8. Defective CFTR genes have been linked to the development of chronic genetic diseases such as cystic fibrosis and pancreatitis. Among patients with cystic fibrosis, the most prevalent mutation is ∆F508-CFTR, where phenylalanine at position 508 of the CFTR protein is deleted. The synthesis of the ∆F508-CFTR mutant protein leads to structural and folding abnormalities, which subsequently cause a trafficking deficiency in ER exit and degradation through the ER-associated degradation (ERAD) mechanism9-11. Consequently, in patients with the ∆F508-CFTR mutation, there is an absence of CFTR protein at the cell membrane, which impairs anion transport and ultimately leads to disease development. Previous studies have shown that, under certain circumstances, such as the induction of ER stress or blockade of ER-to-Golgi transport, the ER core-glycosylated form of CFTR, including the ΔF508 mutant, can be transported to the cell membrane via the UPS pathway, bypassing the Golgi apparatus12-15. Notably, the unconventionally transported ∆F508-CFTR retains a certain level of functional activity at the cell surface12-15, suggesting that activation of the UPS pathway has the potential to treat the disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, has a spike protein in its envelope that plays a critical role in the infection of host cells16. It has been suggested that UPS pathways are employed in the viral egress process and also the trafficking of spike proteins throughout SARS-CoV-2 infection. For example, the Golgi apparatus becomes fragmented during SARS-CoV-2 infection17,18, indicating a potential for the activation of Golgi-independent pathways. Furthermore, SARS-CoV-2 uses an endo-lysosomal system for egress, rather than the conventional secretory pathway19.

Despite considerable progress made recently, the molecular mechanisms of UPS remain largely elusive6,20. For example, several studies have proposed a link between recycling endosomes and UPS or secretory autophagy, a non-degradative form of autophagy that share many common aspects with, or is regarded as a form of, UPS14,21. However, the key regulatory factors and molecular machinery involved in the interplay between endosomes and UPS or secretory autophagy remain unclear. In this study, we performed a CRISPR knockout (KO) screen using a custom single-guide RNA (sgRNA) library to identify molecules essential for the regulation of the UPS in ∆F508-CFTR. This initial analysis identified VPS26A, a component of the retromer complex, as exerting a strong regulatory influence on CFTR UPS. Subsequent investigation revealed that the trimeric VPS26A-VPS35-VPS29 retromer complex, together with the PDZ (postsynaptic density 95/discs large/zonula occludens-1) domain-containing sorting nexin 27 (SNX27), known to direct the retromer to early and recycling endosomes22, participates in the Golgi-independent cell surface trafficking of ∆F508-CFTR and SARS-CoV-2 spike protein, particularly under certain cellular stress conditions. These findings broaden our understanding of how endosomal sorting machinery, classically linked to recycling and retrograde transport, can be repurposed to facilitate the stress-induced Golgi-independent trafficking.

Results
The VPS26A retromer complex participates in the UPS of ∆F508-CFTR

We initially performed genetic screening using the CRISPR KO system to identify factors involved in the UPS of the transmembrane protein CFTR (Fig.1a). Genes involved in vesicular transport, autophagy, and ER stress were selected for CRISPR/Cas9 KO screening in Cas9-expressing HeLa cells. A total of 9,072 single-guide (sg) RNAs targeting 1,082 genes were designed, as described in the Materials and Methods section (Supplementary Fig. 1 and Supplementary Data 1, 2). To monitor cell-surface expression of CFTR, cells were transfected with plasmids encoding extracellular HA-tagged CFTR and stained with phycoerythrin (PE)-conjugated anti-HA antibodies. Cells were then analyzed and sorted by fluorescence-activated cell sorting (FACS) (Fig. 1a, b). To establish a gating threshold for PE-positive cells, we first measured PE intensity in cells expressing wild-type CFTR, which efficiently traffics to the plasma membrane via the conventional secretory pathway (Fig. 1b left). In contrast, cells expressing ∆F508-CFTR alone showed minimal PE signal (Fig. 1b middle), confirming that background surface expression is negligible without rescue. Based on this comparison, we defined a PE intensity threshold (P4 gate) to identify cells with significant CFTR surface expression. To induce UPS activation, cells were co-expressed with the dominant-inhibitory form of ADP-ribosylation factor 1 (ARF1-Q71L, Fig. 1b right), which blocks the conventional ER-to-Golgi transport pathway and thereby activates alternative secretion pathways23.

Next-generation sequencing (NGS) was performed on PE-positive (rescued) and total populations of ∆F508-CFTR-expressing cells co-transfected with ARF1-Q71L to identify genes regulating UPS. Analysis of sgRNA sequences using the MAGeCK algorithm identified VPS26A, a core component of the retromer complex, as the top-ranked factor required for CFTR UPS (Fig. 1c and Supplementary Data 3). The involvement of VPS26A in ∆F508-CFTR UPS was also verified via cell-surface biotinylation assay, in which knockdown of VPS26A greatly reduced the ARF1-Q71L-induced cell-surface expression of ∆F508-CFTR (Fig. 1d, e). In addition to ARF1-Q71L, the blockade of ER-to-Golgi transport using the dominant-inhibitory form of the secretion-associated and Ras-related protein 1 (SAR1-T39N), along with the overexpression of the cis-Golgi target SNARE protein syntaxin 5 (STX5), has been shown to induce UPS of the trafficking-deficient ΔF508-CFTR via the activation of an ER stress-associated mechanism12,13. As shown in Fig. 1f-i, the knockdown of VPS26A also greatly reduced ΔF508-CFTR UPS induced by SAR1-T39N and STX5 overexpression, indicating a common requirement for VPS26A under ER stress-associated conditions.

Retromer mediates the sorting of cargo proteins within endosomes, generally directing them either to be transported back to the trans-Golgi network or recycled to the plasma membrane in animal cells, rather than being targeted for degradation in lysosomes22,24. The core retromer complex is composed of three vacuolar protein sorting (VPS) proteins in mammalian cells: VPS26, VPS29, and VPS3522,24. We thus examined whether VPS29 and VPS35 are involved in the UPS of ∆F508-CFTR. Notably, the silencing of both VPS29 and VPS35 resulted in inhibition of the UPS of ΔF508-CFTR induced by ARF1-Q71L, indicating that a whole retromer complex is necessary for the ER stress-associated UPS (Fig. 2a-d). Since VPS26B, like VPS26A, is a known component of a retromer complex25, we also investigated its potential involvement in CFTR UPS. As shown in Supplementary Fig. 2, silencing of VPS26B reduced the surface level of ΔF508-CFTR (surface biotinylation; Supplementary Fig. 2a, b); however, it also significantly decreased the total cellular level of ΔF508-CFTR (cell lysate; Supplementary Fig. 2a, c), which hampers the precise determination of trafficking effects. We therefore conducted all subsequent experiments using VPS26A to focus on trafficking events without affecting protein stability.

Next, we examined whether these proteins are also involved in the conventional Golgi-mediated trafficking of wild-type CFTR. As CFTR is an N-glycosylated protein, its core glycosylation occurs in the ER, while complex glycosylation takes place in the Golgi apparatus. The complex-glycosylated form, commonly referred to as band C, has a higher molecular weight than the core-glycosylated form, referred to as band B. Wild-type CFTR primarily appears as band C in immunoblots, as it passes through the Golgi, whereas the ∆F508-CFTR variant appears as band B because it is retained in the ER12. Interestingly, knockdowns of VPS26A, VPS29, and VPS35 did not affect the conventional Golgi-mediated trafficking of the band C form of wild-type CFTR to the cell surface (Fig. 2e-j). The blockade of ER-to-Golgi transport by ARF1-Q71L also induced the UPS of wild-type CFTR, resulting in the appearance of the band B form of CFTR on the cell surface due to trafficking via a Golgi-bypass route. Notably, knockdowns of VPS26A, VPS29, and VPS35 significantly inhibited the UPS of ER form (band B) of wild-type CFTR (Fig. 2e-j). These findings indicate that the retromer complex is more crucial for the Golgi-independent UPS of CFTR rather than conventional Golgi-mediated transport.

SNX27 recruits CFTR to the retromer complex through PDZ-based interaction

The mammalian retromer complex functions as a coat complex that regulates intracellular trafficking in general by interacting with sorting nexins (SNXs), which serve as adaptor proteins that link retromer to specific cargo or membrane domains24,26-28. We then examined whether sorting nexins are potentially involved in the regulation of CFTR UPS in conjunction with the retromer complex. We focused primarily on SNX27, which has been shown to recruit transmembrane protein cargos containing a PDZ-binding motif at their C-terminus, such as the β2-adrenergic receptor29, the glucose transporter GLUT130, and the AMPA receptor subunit GluA131. SNX27 is composed of PDZ, PX (phosphoinositide-binding Phox homology), and FERM (4.1/ezrin/radixin/moesin) domains. Notably, structural analyses have revealed that the PDZ domain of SNX27 plays a critical role in recruiting cargo to the retromer complex by simultaneously binding the PDZ-binding motifs of membrane proteins, while an exposed β-hairpin within the SNX27 PDZ domain engages a groove in VPS2632. Because CFTR is also a plasma membrane protein bearing a C-terminal PDZ-binding motif33,34, we prioritized SNX27 as a mechanistically plausible adaptor for CFTR UPS. As shown in Fig. 3a and 3b, silencing of SNX27 resulted in a strong reduction in the ΔF508-CFTR UPS induced by ARF1-Q71L. Like the retromer components, silencing of the SNX27 gene reduced UPS of wild-type CFTR (band B) but not its conventional trafficking (band C) (Fig. 3c, d).

We next investigated whether SNX27 functions as a cargo receptor for CFTR by assessing its ability to bind to CFTR using a co-immunoprecipitation (co-IP) assay. The co-IP results indicate that SNX27 associates with ΔF508-CFTR (Fig. 3e, f). It is noteworthy that the SNX27-CFTR association was augmented when UPS was induced by ARF1-Q71L, implying that SNX27 may play a prominent role during the UPS process. Subsequent immunocytochemistry experiments revealed that SNX27 co-localizes with ΔF508-CFTR during UPS (Fig. 3g, h), which is in line with the co-IP results. Based on these findings, which demonstrate the potential role of SNX27 in CFTR UPS, we examined the impact of SNX27 upregulation on the induction of ΔF508-CFTR UPS. Notably, SNX27 overexpression partially restored mutant CFTR expression at the plasma membrane and further enhanced ARF1-mediated rescue of its surface localization (Fig. 3i, j), suggesting that SNX27 promotes vesicular membrane trafficking of ΔF508-CFTR via a route bypassing the Golgi.

We next tested whether SNX27 engages CFTR in a PDZ-dependent manner and thereby couples CFTR to the retromer complex via VPS26. In the co-IP experiments shown in Fig. 4a and 4b, the deletion of four amino acids from the CFTR C-terminus (CFTR-ΔC), which disrupted its PDZ-binding motif, prevented its interaction with SNX27. This strongly indicates that the CFTR-SNX27 association is mediated by a PDZ-based interaction. To further validate this notion, we fragmented and purified each of the three domains of SNX27 (PDZ, PX, and C-terminal FERM) as illustrated in Fig. 4c and then performed pull-down assays using a GST-tagged peptide containing the C-terminal 30 amino acids of CFTR (CFTR-Ct30). The results evidently demonstrate that the PDZ domain of SNX27, and not its other domains, binds to CFTR-Ct30 (Fig. 4d).

Subsequently, we investigated the overall physical interactions of SNX27, VPS26A, and CFTR. In co-IP experiments, VPS26A and SNX27 were found to interact with each other (Fig. 4e, f) consistent with previous reports30,32. Notably, this interaction was significantly enhanced by UPS induction via ARF1-Q71L and the co-expression of the cargo protein CFTR, resulting in a tenfold increase in SNX27-VPS26A interaction when ARF1-Q71L and CFTR were co-expressed (Fig. 4e, f). Additionally, the interaction between SNX27 PDZ and VPS26A increased proportionally with the amount of CFTR-Ct30 (Fig. 4g, h). Furthermore, in semi-in vivo pull-down assays using purified SNX27 PDZ and CFTR-Ct30, VPS26A failed to interact with the CFTR C-terminus in the absence of the SNX27 PDZ domain (Fig. 4i). These findings indicate that SNX27 is essential for the association of the retromer complex with the UPS cargo protein CFTR.

To quantitatively assess the SNX27-VPS26A interaction, we performed surface plasmon resonance (SPR) analysis using immobilized VPS26A and soluble SNX27-PDZ, either alone or pre-incubated with CFTR-Ct30. Consistent with our co-IP and pull-down results, the presence of CFTR markedly increased the affinity of SNX27-PDZ for VPS26A. SNX27-PDZ alone exhibited a dissociation constant (KD) of approximately 51.4 μM, whereas the SNX27-PDZ–CFTR-Ct30 complex bound VPS26A with substantially higher affinity (KD = 3.8 μM; Fig. 4j; Supplementary Fig. 3). These findings provide additional biophysical evidence that CFTR binding strengthens the SNX27-VPS26A interaction.

We then examined whether the VPS26A- and SNX27-mediated route represents a common pathway for the UPS of CFTR. It has been shown that induction of ER stress by thapsigargin treatment triggers the UPS of ΔF508-CFTR12. Notably, knockdowns of VPS26A and SNX27 significantly reduced thapsigargin-induced ΔF508-CFTR UPS (Fig. 5a, b), indicating that VPS26A and SNX27 participate in the UPS pathway under multiple ER stress conditions.

GRASPs, also known as GLORASPs, are involved in the UPS of many cargo molecules across a wide variety of organisms4,35. Having identified SNX27 as a PDZ-dependent adaptor for CFTR UPS, and given that mammalian GRASP55 (GORASP2) is a well-established UPS modulator and a PDZ-containing protein12,20, we further investigated how these two adaptors function within the UPS pathway. Combined knockdown of SNX27 and GRASP55 further decreased ARF1-Q71L-induced UPS of ΔF508-CFTR compared with either single knockdown (Fig. 5c, d), indicating that the two PDZ adaptors act in a non-redundant manner. In cellular immunostaining experiments, GRASP55 localized to perinuclear Golgi regions. Notably, upon UPS activation by ARF1-Q71L, GRASP55 redistributed toward peripheral structures, likely tubular ER regions, leading to a marked increase in its co-localization with ΔF508-CFTR (from 22% to 68%, Supplementary Fig. 4), consistent with previous findings12,36. Interestingly, activation of the UPS by ARF1-Q71L did not significantly increase its co-localization with SNX27, despite the peripheral redistribution of GRASP55 (Fig. 5e, f). Taken together, these findings imply that GRASP55 and SNX27 contribute to the UPS pathway in distinct yet complementary manners, functioning at different stages of the trafficking process.

In addition to SNX27, we examined the potential involvement of SNX2 and SNX17 in the UPS of ΔF508-CFTR, given that these have previously been shown to be involved in the endosomal transport of transmembrane protein cargos37,38. SNX2 is a member of SNX-BAR (Bin/Amphiphysin/Rvs) proteins, which are responsible for tubular-based endosomal sorting, and it has been shown to interact with SNX2737. Furthermore, SNX2 has been implicated in ER-endosome contact regulation, which may be relevant that promote Golgi-bypass trafficking39. Similarly to SNX27, SNX17 possesses PX and FERM domains; however, it lacks the PDZ and is known as an adaptor for PDZ-independent endosomal recycling pathways38,40. While SNX2 gene knockdown resulted in reduced ΔF508-CFTR surface expression via UPS, SNX17 knockdown did not (Supplementary Fig. 5). These findings suggest that SNX-BAR-related mechanisms may also be involved in this process (see Discussion) and that the PDZ domain of SNX27 may play a role in CFTR cargo recognition.

Arginine 68 of SNX27 PDZ is a key residue in the VPS26A-SNX27-CFTR assembly

Next, we conducted structural analyses of the interactions involving VPS26A, the SNX27 PDZ domain, and CFTR using the AlphaFold system, along with co-IP and site-directed mutagenesis. We generated VPS26A/SNX27-PDZ models using both AlphaFold241 and AlphaFold342. Although the two models were broadly similar, the AlphaFold2-V2 predictions exhibited more favorable confidence metrics, including higher pLDDT scores and lower predicted aligned error (PAE) values (Supplementary Fig. 6a-d). Ramachandran plot analysis comparing the models with the experimentally determined crystal structure further indicated that the AlphaFold2 model more closely resembled the known structure (Supplementary Fig. 6e-g). Consistent with this, the AlphaFold2-predicted structure of human VPS26A and the SNX27 PDZ domain was nearly identical to the previously reported crystal structure of mouse VPS26A and rat SNX27 PDZ32, with a root mean square deviation (RMSD) of less than 1 Å (Supplementary Fig. 6h). Based on these results, we selected the AlphaFold2-V2 model for all subsequent analyses. We then incorporated CFTR PDZ-binding motif into the structure, and the predicted model showed that the last four residues CFTR ‘DTRL’, known as key residues of the PDZ-binding motif, fit into the binding pocket of the SNX27 PDZ domain (Fig. 6a, Supplementary Data 4 and 5).

After successful construction of the VPS26A/SNX27 PDZ/CFTR PDZ-binding motif assembly, we performed molecular dynamics (MD) simulations to determine whether the binding of CFTR PDZ-binding motif to the SNX27 PDZ domain affected the interaction between VPS26A and SNX27 PDZ. Due to the high intrinsic flexibility of the CFTR C terminus, 6 residues that were stably bound to the SNX27 PDZ domain (VQDTRL) were included for the simulation. Eight independent 100 ns-long MD simulations were conducted with and without CFTR PDZ-binding motif. The RMSD of SNX27 PDZ and VPS26A stabilized after 20 ns of simulation, indicating that the overall protein complex had reached an equilibrated state (Fig. 6b, c). Next, we performed principal component analysis (PCA) separately on SNX27 PDZ and VPS26A during the last 50 ns of the simulation. While the structural distribution of SNX27 PDZ remained largely unchanged with or without CFTR PDZ-binding motif, VPS26A exhibited structural differences in the presence of CFTR PDZ-binding motif (Fig. 6d, e). To further investigate the interaction between VPS26A and SNX27 PDZ, we calculated their interaction energies using the molecular mechanics with generalized Born and surface area solvation (MM/GBSA) method43. Notably, in the presence of CFTR PDZ-binding motif, the interaction energy between VPS26A and SNX27 PDZ increased significantly (Fig. 6f), which was consistent with the co-IP and pull-down experiments showing that CFTR PDZ-binding motif enhanced VPS26A-SNX27 interaction (Fig. 4e-i).

To identify key residues responsible for the increased interaction, we performed residue decomposition analysis based on the MM/GBSA results. By comparing the interaction energy differences in the absence and presence of CFTR PDZ-binding motif (Fig. 6g, h), we identified R68, D70, and E110 in SNX27 PDZ as residues that interacted more strongly with VPS26A when CFTR PDZ-binding motif was present. Similarly, in VPS26A, D44 and R303 were identified as residues responsive to the CFTR PDZ-binding motif. As illustrated in Fig. 6i, R68 from SNX27 and D44 from VPS26A were positioned closely, forming a salt bridge that stabilizes the interaction interface. To assess the functional relevance of these predicted interactions, we experimentally validated the VPS26A-SNX27-CFTR interaction using the SNX27-R68A mutant (Fig. 6j, k), as R68 exhibited the greatest difference in interaction energy in the presence and absence of the CFTR PDZ-binding motif (Fig. 6g, h). Co-IP experiments in HEK293 cells expressing whole proteins of VPS26A, SNX27, and CFTR revealed that the SNX27-R68A substitution significantly reduced the interaction between VPS26A and SNX27 in the presence of CFTR (Fig. 6j, k), consistent with the MD simulation results.

VPS26A, SNX27, and RAB11A contribute to anterograde CFTR trafficking via the UPS

The steady-state cell-surface abundance of CFTR proteins observed in surface biotinylation experiments reflects a dynamic equilibrium among: 1) newly synthesized proteins arriving at the plasma membrane via anterograde transport, 2) retrograde removal through endocytosis, and 3) recycling of endocytosed proteins back to the cell surface. In contrast to wild-type CFTR, for which approximately 40% of endocytosed proteins are recycled in 10 min, recycling of cell-surface rescued ΔF508-CFTR is markedly reduced (~5% in 10 min) due to peripheral protein quality control-mediated ubiquitination and subsequent lysosomal targeting44. Therefore, inhibition of endocytic removal largely reflects de novo anterograde transport in the case of ΔF508-CFTR. Accordingly, to minimize the contribution of endocytic retrieval to cell-surface abundance and to more accurately interpret CFTR surface delivery under UPS-inducing conditions, we inhibited endocytic removal using dynasore, a dynamin GTPase inhibitor that blocks clathrin- and dynamin-dependent endocytosis45, and assessed the effects of retromer and SNX27 silencing. Treatment with dynasore increased the cell-surface expression of ΔF508-CFTR induced by ARF1-Q71L (Fig. 7a, b), which correlates with the previous report that endocytic removal of cell-surface ΔF508-CFTR is augmented due to the peripheral quality control systems44. Importantly, knockdown of VPS26A or other retromer complex components still significantly reduced surface levels of ∆F508-CFTR even under dynasore treatment (Fig. 7a, b), indicating that the VPS26A-SNX27 complex contributes to an anterograde route to promote Golgi-bypass delivery of CFTR to the plasma membrane. Unlike the UPS of ∆F508-CFTR, the conventional Golgi-mediated trafficking of wild-type CFTR was not significantly affected by silencing of retromer complex components (Fig. 7c, d), highlighting the specificity of the complex in the UPS pathway.

To better understand the directionality and vesicular route of this anterograde trafficking, we examined whether the CFTR UPS involves recycling endosomes. Specifically, we investigated the role of Rab11A, a well-characterized regulator of canonical recycling endosomes46. As shown in Supplementary Fig. 7, RAB11A knockdown significantly reduced ARF1-Q71L-induced UPS of both WT- and ΔF508-CFTR (band B), while leaving conventional WT-CFTR trafficking (band C) largely unaffected. Notably, the effect of RAB11A silencing persisted under dynasore-treated conditions, providing further evidence for the involvement of Rab11A-mediated transport in ΔF508-CFTR UPS (Fig. 7e, f).

UPS activation by ARF1-Q71L increased the spatial overlap between ∆F508-CFTR and Rab11A (Fig. 7g, h), and similar trends were observed under additional UPS-inducing conditions, including STX5 or SAR1-T39N overexpression and thapsigargin treatment (Fig. 7h; Supplementary Fig. 8). Likewise, UPS induction also increased overlap between SNX27 and Rab11A (Fig. 7i, j; Supplementary Fig. 9). Collectively, these results suggest increased engagement of Rab11A-positive recycling compartments during stress-induced Golgi-bypass of ΔF508-CFTR.

VPS26A and SNX27 participate in the UPS of SARS-CoV-2 spike protein

We next examined the roles of VPS26A and SNX27 in the intracellular trafficking of the viral envelope protein SARS-CoV-2 spike, which plays a critical role in host cell entry16. The spike protein can be cleaved into S1 and S2 fragments by furin, a cellular protease predominantly present in the Golgi apparatus47. Thus, the presence of cleaved spike at the plasma membrane or the viral envelope is consistent with Golgi-mediated transport, whereas the presence of uncleaved spike is compatible with Golgi-independent trafficking. Notably, it has been reported that authentic SARS-CoV-2 particles contain substantial amounts of uncleaved spike protein, and that the spike protein can be transported via a Golgi-bypass pathway15,19,48

When the spike protein is exogenously expressed in HEK293 cells using plasmid vectors under non-stress inducing conditions, the majority of cell surface-expressed spike proteins are cleaved, indicating that they have been transported via a Golgi-mediated route (Fig. 8a). Silencing VPS26A or SNX27 had no effect on the Golgi-dependent surface trafficking of these cleaved spike proteins (Fig. 8a, b), suggesting that VPS26A and SNX27 are dispensable for this conventional Golgi-dependent route. We then examined whether VPS26A and SNX27 are required when UPS is activated by blockade of ER-to-Golgi trafficking. Blocking ER-to-Golgi trafficking with ARF1-Q71L induced Golgi-independent cell surface expression of uncleaved spike protein (Fig. 8c). Notably, knockdown of VPS26A or SNX27 significantly reduced the surface delivery of uncleaved spike (Fig. 8c, d), indicating that VPS26A and SNX27 are required for Golgi-bypass trafficking of the spike protein. The effects of VPS26A and SNX27 silencing remained unchanged following dynasore treatment, similar to that observed for ΔF508-CFTR UPS.

We next assessed the physiological relevance of these findings using authentic SARS-CoV-2 infection in HeLa-ACE2 cells. Authentic SARS-CoV-2 viral particles harvested from the supernatant of HeLa-ACE2 cells infected with SARS-CoV-2 contain a substantial proportion of uncleaved spike proteins. Notably, the silencing of VPS26A or SNX27 resulted in a significant reduction in uncleaved spike proteins in viral particles (Fig. 8e, f). To determine the pathophysiological impact of reducing spike protein content in viral particles, a virus plaque assay was performed. Inhibition of either VPS26A or SNX27, or their combined inhibition, resulted in decreased levels of active virus secretion (Fig. 8g). Additionally, analysis of secreted viral mRNA levels showed results consistent with those of the plaque assay (Fig. 8h). A recent report suggests that SNX27 affects SARS-CoV-2 infection by inhibiting cell surface expression of the viral receptor protein ACE249. However, in our system, VPS26A or SNX27 knockdown did not alter ACE2 surface expression (Supplementary Fig. 10). Taken together, these findings indicate that VPS26A- and SNX27-mediated UPS of the spike protein contributes to maintaining proper viral infectivity and replication of SARS-CoV-2.

Methods
Cell culture, plasmids, siRNAs, and transfection

HEK293T cells (ATCC, CRL-3216) and HeLa cells (ATCC, CCL-2) were obtained from the American Type Culture Collection (ATCC). HeLa and HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, #11995-065, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, #26140-079, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco, #15140-122, Carlsbad, CA, USA). The cells were incubated in a 5% CO2 environment at 37°C. ACE2-expressing HeLa cells were generated by lentiviral transduction with hACE2-P2A-BSD followed by blasticidin selection (10 μg/mL), as reported previously59.

Plasmids encoding human pCMV-ΔF508-CFTR, pCMV-WT-CFTR (pCMVNot6.2), HA-ΔF508-CFTR, HA-WT-CFTR, HA-ΔC-WT-CFTR, pGEX-4T1-CFTR-Ct-30, Myc-SAR1-T39N, HA-Syntaxin5 and mammalian expressible extracellular-tagged full-length HA-ΔF508-CFTR have been described previously12. The coding sequence for human ARF1-Q71L was synthesized and incorporated into the pcDNA3.1-HA vector using NEBuilder HiFi DNA Assembly Kit (NEB Biolabs) with a Myc-tag or HA-tag. The commercially obtained vectors pCMV6-VPS26-GFP and pCMV-SNX27-GFP (Origene #NM_004896, #NM_030918, Rockville, MD, USA) contained the coding sequences for VPS26 and SNX27, which were inserted into the pCMV6-Entry-Myc-DYK vector using the BamHI and XhoI restriction enzyme sites. To generate HA-VPS26 construct, the sequence for VPS26 was amplified from pCMV6-VPS26-GFP and the amplicons were cloned into pcDNA3.1-HA vector using the EcoRI and XhoI restriction enzyme sites. To create domain-specific SNX27 constructs with a His6 tag, the PCR fragments (full-length SNX27 [a.a. 1-525], PDZ [a.a. 1-149], PX [a.a. 153-265], FERM [a.a. 266-525], and ∆PDZ [a.a. 153-525]) were inserted into the pET-28a (+) by using the NcoI and XhoI restriction enzyme sites. The SNX27-R68A mutant construct was generated using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, #E0554S) with the forward primer 5'- CGG GCA ACT GGC GAG CAT CAA CGG GGA G -3', and reverse primer 5'- CCC TCG CTC ACT TGG CCC CGC ACG TTG AA -3'. The mammalian expressible pcDNA3.1-spike plasmid was generated by inserting a stop codon before the C9 tag of a pcDNA3.1-SARS2-spike plasmid (Addgene #145032). The plasmids were transfected into HeLa and HEK293 cells using either Lipofectamine LTX Reagent (Invitrogen, #15338-100, Carlsbad, CA, USA), TransIT-X2 Dynamic Delivery System (Mirus Bio LLC, #MIR6006, Madison, WI, US) or JetPRIME Reagent (Polyplus, #101000001, Illkirch-Graffenstaden, France), following the manufacturer’s protocol. siRNAs (Supplementary Data 8) were transfected into HEK293 cells or ACE2-overexpressing HeLa cells using RNAiMAX transfection reagent (Invitrogen, #13778-150, Carlsbad, CA, USA), following the instructions provided by the manufacturer. All newly generated plasmids are available from the corresponding author upon reasonable request.

High-throughput genetic screening using CRISPR/Cas9 knockout library

To generate HeLa cell lines expressing Cas9 stably, HEK293T cells were co-transfected with the lentiCas9-Blast plasmid, psPAX2 packaging plasmid and the pMD2.G envelope plasmid using the TransIT-X2® Dynamic Delivery System (Mirus Bio LLC, #MIR6006) as per the manufacturer’s instructions. Subsequently, the HeLa cells were transduced with lentiviral particles obtained from supernatants of HEK293T cells expressing Cas9. After 24 h, the supernatant was replaced with complete medium supplemented with 3 μg/mL blasticidin, and the cells were incubated for an additional 48 h.

For genetic screening, customized single-guide RNA libraries targeting 1,082 genes expected to be involved in unconventional protein secretion were selected and generated. As indicated in Supplementary Fig. 1b, eight single-guide RNA (sgRNA) sequences per each gene were obtained from the Brunello library (https://portals.broadinstitute.org/gppx/crispick/public) as the default option, while the remaining four were designed using an online tool called GUIDES (http://guides.sanjanalab.org/) provided by the Sanjana Lab, the Genome-Scale CRISPR Knock-Out (GeCKO) library (https://www.addgene.org/pooled-library/zhang-human-gecko-v2), or CRISPR knockout (CRISPRko) library (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). The sgRNA sequences and sources for all genes are provided in Supplementary Data 2.

Lentiviral pooled sgRNA library was prepared, as previously described in detail60. To obtain lentiviral particles, libraries were co-transfected with the psPAX2 packaging plasmid and the pMD2.G envelop plasmid into HeLa-Cas9 stable cells. After 24 h, the supernatant was replaced with a complete medium supplemented with 3 μg/mL puromycin for selection. HeLa cells expressing Cas9 were transduced with a lentiviral pooled sgRNA library at a multiplicity of infection (MOI) of approximately 0.3. This process enabled each cell to receive a single sgRNA. Subsequently, the cells were subjected to a six-day puromycin selection process.

After 48 h, stable cells were transfected with CFTR plasmids tagged with the human influenza hemagglutinin (HA) at the extracellular loop region. Unconventional secretion of CFTR protein was induced by co-expression of the ARF1-Q71L, as previously reported12. After 24 h, cells were fixed with a 4% paraformaldehyde solution for 10 min, and then washed twice with FACS buffer containing 10% FBS in PBS. The cells were stained with the PE conjugated anti-HA antibodies (BioLegend, #901517, San Diego, CA, USA) for 1 h at 37°C, followed by three washes with FACS buffer. Cells expressing CFTR at the cell surface were collected by flow cytometry. Debris and cell clumps were excluded based on forward scatter area (FSC-A) and side scatter area (SSC-A) profiles. Single cells were then identified by gating on forward scatter height (FSC-H) versus forward scatter area (FSC-A). Within the singlet population, the frequencies of HA-positive and HA-negative cells were determined. Data were acquired using a FACS sorter (BD FACS Aria III, Franklin Lakes, New Jersey, USA) and analyzed using FlowJo software (BD Biosciences, Franklin Lakes, New Jersey, USA). Subsequently, genomic DNA was extracted from the obtained cells using the GeneRead DNA FFPE Kit (Qiagen, #180134, Hilden, Germany) following the instructions provided by the manufacturer. The obtained genomic DNA was amplified using polymerase chain reaction with NEB Next® High-Fidelity 2X PCR Master Mix (New England Biolabs, #M0541, MA, USA). Barcoded next-generation sequencing (NGS) libraries were generated using a two-step PCR strategy following the protocol described by Joung et al60. The resulting amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen, #28706, Valencia, CA, USA) and sequenced on an Illumina NovaSeq 6000 platform. NGS data were analyzed using MAGeCK (version 0.5.4), and gene enrichment/depletion was assessed with the MAGeCK test based on the robust rank aggregation (RRA) framework. sgRNA-level p-values were estimated using a negative binomial model comparing treatment and control read counts. Gene-level p-values were then derived by α-RRA ranking of significant sgRNAs followed by permutation testing.

RNA extraction and quantitative polymerase chain reaction (qPCR)

Cellular RNA was extracted using an RNA extraction kit (Bioneer, #K-3140, Daejeon, Korea) following the provided instructions. Subsequently, cDNA synthesis from the isolated RNA was carried out using cDNA EcoDry Premix (Takara, #639549, San Jose, CA, USA) in accordance with the manufacturer's guidelines.

Quantitative PCR (qPCR) was conducted using the Applied Biosystem StepOne System (Applied Biosystems, Forster City, CA, USA). The real-time PCR reactions were quantified by detecting the binding of fluorescent SYBR Green dye to double-stranded DNA. For PCR amplification, the total reaction volume was set to 20 μL with RNase-free water, after combining with 100 ng of cDNA, 2 μL of primer sets, 10 μL of 2× SYBR premix Ex Taq, ROX Plus (Takara, #RR42LR). The amplification protocol included an initial step of incubation at 95 °C for 15 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 40 s. Each cDNA was analyzed in duplicate. Relative mRNA expression levels were normalized to the housekeeping gene GAPDH, and the ΔCt value was determined as follows: ΔCt = Ct (GAPDH) − Ct (target gene). The average ΔCt was subtracted from each experimental condition to obtain the ΔΔCt value. Fold changes in gene expression were calculated as 2−ΔΔCt relative to the control sample, normalized to GAPDH. The primer sequences used in qPCR for each gene are listed in Supplementary Data 5.

Surface biotinylation and immunoblotting

To conduct the biotinylation assay, HEK293 cells cultured in 0.02 mg/mL poly-D-lysine (Sigma Aldrich, #P6407)-coated 6-well plates were incubated for 5 min at 4 °C and washed twice with cold phosphate-buffered saline (PBS). The transmembrane proteins of the cells were biotinylated using Sulfo-NHS-SS-Biotin (Thermo Scientific, #21331, Waltham, MA, USA) in cold PBS (0.3 mg/ml biotin in PBS) on ice for 30 min in darkness. Subsequently, the cells were treated with a quenching buffer consisting of 1% BSA in 0.5X PBS for 10 min on ice in darkness, followed by two washes with cold PBS. The cells were then collected using a lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 7.4), 1 mM EDTA, 1% (v:v) NP40, 0.5% (v:v) sodium deoxycholate, and a protease inhibitor (Roche, #04693159001, Mannheim, Germany). The harvested cells were homogenized using a sonicator for 20 s and centrifuged at 16,000 x g for 20 min at 4°C. The supernatant of the lysate was collected, and 300 µg of the lysate was incubated overnight at 4°C with 200 µl of 10% streptavidin agarose resin (Thermo Scientific™, #20349, Waltham, MA, USA). The resin bound to the biotinylated proteins was then subjected to centrifugation and washed four times using lysis buffer. The biotinylated proteins were eluted with 2x sodium dodecyl sulfate (SDS) sample buffer containing dithiothreitol (DTT, 0.02 g/ml) at 38 °C for 30 min and subsequently separated through SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred to a nitrocellulose membrane (GE Healthcare, Amersham, #10600004, Chicago, IL, USA) and the membrane was blotted using suitable primary antibodies and HRP-conjugated secondary antibodies in 5% skim milk. Protein bands were visualized using enhanced chemiluminescence (GE Healthcare, Amersham #RPN2105), and the density of each protein band was quantified with imaging software (Multi Gauge ver. 3.0; Fujifilm, Tokyo, Japan).

Immunoprecipitation assay

The cells were washed twice with cold phosphate-buffered saline (PBS). Subsequently, the cells were harvested using lysis buffer consisting of 150 mM NaCl, 20 mM Tris (pH 7.4), 1 mM EDTA, 1% (v:v) NP-40, and a protease inhibitor (Roche, #04693159001, Mannheim, Germany). The harvested cells were then homogenized using a sonicator for 20 s and centrifuged at 16,000 x g for 20 min at 4°C. The supernatant of the lysate was collected, and 400 µg of the lysate was incubated for 16 h at 4°C with 200 µl of lysis buffer containing 5% EZview™ red anti-HA affinity gel (Millipore, #E6779, Billerica, MA, USA). The resin bound to the protein was subjected to centrifugation, washed four times with lysis buffer, and eluted using 2x sodium dodecyl sulfate (SDS) sample buffer containing dithiothreitol (DTT, 0.02 g/ml) at 38°C for 30 min. Subsequently, the eluate was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to immunoblotting as previously described.

Immunofluorescence assay

HeLa cells were cultured on 18-mm round coverslips. Subsequently, the cells were fixed using a solution of 4% paraformaldehyde for 7 min, and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. Then, the cells were washed three times with PBS and incubated with a blocking buffer containing 1% BSA and 5% horse serum for 1 h at room temperature. Following the blocking step, the cells were exposed to the appropriate primary antibodies for 1 h at room temperature, and then washed three times with PBS. Subsequently, the cells were stained with fluorescent-dye conjugated secondary antibodies for 30 min at room temperature, and again washed three times with PBS. The following antibodies were used for staining: anti-CFTR ACL006 (Alomone Labs, #ACL006), anti-SNX27 (Abcam, #ab77799), anti-HA (Novus #NB600-362), Alexa Fluor 488-conjugated polyclonal donkey anti-rabbit (Invitrogen, #A21206), Alexa Fluor 568-conjugated polyclonal donkey anti-mouse (Invitrogen, #A10037), and Alexa Fluor 680-conjugated polyclonal donkey anti-goat (Invitrogen, #A21084) antibodies. The sample was mounted onto a slide glass using a mounting medium (Agilent Dako, #S3025, Santa Clara, CA, USA), and the images were captured using a confocal microscope (LSM 980, Carl Zeiss, Berlin, Germany) equipped with a 63×/1.4 numerical aperture oil objective lens.

The degree of colocalization between CFTR, SNX27 and Rab11A was assessed using Manders' colocalization coefficients (MCC), using the colocalization module of the ZEN 2012 software (black edition; Carl Zeiss, Berlin, Germany). For measuring MCC under each condition, pixels with fluorescence intensity above the threshold level of 50 in both channels (red and green) were considered to represent overlapping signals. Subsequently, the mean MCC from the regions of interest was determined. For the two target proteins, referred to as A and B, two distinct MCC values were computed as follows:
MCCA colocalized with B∕Total A = ΣiAi,colocal / ΣiAi (1)
MCCB colocalized with A∕Total B = ΣiBi,colocal / ΣiBi (2)
To visualize spatial colocalization between channels, line-scan intensity profiles were generated using ZEN v3.12 Blue edition (Blue edition; Carl Zeiss, Berlin, Germany). Straight lines were drawn across representative regions containing overlapping fluorescence signals in both channels (red and green) merged images. Fluorescence intensity values (0-255) along the line were extracted for each channel and plotted as a function of distance using GraphPad Prism (GraphPad Software).

Pull-down assay

The Rosetta BL-21(DE3) strain of Escherichia coli (Enzynomics, #CP1010, Daejeon, Korea) was used for the production of recombinant proteins. Expression of the recombinant fusion proteins was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and followed by incubation at 30°C for 6 h. The proteins were subsequently purified using either Glutathione Sepharose™ beads (GE Healthcare, #17-0756, Chicago, IL, US) or nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (QIAGEN, #30210, Valencia, CA), according to the manufacturer’s instructions. For the pull-down assay, the buffer containing His6-fusion proteins was dialyzed against a buffer composed of 50 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% (v:v) NP40. Subsequently, 10 µg of the eluted His6-fusion proteins were incubated with Glutathione Sepharose™ beads pre-loaded with GST-fusion proteins overnight at 4°C. The protein-bound beads were washed four times with the dialysis buffer and subsequently eluted in 2x SDS sample buffer. The eluted proteins were subjected to immunoblotting, following the previously described protocol.

Surface plasmon resonance (SPR) analysis

The cDNA fragments encoding the PDZ domain (residues 1-149) of human SNX27 (UniProt: Q96L92) and the full-length human VPS26A (residues 1-327; UniProt: O75436) were cloned into the pET28a vector with an N-terminal 6×His tag. The construct pET28a-6×His-SNX27-PDZ was transformed into E. coli BL21(DE3) cells and cultured in LB medium at 37 °C until the optical density at 600 nm (OD₆₀₀) reached 0.5-0.6. Protein expression was induced with 0.5 mM IPTG and carried out at 17 °C overnight. Similarly, the pET28a-6×His-VPS26A construct was transformed into E. coli C41(DE3) cells, induced at OD₆₀₀ = 0.5-0.6 with 0.3 mM IPTG, and expressed at 17 °C overnight. The cells were harvested by centrifugation at 3,000 × g for 15 min and stored at −80 °C until use.

For purification, the cell pellets were resuspended in lysis buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, and 30 mM imidazole, supplemented with DNase I and a protease inhibitor cocktail (500 μM AEBSF, 1 μM E-64, 1 μM leupeptin, and 150 nM aprotinin). Cells were disrupted by sonication and the lysate was clarified by centrifugation at 35,000 × g for 60 min at 4 °C, followed by filtration through a 0.45 μm membrane. The supernatant was incubated with Ni-NTA resin (Thermo Fisher Scientific), washed with the same buffer containing 30 mM imidazole, and eluted with buffer containing 300 mM imidazole. Eluted proteins were concentrated using Amicon® Ultra centrifugal filters (10 kDa or 30 kDa MWCO) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 200 prep-grade column (Cytiva) equilibrated with 20 mM HEPES (pH 7.5) and 150 mM NaCl.

All SPR experiments were performed using a Biacore T200 system (Cytiva) equipped with a Series S CM5 sensor chip. Purified VPS26A protein was covalently immobilized onto the chip surface via standard amine coupling according to the manufacturer’s protocol. For pH scouting, 10 mM sodium acetate (pH 5.5) was used, and the immobilization level was adjusted to approximately 3,900 RU. A reference flow cell was activated and blocked without ligand for background subtraction. Binding experiments were carried out in HBS-EP⁺ running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% [v/v] Surfactant P20) at 25 °C. SNX27-PDZ was injected over the VPS26A-immobilized surface either alone or in complex with the CFTR-Ct30 peptide at a PDZ-to-peptide molar ratio of 1:6, at a flow rate of 30 μL min⁻¹. Association and dissociation phases were recorded, and after each cycle the surface was regenerated with 10 mM glycine-HCl (pH 2.0) for 30 s to remove bound analyte. Sensorgrams were reference-subtracted and analyzed using Biacore T200 Evaluation Software. The equilibrium binding responses were fitted to a 1:1 Langmuir model using steady-state affinity analysis to derive the dissociation constant (KD).

Protein structure prediction

AlphaFold2 and ColabFold were used to construct VPS26A and SNX27 complex41,61,62. We used AlphaFold2-multimer-V2 algorithm without template or minimization and 3 recycle number parameters in ColabFold. AlphaFold3 structure prediction was run directly on the AlphaFold3 server (https://alphafoldserver.com/)42.The input sequences were as follows: full sequence of human VPS26A; PDZ domain in human SNX27, corresponding to residues 41 to 136; last 30 residues of human CFTR. The last 30 residues of CFTR, when treated as an independent protein chain, did not form any significant secondary structures, nor did independent ColabFold runs suggest a stably bound conformation. This outcome was anticipated, as although AlphaFold2 excels in de novo protein structure prediction, unstructured N- or C-terminal regions or long loops are likely to exhibit significant flexibility, making them challenging to predict accurately41. Therefore, we appended the last 30 residues of CFTR to the C-terminus of the SNX27 PDZ domain, introducing a flexible linker consisting of 30 Gly residues. This Gly-linker approach was widely employed prior to the introduction of AlphaFold2-multimer, as it allows for sufficient structural flexibility without disrupting the overall protein conformation63. Incorporating the Gly-rich linker in this manner resulted in consistent binding of the CFTR C terminus to the PDZ domain of SNX27. ChimeraX software was used for the three-dimensional visualization of the proteins and calculation of the Ramachandran plot64.

Molecular dynamics simulations

The predicted structures of VPS26A-SNX27 and VPS26A-SNX27-CFTR C terminus were used as templates for molecular dynamics (MD) simulations. Since only the last four residues of CFTR were stably bound to the SNX27 PDZ domain, we included only the last six residues (VQDTRL) for the CFTR-bound VPS26A-SNX27 interaction analysis. The system was initially neutralized and solvated in a 150 mM KCl environment using a CHARMM-GUI web server 65,66. The final system was contained in an ~120 × 120 × 120 Å3 box, with ~50,000 water molecules and a total of ~160,000 atoms. All the simulations were performed with the CHARMM36m force field and GROMACS software 67,68.

The particle mesh Ewald (PME) algorithm was used to evaluate the long-range electrostatic interactions. The van der Waals interactions for a smoothing function were between 10 and 12 Å. The system was minimized using the steepest descent algorithm, for 5000 steps. Then, 125 ps equilibration steps were applied to the protein, with the restraints being gradually reduced to zero. A complete electronic evaluation was performed every 2 fs using the SHAKE algorithm69. The pressure and temperature were, maintained at 1 atm and 298 K throughout the simulation using the Nosé-Hoover Langevin piston method and Langevin dynamics, respectively70. No anisotropic cell fluctuations were observed. For each system, eight replicates of 100 ns simulation were performed, and snapshots were saved at 100 ps intervals for further analysis.

The root mean square distance (RMSD) and principal component analysis (PCA) were calculated using GROMACS software67,68. To calculate the interaction energy between VPS26A and SNX27, the molecular mechanics with generalized Born and surface area solvation (MM/GBSA) method was used43. Residue decomposition analysis within the MM/GBSA framework was used to determine the contribution of each residue.

Viral propagation, quantification, and infection of SARS-CoV-2

A clinically isolated SARS-CoV-2 NCCP43344strain (Korean National Culture Collection for Pathogens Collection number: NCCP-43344) was provided by Prof. MS Park of Korea University. For the propagation of viruses, ACE2-overexpressing HeLa cells were infected by SARS-CoV-2 with 10 multiplicity of infection (MOI) and then culture supernatant was harvested 24 h post-infection. The harvested supernatant was filtered and stocked at -80℃. For plaque formation assay, one of the stock vials was used to infect Vero cells which were obtained from the Ministry of Food and Drug Safety (Osong, Republic of Korea). Infected cells then incubated with culture media containing 1 % low gelling temperature agarose (Lonza #50L101, Rockland, ME) for 48 h. Viral plaques were visualized with Neutral Red Solution (Sigma #2889, Sigma-Aldrich, St. Louis, MO) for additional 24 h and were counted to determine the plaque-forming unit of the viral stock. For viral infection assays, target cells were incubated with the virus for 1 h, after which the medium was replaced with fresh medium supplemented with 2% FBS until further experimentation.

For the quantification of the SARS-CoV-2 virus secretion, viral RNA in cell culture supernatant was extracted using a QIAamp Viral RNA Mini Kit (QIAGEN #52906, Valencia, CA), as per the manufacturer's instructions. Quantitative PCR was performed with a Luna® Universal One-Step RT-qPCR Kit (New England Biolabs Inc., Ipswich, MA, USA) using the ABI Prism 7000 detection system (Applied Biosystems, Waltham, MA, USA). For amplification, 2 μL of extracted RNA was added to a mixture of 10 μL of 2X Luna® Universal One-Step Reaction Mix, 0.4 μl of 10 μM primers, 0.2 μl of 10 μM probe and 1 μL of 20X Luna WarmStart® RT Enzyme Mix adjusted for a total reaction volume of 20 μL with RNase-free water. Amplification was carried out using the optimal thermocycling condition outlined in the manufacturer’s protocol for 40 cycles. The following primer-probe sets, targeting nsp14, were used: forward primer 5’-TGGGGYTTTACRGGTAACCT-3’, reverse primer 5’- AACRCGCTTAACAAAGCACTC-3’, probe 5’-56-FAM- FAM-TAGTTGTGA/ZEN/TGCWATCATGACTAG-3IABkFQ-3’. The standard curve was generated by conducting qRT-PCR, using serially diluted virus stock. Then Ct values were converted into viral titer (pfu/mL).

Statistical analysis

The results are presented as the mean ± standard error of the mean (SEM), using the data obtained from multiple experiments. Statistical analyses were conducted using appropriate tests, such as a two-tailed Student's t-test or one-way analysis of variance, followed by Tukey's multiple comparison test. GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) was used for the statistical analysis. A p-value under 0.05 was considered statistically significant.

Chemicals and antibodies

Further materials and reagents used in this study are listed in Supplementary Data 6, 7 and 8.

Data availability

All data supporting the findings of this study are available within the main text, main figures, and Supplementary Information. The original ICC image files generated in this study have been deposited in the EBI BioImage Archive under accession code S-BIAD3018 (https://doi.org/10.6019/S-BIAD3018).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The paper by Kim et al. suggests that the retromer complex, in combination with SNX27, participates in unconventional protein secretion pathways in addition to its well-established role in cargo recycling. The authors investigate intracellular trafficking to the plasma membrane of two unrelated proteins: the cystic fibrosis transmembrane conductance regulator (CFTR), focusing on the disease-causing mutant (ΔF508), and the SARS-CoV-2 spike protein. The introduction gives the impression that the spike protein was included to exemplify a naturally occurring Golgi-bypass mechanism utilized by viruses that employ the same combination of proteins to assist secretion of the S-protein to the plasma membrane of infected cells. However, the main focus of the paper is on CFTRΔF508, as characterization of unconventional protein secretion pathways might provide pharmacological targets.\nIn general, this appears to be a very interesting research direction, and overall, the experimental approach is reminiscent of work published by the Lee group in 2011. Therefore, it is surprising that in the current research, the authors never mention their own previous study focusing on the PDZ-based interaction between GRASP and CFTR that plays a crucial role in unconventional surface trafficking of this protein. Since GRASP and SNX27 are both able to bind the CFTR-PDZ motif and both appear to be important for unconventional surface trafficking under exactly the same conditions—such as when the ER-to-Golgi trafficking route is blocked by dominant-negative ARF1-Q71L or SAR1-T39N mutants, or by syntaxin 5 overexpression—this omission is particularly concerning. However, the authors do not even consider including their previous findings in the discussion, let alone testing experimentally whether these proteins play redundant, competing, or complementary roles, or might have stepwise involvement in ER stress-induced CFTR trafficking to the plasma membrane.\nAnother significant concern is that the authors appear to conflate endosomal membrane trafficking with endocytosis and seem to have limited understanding of retromer function. Unlike clathrin, retromer is not directly involved in endocytosis as a process of internalization from the plasma membrane. Rather, it assists in the sorting of cargo proteins within endosomes, ensuring that they are either recycled back to the trans-Golgi network (TGN) or delivered to other cellular locations, rather than being targeted for degradation in lysosomes. SNX27-retromer is a well-known and unique mammalian retromer complex that participates in direct cargo recycling to the plasma membrane. Therefore, the use of the dynamin-dependent endocytosis inhibitor dynasore to characterize the SNX27-retromer pathway appears to be off-target. The follow-up experiments with Rab proteins, including the selection of tested Rab proteins, raise questions about what the authors aimed to test. Overall, the paper gives the impression of patchwork experimentation without a clear outcome.\nMinor Comments\nLine 95: \"...VPS26A-associated recycling endosomes may serve as a common platform for...\"\nWhat do the authors mean by \"VPS26A-associated recycling endosomes\"? Please clarify. VPS26A is a component of the retromer complex that can localize to early endosomes when it uses SNX3 or SNX27 as an adaptor, whereas in combination with SNX-BAR proteins, it will localize to late endosomes.\nLines 130-131: \"Retromer complexes are multi-protein complexes that play a key role in endocytosis and endosomal recycling of cellular proteins.\"\nPlease provide references for retromer complexes being involved in endocytosis. To my knowledge, retromer is a coat complex involved in endosomal membrane trafficking.\nLines 162-163: \"The mammalian retromer complex functions as a regulator of intracellular trafficking in general by interacting with sorting nexins (SNXs)...\"\nIt would be easier to follow if the authors used conventional terminology such as \"coat complex\" when referring to retromer.\nLine 228: \"...identical to the previously reported crystal structure prediction using mouse VPS26A...\"\nRemove the word \"prediction\"; the structure was experimentally determined.\nLine 271: \"The VPX26A-SNX27 complex mediates CFTR UPS through endocytic recycling\"\nTypo error: VPX26A should be VPS26A.\nFigure 3g: The immunostaining for SNX27 in ΔF508-CFTR cells in the presence of ARF1-Q71L appears more plasma membrane-like and is very different compared to the cells in Figure 6i, where cells received the same treatment but were additionally stained for Rab11A. For all immunostaining co-localization experiments, please provide additional representative images in the supplementary materials to account for staining variation.", "reviewer_2": "The CFTR mutant ΔF508, which is implicated in cystic fibrosis, is known to exhibit defective trafficking from the endoplasmic reticulum (ER) to the plasma membrane, resulting in reduced surface expression of CFTR and, consequently, disease pathology. The authors have previously demonstrated that under conditions where the conventional secretion pathway through the Golgi is inhibited—leading to ER stress and the induction of an unconventional secretion pathway—the transport of ΔF508 to the plasma membrane is enhanced, where it can exert partial CFTR activity. These findings suggest that promoting proper trafficking of ΔF508 to the cell surface could be a viable therapeutic strategy for cystic fibrosis.\nIn the present study, Kim et al. employed a targeted CRISPR knockout screen to identify factors involved in the unconventional trafficking of ΔF508 under conditions favoring activation of this pathway. Their screen revealed key components of the retromer complex—VPS26A, VPS29, VPS35—as well as the sorting nexin SNX27, as contributors to ΔF508 surface transport. Structural modeling using AlphaFold further suggested that the interaction between VPS26A and SNX27 is mediated by the binding of CFTR’s PDZ-binding motif to the PDZ domain of SNX27. The authors showed that these interactions facilitate the unconventional transport of CFTR through an endocytic recycling mechanism. Importantly, they also found that this pathway contributes to the unconventional secretion of the SARS-CoV-2 spike protein, suggesting broader relevance of the mechanism. The study is novel in elucidating the mechanisms underlying unconventional secretion of both CFTR ΔF508 and the SARS-CoV-2 spike protein. The evidence that this pathway may operate in SARS-CoV-2-infected cells in vivo is particularly compelling. The experimental approaches are rigorous, and the quality of the data is high and trustworthy. Nevertheless, several issues require clarification or further exploration:\nMajor Points\n1. The conclusions regarding the unconventional trafficking of ΔF508 are primarily based on experimental conditions involving overexpression of a dominant-active ARF1 mutant (ARF1 QL). It remains unclear under what physiological, pathological, or pharmacological contexts this pathway might be activated in actual cystic fibrosis patients. At a minimum, key experiments should be performed under other ER stress conditions—conditions under which the unconventional pathway is presumably activated—to determine whether ΔF508 is similarly trafficked to the cell surface. Moreover, it is critical to discuss whether such ER stress-inducing conditions could realistically occur in cystic fibrosis patients and whether ΔF508 trafficking through this pathway is plausible in vivo. Without such insight, the study risks being interpreted as a detailed analysis of an artificially induced transport mechanism specific to ARF1QL-overexpressing cells.\n2. In Fig. 6g and 6i, it is unclear whether the observed immunostaining patterns are specific to ARF1QL overexpression or whether they can also be reproduced by activating the unconventional pathway through alternative means.\nMinor Point\nThe details of the CRISPR KO screen are difficult to follow. It is not clearly stated what comparisons were made. While it appears that PE intensity was measured under three different conditions, the specific comparisons—particularly in the volcano plot in Fig. 1c—are not clearly described in the figure legend, making it difficult for the reader to interpret the data without guessing.", "reviewer_3": "This manuscript by Ye Jim Kim et al. presents a comprehensive and well-executed study describing a Golgi-independent unconventional protein secretion (UPS) pathway involving SNX27 and the VPS26A-containing retromer complex, which is shown to participate in the trafficking of both ∆F508-CFTR and SARS-CoV-2. The identification of the retromer complex as a component of the UPS pathway is novel and highly relevant to researchers studying unconventional secretion mechanisms as well as the retromer complex and its associated proteins.\nI support publication of this manuscript with the following suggestions:\n1. For the AlphaFold model of VPS26A:SNX27-PDZ:CFTR-C-terminus, the authors should include both the PAE plot and the pLDDT-colored model in Supplementary Figure 4. This would provide crucial information on the model's confidence and predicted errors. Additionally, the authors should clarify why AlphaFold3 was not used for this analysis. Have they obtained similar results with this newer version? The model should be deposited in a publicly accessible database or provided as supplementary material for verification and to facilitate further research.\n2. The authors did not include a control to verify SNX2 silencing in Supplementary Fig. 3a. They should use a technique such as immunofluorescence, western blotting, or RT-qPCR to confirm gene knockdown.\n3. The authors claim in the Discusion that the binding affinity between SNX27 and VPS26A increases in the presence of CFTR. This is a novel and potentially important finding, as prior work has only demonstrated allosteric enhancement of SNX27 binding to cargo by VPS26. Their pull-down experiments show that SNX27 binds to GST-CFTR, while VPS26A interacts only with the pre-formed SNX27-CFTR complex. Their molecular dynamics simulations adequately complement their structural predictions and support the hypothesis that CFTR binding enhances the SNX27-VPS26A interaction. However, to conclusively demonstrate an increase in binding affinity, quantitative interaction assays, such as isothermal titration calorimetry (ITC), should be conducted, comparing VPS26A titration against SNX27-PDZ alone versus SNX27-PDZ pre-incubated with CFTR-Ct30.\nOther Comments:\n1. Line 261: The phrase \"forming electrostatic interactions with hydrogen bonds\" is unclear. A more accurate term would be \"salt bridge\" o \"bidentate salt bridge\".\n2. Line 271: Correct \"VPX26A\" to \"VPS26A\".\n3. Line 380: \"(Fig.2)\" should be corrected to \"(Figs. 2 and 3)\" as both figures support the referenced claim.\n4. Line 457, 459, 463: Restriction enzyme names should use Roman numerals by convention: BamHI, EcoRI, XhoI, NcoI.\n5. Lines 516-521: The section describing the high-throughput genetic screening using the CRISPR/Cas9 knockout library does not specify how barcoded NGS libraries were generated or which NGS platform was used for sequencing. This information should be included for clarity and reproducibility.\n6. Line 539: The expression \"−ΔΔCt\" should be formatted as a superscript.\n7. Line 1017: In the legend for Figure 5, panels c and d appear to be mislabeled or switched, this should be corrected.\n8. Line 1122: In Supplementary Figure 2b, one of the statistical comparisons is missing a significance asterisk.\n9. Line 1153, 1155, 1157: In Supplementary Fig. 4 the term \"prediction\" should be removed when referring to the \"crystal structure (4p2a)\", as this is an experimentally determined structure.\n10. Line 1156: Correct \"Ramachandra\" to \"Ramachandran\".\n11. Line 1159: In Supplementary Figure 5b, the Y-axis label overlaps with the X-axis and should be repositioned for clarity.\n12. The concentrations of antibodies used in the study are not indicated in the Methods section or in Supplementary Table 6. The authors should specify whether they used the manufacturer’s recommended concentrations or an alternative to ensure experimental reproducibility.\nOverall, this is a well-executed study that significantly advances our understanding of the broad implications of the retromer complex in intracellular trafficking and introduces new mechanistic insights into UPS. I look forward to seeing the authors’ revised manuscript.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-Point Response to Reviewer’s Comments\nReviewer #1:\nThe paper by Kim et al. suggests that the retromer complex, in combination with SNX27,\nparticipates in unconventional protein secretion pathways in addition to its well-established\nrole in cargo recycling. The authors investigate intracellular trafficking to the plasma\nmembrane of two unrelated proteins: the cystic fibrosis transmembrane conductance regulator\n(CFTR), focusing on the disease-causing mutant (ΔF508), and the SARS-CoV-2 spike protein.\nThe introduction gives the impression that the spike protein was included to exemplify a\nnaturally occurring Golgi-bypass mechanism utilized by viruses that employ the same\ncombination of proteins to assist secretion of the S-protein to the plasma membrane of infected\ncells. However, the main focus of the paper is on CFTRΔF508, as characterization of\nunconventional protein secretion pathways might provide pharmacological targets.\nResponse: We appreciate the reviewer’s constructive comments and suggestions.\nQuestion 1: In general, this appears to be a very interesting research direction, and overall,\nthe experimental approach is reminiscent of work published by the Lee group in 2011.\nTherefore, it is surprising that in the current research, the authors never mention their own\nprevious study focusing on the PDZ-based interaction between GRASP and CFTR that plays\na crucial role in unconventional surface trafficking of this protein. Since GRASP and SNX27\nare both able to bind the CFTR-PDZ motif and both appear to be important for unconventional\nsurface trafficking under exactly the same conditions—such as when the ER-to-Golgi\ntrafficking route is blocked by dominant-negative ARF1-Q71L or SAR1-T39N mutants, or by\nsyntaxin 5 overexpression—this omission is particularly concerning.\nHowever, the authors do not even consider including their previous findings in the discussion,\nlet alone testing experimentally whether these proteins play redundant, competing, or\ncomplementary roles, or might have stepwise involvement in ER stress-induced CFTR\ntrafficking to the plasma membrane.\nSNX27 relates to GRASP55 within the UPS pathway. To determine whether GRASP55 and\nSNX27 function redundantly or complementarily in UPS, we performed surface biotinylation\nexperiments with silencing of both proteins, followed by immunocytochemistry and subcellular\nlocalization analyses under UPS inducing conditions.\nOur cell-surface biotinylation results show that the combined depletion of SNX27 and\nGRASP55 caused a greater reduction in ΔF508-CFTR UPS than either single knockdown,\nsuggesting that the two PDZ adaptors have non-redundant roles within the UPS pathway (Fig.\n4c, d).\nThe imaging analyses revealed that GRASP55 primarily localizes to perinuclear Golgi\nregions under basal conditions. Notably, upon UPS activation by ARF1-Q71L, GRASP55\nredistributed toward peripheral structures, putatively tubular ER regions, resulting in a strong\nincrease in its co-localization with ΔF508-CFTR (from 22% to 68%, Supplementary Fig.3),\nconsistent with our previous findings1,2. While both SNX27 and GRASP55 were enriched in\nperinuclear regions and partially co-localized under basal conditions, UPS activation by ARF1-\nQ71L resulted in no significant increase in their co-localization despite the peripheral\nredistribution of GRASP55 (Fig. 4e, f).\nTaken together, these results suggest that GRASP55 and SNX27 contribute to the UPS\npathway in distinct yet complementary ways, acting at different stages of the trafficking\nprocess. We propose that GRASP55 facilitates the ER exit of ΔF508-CFTR upon UPS\nactivation, particularly at ER-tubular bodies3, whereas SNX27 participates in the cell-surface\ntrafficking of ΔF508-CFTR from recycling endosomes. These findings and interpretations have\nbeen incorporated into the main text (Lines 207-223) and Discussion (Lines 469-478) have\nbeen updated accordingly.\nQuestion 2: Another significant concern is that the authors appear to conflate endosomal\nmembrane trafficking with endocytosis and seem to have limited understanding of retromer\nfunction. Unlike clathrin, retromer is not directly involved in endocytosis as a process of\n2\ninternalization from the plasma membrane. Rather, it assists in the sorting of cargo proteins\nwithin endosomes, ensuring that they are either recycled back to the trans-Golgi network (TGN)\nor delivered to other cellular locations, rather than being targeted for degradation in lysosomes.\nSNX27-retromer is a well-known and unique mammalian retromer complex that participates\nin direct cargo recycling to the plasma membrane. Therefore, the use of the dynamin-\ndependent endocytosis inhibitor dynasore to characterize the SNX27-retromer pathway\nappears to be off-target.\nResponse: We have updated the text to avoid any potential misinterpretation.\n1) We have revised all text that might give the impression that retromer is involved in the\ninternalization process from the plasma membrane, and we now clearly state that “retromer\nmediates the sorting of cargo proteins within endosomes, directing them either to be recycled\nback to the trans-Golgi network (TGN) or transported to alternative intracellular destinations,\nrather than being targeted for degradation in lysosomes” (Lines 96–104, 145-148, and 178-\n181).\n2) The reason we performed the dynasore experiments was to confirm that the retromer effect\nwe observed is related to the endosome-associated recycling pathway, rather than to\nalterations in the retrograde removal of CFTR from the plasma membrane. This is particularly\nrelevant because ΔF508-CFTR is known to be vulnerable to endocytosis and subsequent\nlysosomal degradation due to the peripheral protein quality control system4. As shown in our\nresults (Fig. 7a), dynasore treatment markedly increased the cell-surface expression of\nΔF508-CFTR under UPS-inducing conditions, indicating that endocytosis of ΔF508-CFTR\nfrom the plasma membrane is indeed substantial. However, silencing of retromer components,\nSNX27, and RAB11 significantly reduced the UPS-induced cell-surface expression of ΔF508-\nCFTR even under dynasore-treated conditions (Fig. 7a-f), further supporting the conclusion\nthat retromer participates in anterograde trafficking via recycling endosomes. We believe\nthese results remain important and of value to readers. We have revised the relevant section\nof the manuscript to clarify this rationale (lines 328–334).\nRab proteins, raise questions about what the authors aimed to test. Overall, the paper gives\nthe impression of patchwork experimentation without a clear outcome.\nResponse: Our intent in performing the Rab protein experiments was to determine whether\nthe endosomal recycling pathway is involved in CFTR UPS, rather than to comprehensively\ncharacterize all ~60 human Rab proteins. Given that the UPS pathway bypasses the Golgi\napparatus, we initially hypothesized that endosomal compartments might mediate membrane\ndelivery and therefore selected a panel of Rab proteins known to regulate distinct endosome-\nassociated vesicular compartments, including early endosomes (Rab5A), recycling\nendosomes (Rab11A, Rab4A), secretory endosomes (Rab27A), and lysosomes (Rab38).\nAlthough our previous surface-biotinylation data implied that recycling and secretory\nendosomal pathways, but not the lysosomal route, may contribute to CFTR UPS, we agree\nwith the reviewer that these data alone did not provide comprehensive insight into the role of\nRab proteins in UPS pathways. Therefore, we removed the data previously included in\nSupplementary Fig. 6. Instead, we now provide more detailed evidence showing that Rab11,\na representative Rab protein involved in endosomal recycling5,6, participates in CFTR UPS,\nfurther supporting that the SNX27-mediated pathway indeed promotes UPS via an endosomal\nrecycling route (Fig. 7e-j; Supplementary Figs. 8, 9, and 11c-d). The relevant Results sections\n(Lines 343–347 and 353–360) have been revised accordingly.\nMinor Comments\nQuestion 4: Line 95: \"...VPS26A-associated recycling endosomes may serve as a common\nplatform for...\"\nWhat do the authors mean by \"VPS26A-associated recycling endosomes\"? Please clarify.\nVPS26A is a component of the retromer complex that can localize to early endosomes when\nit uses SNX3 or SNX27 as an adaptor, whereas in combination with SNX-BAR proteins, it will\nlocalize to late endosomes.\nResponse: We rewrote the text for clarity. It now reads: “These findings broaden our\n4\nunderstanding of how endosomal sorting machinery, classically linked to recycling and\nretrograde transport, can be repurposed to facilitate the stress-induced Golgi-independent\ntrafficking.” (Lines 101-104)\nQuestion 5: Lines 162-163: \"The mammalian retromer complex functions as a regulator of\nintracellular trafficking in general by interacting with sorting nexins (SNXs)...\"\nIt would be easier to follow if the authors used conventional terminology such as \"coat\ncomplex\" when referring to retromer.\nResponse: As recommended, we revised the text. It now reads: “The mammalian retromer\ncomplex functions as a coat complex that regulates intracellular trafficking in general by\ninteracting with sorting nexins (SNXs)…” (Lines 178-181)\nQuestion 6: Line 228: \"...identical to the previously reported crystal structure prediction using\nmouse VPS26A...\"\nRemove the word \"prediction\"; the structure was experimentally determined.\nResponse: Regarding your suggestion, and also taking Reviewer 3, Question 1 into\nconsideration, we have revised the text. It now reads: “…the AlphaFold2-predicted structure\nof human VPS26A and the SNX27 PDZ domain was nearly identical to the previously reported\ncrystal structure derived from mouse VPS26A and the rat SNX27 PDZ…” (Lines 281–285).\nQuestion 7: Line 271: \"The VPX26A-SNX27 complex mediates CFTR UPS through endocytic\nrecycling\", Typo error: VPX26A should be VPS26A.\nResponse: Thank you. The error has been corrected (Line 327).\nQuestion 8: Figure 3g: The immunostaining for SNX27 in ΔF508-CFTR cells in the presence\nof ARF1-Q71L appears more plasma membrane-like and is very different compared to the\ncells in Figure 6i, where cells received the same treatment but were additionally stained for\nRab11A. For all immunostaining co-localization experiments, please provide additional\n5\nrepresentative images in the supplementary materials to account for staining variation.\nResponse: We thank the reviewer for this constructive comment. As requested, we have now\nincluded additional representative images for the immunocytochemistry (ICC) data presented\nin the main figures (Figs. 3, 4, and 7) in Supplementary Fig. 11. In addition, we provide multiple\nrepresentative images for the newly performed ICC experiments, including Supplementary\nFigs. 3, 8, and 9, to minimize any potential misinterpretation arising from image-to-image\nvariation. We believe that these additional images further strengthen our main conclusions,\ndespite minor variability among individual images.\nThe CFTR mutant ΔF508, which is implicated in cystic fibrosis, is known to exhibit defective\ntrafficking from the endoplasmic reticulum (ER) to the plasma membrane, resulting in reduced\nsurface expression of CFTR and, consequently, disease pathology. The authors have\npreviously demonstrated that under conditions where the conventional secretion pathway\nthrough the Golgi is inhibited—leading to ER stress and the induction of an unconventional\nsecretion pathway—the transport of ΔF508 to the plasma membrane is enhanced, where it\ncan exert partial CFTR activity. These findings suggest that promoting proper trafficking of\nΔF508 to the cell surface could be a viable therapeutic strategy for cystic fibrosis.\nIn the present study, Kim et al. employed a targeted CRISPR knockout screen to identify\nfactors involved in the unconventional trafficking of ΔF508 under conditions favoring activation\nof this pathway. Their screen revealed key components of the retromer complex—VPS26A,\nVPS29, VPS35—as well as the sorting nexin SNX27, as contributors to ΔF508 surface\ntransport. Structural modeling using AlphaFold further suggested that the interaction between\nVPS26A and SNX27 is mediated by the binding of CFTR’s PDZ-binding motif to the PDZ\ndomain of SNX27. The authors showed that these interactions facilitate the unconventional\ntransport of CFTR through an endocytic recycling mechanism. Importantly, they also found\nthat this pathway contributes to the unconventional secretion of the SARS-CoV-2 spike protein,\nsuggesting broader relevance of the mechanism. The study is novel in elucidating the\nmechanisms underlying unconventional secretion of both CFTR ΔF508 and the SARS-CoV-2\nspike protein. The evidence that this pathway may operate in SARS-CoV-2-infected cells in\nvivo is particularly compelling. The experimental approaches are rigorous, and the quality of\nthe data is high and trustworthy. Nevertheless, several issues require clarification or further\nexploration:\nResponse: We thank the reviewer for the positive comments and constructive suggestions.\nQuestion 1: The conclusions regarding the unconventional trafficking of ΔF508 are primarily\nbased on experimental conditions involving overexpression of a dominant-active ARF1 mutant\n(ARF1 QL). It remains unclear under what physiological, pathological, or pharmacological\ncontexts this pathway might be activated in actual cystic fibrosis patients. At a minimum,\nkey experiments should be performed under other ER stress conditions—conditions under which\nthe unconventional pathway is presumably activated—to determine whether ΔF508 is similarly\ntrafficked to the cell surface. Moreover, it is critical to discuss whether such ER stress-inducing\nconditions could realistically occur in cystic fibrosis patients and whether ΔF508 trafficking\nthrough this pathway is plausible in vivo. Without such insight, the study risks being interpreted\nas a detailed analysis of an artificially induced transport mechanism specific to ARF1QL-\noverexpressing cells.\nResponse:\n1) As requested, we additionally performed experiments using the pharmacological ER stress\ninducer thapsigargin (Tg). As shown in Fig. 4a and 4b, treatment with Tg, which induced\nupregulation of the ER stress marker BiP, significantly increased ∆F508-CFTR cell-surface\nexpression, confirming activation of the UPS. Notably, knockdown of VPS26A or SNX27\nmarkedly suppressed Tg-induced UPS of ∆F508-CFTR. Furthermore, Tg also increased the\nco-localization of Rab11A with ∆F508-CFTR, as well as that of SNX27 with Rab11A (Fig. 7h,\nj; Supplementary Figs. 8 and 9). These results extend the involvement of SNX27 and Rab11A\nin the UPS mechanism beyond ARF1-Q71L and link it to a broader stress-responsive pathway.\nThe relevant descriptions have been added to the revised texts (Lines 201-206 and 353-360).\n2) It is generally accepted that cellular stress conditions, particularly ER stress, activate the\nUPS7,8. However, ER stress itself can be detrimental to the pathogenesis of cystic fibrosis9.\nTherefore, our strategy is not to directly activate the cellular stress but rather to rescue the\ncell-surface expression of ∆F508-CFTR through a specific activation of UPS without causing\nstress, following molecular identification of the UPS machinery. For example, based on the\npresent findings, selective activation of the SNX27-retromer pathway without inducing cellular\n8\nstress may represent a potential therapeutic approach, particularly when combined with\ninterventions that enhance ER exit of ∆F508-CFTR. This point is now discussed in the revised\nmanuscript (Lines 141-144, 484-495).\nQuestion 2: In Fig. 6g and 6i, it is unclear whether the observed immunostaining patterns are\nspecific to ARF1QL overexpression or whether they can also be reproduced by activating the\nunconventional pathway through alternative means.\nResponse: As suggested, we performed additional experiments to determine whether these\nfindings can be reproduced under other UPS-activating conditions (Fig. 7g–j; Supplementary\nFigs. 8 and 9). Specifically, we tested STX5 or SAR1-T39N overexpression, which induces ER\nstress via blockade of ER-to-Golgi transport, as well as pharmacological ER stress induced\nby thapsigargin, all of which have been shown to activate the UPS of ΔF508-CFTR1. The\nresults showed that these conditions also produced co-localization patterns similar to those\nobserved with ARF1-Q71L, suggesting that Rab11A is commonly involved in SNX27-mediated\nUPS of ΔF508-CFTR under various stimuli that evoke ER stress-associated UPS. The\nmanuscript has been revised to incorporate this additional information (Lines 353-360).\nMinor Point\nQuestion 3: The details of the CRISPR KO screen are difficult to follow. It is not clearly stated\nwhat comparisons were made. While it appears that PE intensity was measured under three\ndifferent conditions, the specific comparisons—particularly in the volcano plot in Fig. 1c—are\nnot clearly described in the figure legend, making it difficult for the reader to interpret the data\nwithout guessing.\nResponse: To address the concern, we revised Fig. 1b-c and added clarifying information to\nthe text (Lines 115–128, Legends for Fig. 1).\nWe first analyzed cells expressing extracellular HA-tagged wild-type (WT) CFTR, which\nefficiently traffics to the plasma membrane. The resulting PE signal served as a reference for\ndefining a gating threshold for surface-expressed HA-tagged CFTR (Fig. 1b left). In contrast,\n9\ncells expressing HA-tagged ΔF508-CFTR alone, which fails to exit the ER and reach the cell\nsurface under normal conditions, exhibited negligible PE signal (Fig. 1b middle), confirming\nboth the specificity of our detection system and the absence of background or leaky HA\nexpression.\nUsing this empirically determined PE threshold, we gated cells in the CRISPR screen\nexpressing HA-tagged ΔF508-CFTR together with ARF1-Q71L, a condition that activates the\nUPS pathway. Cells with PE signal above the WT-derived threshold were classified as\n“surface-rescued” and sorted accordingly (Fig. 1b right). These cells were then compared with\nthe total population to identify sgRNAs enriched or depleted in the surface-expressing fraction.\nThe volcano plot in Fig. 1c illustrates this comparison, showing sgRNA abundance in the\nsurface-rescued population relative to the total population and highlighting candidate genes\nthat regulate ΔF508-CFTR UPS.\nThis manuscript by Ye Jin Kim et al. presents a comprehensive and well-executed study\ndescribing a Golgi-independent unconventional protein secretion (UPS) pathway involving\nSNX27 and the VPS26A-containing retromer complex, which is shown to participate in the\ntrafficking of both ∆F508-CFTR and SARS-CoV-2. The identification of the retromer complex\nas a component of the UPS pathway is novel and highly relevant to researchers studying\nunconventional secretion mechanisms as well as the retromer complex and its associated\nproteins.\nI support publication of this manuscript with the following suggestions:\nResponse: We thank the reviewer for the encouraging comments and constructive\nsuggestions.\nQuestion 1: For the AlphaFold model of VPS26A:SNX27-PDZ:CFTR-C-terminus, the authors\nshould include both the PAE plot and the pLDDT-colored model in Supplementary Figure 4.\nThis would provide crucial information on the model's confidence and predicted errors.\nAdditionally, the authors should clarify why AlphaFold3 was not used for this analysis. Have\nthey obtained similar results with this newer version? The model should be deposited in a\npublicly accessible database or provided as supplementary material for verification and to\nfacilitate further research.\nResponse: As suggested, we have now included requested information and results generated\nusing AlphaFold3.\n1) In the revised manuscript, we have expanded Supplementary Fig. 6 to include both the PAE\nplots (Supplementary Fig. 6a-b) and the pLDDT-colored models (Supplementary Fig. 6c-d) for\nthe AlphaFold2-V2 and AlphaFold3-predicted VPS26A/SNX27-PDZ complexes, providing a\ndirect visualization of predicted local errors and overall model confidence. Although the\nAlphaFold2-V2 and AlphaFold3 models were broadly similar, the AlphaFold2-V2 predictions\nexhibited more favorable confidence metrics (lower PAE and higher pLDDT).\n11\n2) We also generated VPS26A/SNX27-PDZ models using AlphaFold2-V2 and AlphaFold3 and\ncompared them with the experimentally determined crystal structure (Supplementary Fig. 6e–\ng). The AlphaFold2-V2 predictions produced Ramachandran plots that more closely matched\nthe crystal structure.\nBased on these results, we selected the AlphaFold2-V2 model for all subsequent\nanalyses. The AlphaFold3 results have now been included in Supplementary Fig. 6 for\ncomparison. In addition, the AlphaFold2-V2 VPS26A/SNX27-PDZ and VPS26A/SNX27-\nPDZ/CFTR C-terminal models are provided as a Supplementary Data file for verification and\nfurther research. These additions are now described in the revised text (lines 275–286).\nQuestion 2: The authors did not include a control to verify SNX2 silencing in Supplementary\nFig. 3a. They should use a technique such as immunofluorescence, western blotting, or RT-\nqPCR to confirm gene knockdown.\nResponse: As requested, we have now included the RT-qPCR data for SNX2 and SNX17\nknockdown in Supplementary Fig. 4i, with the corresponding description added to the figure\nlegend in the revised manuscript. We appreciate the reviewer’s attention to this detail.\nQuestion 3: The authors claim in the Discussion that the binding affinity between SNX27 and\nVPS26A increases in the presence of CFTR. This is a novel and potentially important finding,\nas prior work has only demonstrated allosteric enhancement of SNX27 binding to cargo by\nVPS26. Their pull-down experiments show that SNX27 binds to GST-CFTR, while VPS26A\ninteracts only with the pre-formed SNX27-CFTR complex. Their molecular dynamics\nsimulations adequately complement their structural predictions and support the hypothesis\nthat CFTR binding enhances the SNX27-VPS26A interaction. However, to conclusively\ndemonstrate an increase in binding affinity, quantitative interaction assays, such as isothermal\ntitration calorimetry (ITC), should be conducted, comparing VPS26A titration against SNX27-\nPDZ alone versus SNX27-PDZ pre-incubated with CFTR-Ct30.\nResponse: As suggested, we conducted a quantitative biophysical assay using Surface\nVPS26A interaction. Specifically, we compared VPS26A binding to SNX27-PDZ alone versus\nSNX27-PDZ pre-incubated with CFTR-Ct30. The results revealed a clear increase in affinity\nin the presence of CFTR, with the K decreasing from 51.4 μM to 3.8 μM. These findings\nD\nfurther support our co-IP, pull-down, and MD simulation results, demonstrating that CFTR\nbinding enhances the SNX27-VPS26A interaction. We have incorporated these new data into\nthe revised manuscript (Fig. 5j and Supplementary Fig. 5) and updated the text accordingly\n(Lines 261-269 and 689-725).\nOther Comments:\nQuestion 4: Line 261: The phrase \"forming electrostatic interactions with hydrogen bonds\" is\nunclear. A more accurate term would be \"salt bridge\" or \"bidentate salt bridge\".\nResponse: We have revised the sentence to describe the interaction as a “salt bridge,” as\nsuggested, which more accurately reflects the electrostatic interaction between the charged\nresidues in the structural model (Lines 317-318).\nQuestion 5: Line 271: Correct \"VPX26A\" to \"VPS26A\".\nResponse: This typographical error has been corrected (Line 327). Thank you.\nQuestion 6: Line 380: \"(Fig.2)\" should be corrected to \"(Figs. 2 and 3)\" as both figures support\nthe referenced claim.\nResponse: The citation has been updated as suggested (Line 429).\nQuestion 7: Line 457, 459, 463: Restriction enzyme names should use Roman numerals by\nconvention: BamHI, EcoRI, XhoI, NcoI.\nResponse: Thank you for the careful correction. We have revised all the restriction enzyme\nnames as indicated (Lines 517-523).\nusing the CRISPR/Cas9 knockout library does not specify how barcoded NGS libraries were\ngenerated or which NGS platform was used for sequencing. This information should be\nincluded for clarity and reproducibility.\nResponse: The requested information has been provided in the text. It now reads: “Barcoded\nnext-generation sequencing (NGS) libraries were generated using a two-step PCR strategy\nfollowing the protocol described by Joung et al10. The resulting amplicons were purified using\nthe QIAquick Gel Extraction Kit (Qiagen, #28706, Valencia, CA, USA) and sequenced on an\nIllumina NovaSeq 6000 platform. NGS data were analyzed using MAGeCK (version 0.5.4).”\n(Lines 578-582)\nQuestion 9: Line 539: The expression \"−ΔΔCt\" should be formatted as a superscript.\nResponse: This has now been corrected (Line 600).\nQuestion 10: Line 1017: In the legend for Figure 5, panels c and d appear to be mislabeled\nor switched, this should be corrected.\nResponse: We thank the reviewer for pointing this out. The panel labels have been corrected\n(Legends for Fig. 6).\nQuestion 11: Line 1122: In Supplementary Figure 2b, one of the statistical comparisons is\nmissing a significance asterisk.\nResponse: We have revised Supplementary Fig. 2b to include the missing asterisk indicating\nstatistical significance. Thank you.\nQuestion 12: Line 1153, 1155, 1157: In Supplementary Fig. 4 the term \"prediction\" should be\nremoved when referring to the \"crystal structure (4p2a)\", as this is an experimentally\ndetermined structure.\nResponse: As pointed out, the term “prediction” has been removed (Legends for\nQuestion 13: Line 1156: Correct \"Ramachandra\" to \"Ramachandran\".\nResponse: This typographical error has now been corrected (Legends for Supplementary\nFig.6). Thank you.\nQuestion 14: Line 1159: In Supplementary Figure 5b, the Y-axis label overlaps with the X-\naxis and should be repositioned for clarity.\nResponse: We have adjusted the layout of the revised figure (Supplementary Fig. 7b) to\nprevent label overlap.\nQuestion 15: The concentrations of antibodies used in the study are not indicated in the\nMethods section or in Supplementary Table 6. The authors should specify whether they used\nthe manufacturer’s recommended concentrations or an alternative to ensure experimental\nreproducibility.\nResponse: We have now added all relevant antibody concentrations and dilution ratios to\nSupplementary Table 6.\nOverall, this is a well-executed study that significantly advances our understanding of the\nbroad implications of the retromer complex in intracellular trafficking and introduces new\nmechanistic insights into UPS. I look forward to seeing the authors’ revised manuscript.\nResponse: We sincerely thank the reviewer for the detailed and constructive feedback. We\nhope the revised version fully addresses these points and look forward to the possibility of\npublication.\n1. Gee, H. Y., Noh, S. H., Tang, B. L., Kim, K. H. & Lee, M. G. Rescue of DeltaF508-CFTR\ntrafficking via a GRASP-dependent unconventional secretion pathway. Cell 146, 746-\n760 (2011).\n2. Kim, J. et al. Monomerization and ER Relocalization of GRASP Is a Requisite for\nUnconventional Secretion of CFTR. Traffic 17, 733-753 (2016).\n3. Song, M. S. et al. Tubular ER structures shaped by ER-phagy receptors engage in\nstress-induced Golgi bypass. Dev Cell 60, 1568-1585.e1511 (2025).\n4. Okiyoneda, T. et al. Peripheral protein quality control removes unfolded CFTR from the\nplasma membrane. Science 329, 805-810 (2010).\n5. Takahashi, S. et al. Rab11 regulates exocytosis of recycling vesicles at the plasma\nmembrane. J Cell Sci 125, 4049-4057 (2012).\n6. Welz, T., Wellbourne-Wood, J. & Kerkhoff, E. Orchestration of cell surface proteins by\nRab11. Trends Cell Biol 24, 407-415 (2014).\n7. Park, H., Shin, D. H., Sim, J. R., Aum, S. & Lee, M. G. IRE1α kinase-mediated\nunconventional protein secretion rescues misfolded CFTR and pendrin. Sci Adv 6,\neaax9914 (2020).\n8. Rabouille, C. Pathways of Unconventional Protein Secretion. Trends Cell Biol 27, 230-\n240 (2017).\n9. Ribeiro, C. M. & Boucher, R. C. Role of endoplasmic reticulum stress in cystic fibrosis-\nrelated airway inflammatory responses. Proc Am Thorac Soc 7, 387-394 (2010).\n10. Joung, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation\nscreening. Nat Protoc 12, 828-863 (2017).\nReviewer #1:\nQuestion 1: SNX27 mediates the recycling of transmembrane proteins from\nendosomes to the plasma membrane by linking PDZ-motif cargo to retromer-mediated\nmembrane trafficking. This is well established role of the SNX27, and the authors\npropose that this pathway is repurposed to facilitate stress-induced Golgi-bypass\ntrafficking of the folding-unstable F508del CFTR to the cell surface as part of an\nunconventional protein secretion pathway.\nUnfortunately, despite the authors' inclusion of several additional experiments, the\nmanuscript remains somewhat disjointed and challenging to follow. A significant\nweakness is that most experiments lack clearly stated hypotheses and rationale for\nwhy they were conducted. The observations are largely limited to descriptive\nstatements without rigorous mechanistic interrogation. I would urge the authors to use\nestablished terminology throughout, rather than vague descriptions of processes that\nforce readers to interpret the intended meaning. Additionally, all colocalization\nexperiments must include graphical presentation of the selected regions used for\nquantification (e.g., histograms of fluorescence intensities in both channels along\nselected line profiles, or scatterplots correlating pixel intensities between channels).\nMore fundamentally, I am not convinced that the SNX27-retromer recycling pathway\nin the case of CFTR should be referred to as an unconventional protein secretion\npathway.\nResponse:\nWe thank the reviewer for the constructive suggestions. We have revised the\nmanuscript to improve clarity by more explicitly stating the rationale and hypotheses,\nand by consistently using established terminology.\nAs requested, we have now included demarcation of the quantified regions, along with\nthe corresponding line-scan intensity profiles, for all colocalization analyses. (Figures\n3, 5, 7, S4, S8, S9, and S11). Relevant descriptions have been added to each figure\nlegend and to the Materials and Methods section (Lines 704–709).\nendosomes, directing them either to be recycled back to the trans-Golgi network (TGN)\nor transported to\nalternative intracellular destinations, rather than being targeted for degradation in\nlysosomes 22,24.\nRetromer was indeed first discovered as a protein complex responsible for the\nretrograde (back to the Golgi) pathway for protein recycling, hence the name retromer.\nHowever, once SNX27 was confirmed as a retromer adaptor, it was also established\nthat the retromer-SNX27 complex is responsible for recycling to the plasma membrane\n(PM). It would be helpful if the authors clarify what 'alternative intracellular destinations'\nthey are referring to and provide appropriate citations to support this statement.\nResponse:\nWhen we referred to “alternative intracellular destinations,” this included late\nendosomes and plant vacuoles1,2, in addition to recycling endosomes and the plasma\nmembrane. However, because providing information that is overly broad would distract\nfrom the main focus of the present study, and because the number of references in\nthis manuscript has already reached the maximum allowed by the Journal (Nat\nCommun, 70 refs), we have revised the sentence to specify the two major retromer-\ndependent routes from endosomes in animal cells, retrograde transport to the trans-\nGolgi network and recycling to the plasma membrane. The revised text now reads:\n“Retromer mediates the sorting of cargo proteins within endosomes, generally\ndirecting them either to be transported back to the trans-Golgi network or recycled to\nthe plasma membrane in animal cells, rather than being targeted for degradation in\nlysosomes.” (Lines 146–149)\nQuestion 3: Line 155: …silencing of VPS26B reduced the ΔF508-CFTR UPS\n(Supplementary Fig. 2a, b); however, it also significantly decreased the cytosolic\nprotein levels of ΔF508-CFTR.\nGiven that CFTR is a transmembrane protein, additional explanation regarding the\nexpected presence of a soluble cytosolic pool would strengthen the interpretation. The\n2\nauthors should provide a more precise definition of 'cytosolic levels' in this\nexperimental context.\nResponse:\nAs indicated, we acknowledge that the term ‘cytosolic levels’ could be interpreted as\nreferring specifically to the soluble cytosolic pool. However, our original intention was\nto describe total cellular protein levels, as assessed by immunoblotting of whole-cell\nlysates. Accordingly, we have revised the text by replacing ‘cytosolic levels’ with ‘total\ncellular levels (cell lysate; Supplementary Fig. 2a, c).’ In addition, we have added a\nbrief clarification indicating that the reduction in overall ΔF508-CFTR abundance\nfollowing VPS26B silencing hampers precise assessment of its effects on cell-surface\ntrafficking (Lines 156–160).\nQuestion 4: Line 171: Notably, knockdowns of VPS26A, VPS29, and VPS35\nsignificantly inhibited the UPS of wild-type CFTR (Fig. 2e–j). These findings indicate\nthat the retromer complex is more crucial for the Golgi-independent UPS of CFTR\nrather than conventional Golgi-mediated transport.\nIf there is no difference between WT and mutant CFTR in the case of stress-induced\nGolgi bypass, could the authors clarify the rationale for focusing primarily on the\nmutant? The mechanism of interaction between SNX27 and CFTR is not expected to\ndiscriminate between WT and mutant forms, as it recognizes a PDZ-motif (an\nunstructured sequence of amino acids at the very C-terminus), and the ΔF508\nmutation is not part of this interaction interface. Please clarify: is the focus on the\nmutant primarily driven by therapeutic relevance? If so, how does this Golgi bypass\nmechanism specifically inform therapeutic strategies?\nResponse:\nWhile wild-type CFTR can reach the plasma membrane via conventional Golgi-\nmediated trafficking, the ΔF508-CFTR mutant cannot efficiently traverse this pathway\ndue to trafficking defects arising from structural and folding abnormalities. Accordingly,\nin this study we focused primarily on ΔF508-CFTR, as transport via an unconventional\nGolgi-bypassing route can overcome its trafficking defect, even though SNX27-CFTR\nbinding per se is not expected to distinguish between wild-type and ΔF508-CFTR.\nlatter part of the Discussion section (Lines 516–522). To articulate this rationale more\nclearly, we have revised the corresponding text in the Introduction (Lines 72–79).\nWe have also clarified in several parts of the manuscript that only the ER form (band\nB) of wild-type CFTR, which is not present at the cell surface under normal conditions,\nand not the Golgi form (band C), undergoes the stress-induced UPS pathway (Lines\n74, 175, 198, and 368).\nQuestion 5: The SNX27 recruits CFTR to the retromer complex through PDZ-based\ninteraction\" The section presents information in a somewhat counterintuitive order.\nLine 182: \"We focused primarily on SNX27, which has been shown to recruit\ntransmembrane protein cargos, such as the β2 adrenergic receptor29.\"\nThe rationale could be strengthened by first explaining how CFTR can be compared\nto the β2 adrenergic receptor. The section would be easier to follow if the authors first\nintroduced the functional domains of SNX27 and explained the SNX27-Vps26\nconnection. Only then should they introduce GRASP55 (GORASP2), SNX17, and\nSNX2 and their potential involvement.\nResponse:\nWe thank the reviewer for these helpful suggestions.\n1) As suggested, we have now included a strengthened rationale for prioritizing SNX27\nas an adaptor for CFTR UPS in this study, based on the following evidence: i) CFTR\ncontains a C-terminal PDZ-binding motif, and SNX27 has been shown to recruit\nmultiple transmembrane protein cargos that harbor PDZ-binding motifs at their C-\ntermini including the β2-adrenergic receptor, the glucose transporter GLUT1, and the\nAMPA receptor GluA1; and ii) structural analyses have demonstrated that the PDZ\ndomain of SNX27 plays a critical role in cargo recruitment to the retromer complex by\nsimultaneously binding the PDZ-binding motifs of membrane proteins, while an\nexposed β-hairpin within the SNX27 PDZ domain engages a groove in VPS26 (Lines\n184–195).\n2) As recommended, we have reorganized this section. Specifically, i) we first present\nloss- and gain-of-function data for SNX27; ii) this is followed by mechanistic evidence\n4\ndemonstrating PDZ-dependent CFTR binding and SNX27-mediated coupling to\nVPS26/retromer; and iii) we then introduce the relationships with GRASP55, SNX2,\nand SNX17 (Lines 212–279).\nPlease note that the order of Figs. 4, 5, and S3–S5 has been revised accordingly.\nQuestion 6: It would also help if the authors provided a clearer rationale for why SNX2\nwas selected from the SNX-BAR subfamily (rather than, for example, SNX1,\nSNX5/6/32 – which all possess BAR-domains and can be linked to the retromer\npathway) and why SNX17 was chosen over SNX31.\nResponse:\nThe following are the main rationales for the selection of SNX2 and SNX17 as\nadditional sorting nexins examined in this study.\n1) SNX2: We selected SNX2 as a representative SNX-BAR candidate because SNX2\nis a core component of the ESCPE-1 SNX-BAR complex, which has been reported to\nfunctionally interface with SNX27-dependent recycling pathways3. In addition, SNX2\nhas been linked to the regulation of ER-endosome contact sites4, which may be\nparticularly relevant under Golgi-bypass conditions.\n2) SNX17: We examined SNX17 as a well-established PX–FERM sorting nexin\nadaptor implicated in PDZ-independent endosomal recycling in most cell types5,6,\nwhereas SNX31 expression is restricted to specific tissues, such as the uroepithelium\nand melanoma cells7.\nA more detailed rationale for examining SNX2 and SNX17 has been added to the\nrevised manuscript (Lines 269–275).\nQuestion 7: Line 327: The VPS26A-SNX27 complex mediates CFTR UPS through\nendosomal recycling.\nThis section requires a proper rationale for the experimental work and clear description\nof observations and conclusions.\nThe internalization of the CFTR channel from the plasma membrane (PM) is a\nconstitutive process that occurs predominantly via clathrin-mediated endocytosis;\nhowever, this process is coupled with highly efficient recycling of CFTR. According to\n5\nthe literature, approximately 50% of CFTR is rapidly recycled back to the plasma\nmembrane within a few minutes, while the remaining internalized pool is also\nredirected to the cell surface via a slower recycling route. This compartment can be\nlabeled with Rab11A and is generally referred to as the endosomal recycling\ncompartment (ERC). Thus, both SNX27 and Rab11A are normally responsible for\nprotein recycling to the plasma membrane through pathways that typically do not\ntransit back through the Golgi. Therefore, in the context of CFTR trafficking, Rab11A\nand retromer-SNX27 are performing exactly what is expected of them: recycling\nprotein to the PM once it arrives at the endosome.\nIt appears that by inhibiting endocytosis, the authors were attempting to distinguish\ntwo protein pools: 1) newly synthesized protein arriving at the plasma membrane and\n2) the one that has been recycled from the cell surface. However, the experimental\ndesign and interpretation would benefit from clearer explanation of how these two\npools are definitively separated in the context of Golgi-bypass.\nResponse:\nThe steady-state cell-surface abundance of CFTR proteins observed in surface\nbiotinylation experiments reflects a dynamic equilibrium among: 1) newly synthesized\nproteins arriving at the plasma membrane via anterograde transport, 2) retrograde\nremoval through endocytosis, and 3) recycling of endocytosed proteins back to the\ncell surface. As noted by the reviewer, in the case of WT CFTR, cell-surface CFTR\nundergoes constitutive clathrin-dependent endocytosis (~13% in 5 min)8, followed by\nefficient recycling of these endocytosed proteins (~40% in 10 min)8. In contrast, for\ncell-surface–rescued ΔF508-CFTR, endocytic removal is augmented (~30% in 5 min)8,\nand its recycling is markedly reduced (~5% in 10 min)8, because ΔF508-CFTR is\nsubject to peripheral protein quality control and the resulting ubiquitination promotes\nlysosomal targeting of the protein8,9. Therefore, inhibition of endocytosis largely\nreflects de novo anterograde transport, particularly in the case of ΔF508-CFTR. The\nprimary aim of our dynasore experiments was to reduce the contribution of endocytic\nretrieval to the steady-state surface level, thereby allowing a more accurate\nassessment of surface delivery.\nOverall, this section has been revised to provide this rationale and a more cautious,\n6\ndata-aligned interpretation. The section now begins with an explanation of the protein\npools involved in the steady-state cell-surface abundance of CFTR proteins, followed\nby a step-by-step description of our rationale for performing the dynasore experiments\nIn addition, the section title has been updated to better reflect the present findings,\n“VPS26A, SNX27, and RAB11A contribute to anterograde CFTR trafficking via the\nUPS.” (Lines 337–362).\nQuestion 8: An additional important point: SNX27 and Rab11A normally perform\nprotein recycling independently of each other. Therefore, the statement that 'Rab11A\nis commonly involved in SNX27-mediated UPS of ΔF508-CFTR under various stimuli\nthat induce ER stress-associated UPS' requires further elaboration and supporting\nevidence. The observed increase in partial colocalization at current resolution does\nnot appear sufficient to support this conclusion.\nResponse:\n1) We agree that SNX27 and Rab11A often mediate protein recycling independently,\nand we did not intend to imply that UPS induction establishes a stable functional\ninteraction between SNX27 and Rab11A. Accordingly, we have revised the text to\navoid overinterpretation (Lines 366–378).\n2) In addition, we have expanded the discussion to clarify that our imaging data\ndemonstrating increased spatial proximity between SNX27 and Rab11A do not\nnecessarily indicate an obligate functional relationship between SNX27 and Rab11A.\nNow, the related discussion reads, “In morphological analyses, Rab11A-positive\ncompartments appear to contribute to CFTR surface delivery; however, our imaging\ndata do not necessarily support an obligate functional relationship between SNX27\nand Rab11A. Interestingly, SNX27 has been reported to localize to Rab11-marked\nrecycling endosomes and to exhibit coordinated dynamics with Rab11 in neurons10,\nwhereas other studies have observed limited SNX27-Rab11 overlap in different\ncellular contexts11. These findings suggest that the degree of association between\nSNX27 and Rab11A may vary depending on cargo and cell type, and that further\nstudies will be required to precisely define their respective contributions to UPS.”\n(Lines 469–477).\nspike protein\nAs with the previous section, the authors' justification and logic are extremely difficult\nto follow. The section begins with artificial expression of individual viral protein,\nomits several critical explanatory steps, and concludes with findings about\nSNX27 and Vps26A involvement in viral particle exocytosis rather than internalization.\nResponse:\nAs requested, we have rewritten this section to 1) explain why furin processing of the\nSARS-CoV-2 spike protein serves as a biochemical readout to distinguish Golgi-\nmediated from Golgi-bypass trafficking, 2) explain the rationale for using ectopic spike\nexpression as a model of conventional Golgi-dependent trafficking under non–stress\nconditions, 3) clarify that authentic viral particles acquire spike proteins at their\nenvelope via Golgi-independent routes, and 4) assess the physiological relevance of\nthese findings. We hope that the revised text improves the clarity of the rationale and\nthe experimental flow (Lines 380–419).\nQuestion 10: Line 438: However, silencing VPS26A, VPS29, VPS35, and SNX27\nsignificantly reduced ΔF508-CFTR UPS even under dynasore-treated conditions,\nsuggesting that these proteins primarily function in anterograde trafficking to the\nplasma membrane rather than in retrograde retrieval from the plasma membrane.\nThis statement is inconsistent with established literature. The SNX27-retromer\ncomplex mediates protein recycling from endosomes back to the plasma membrane,\noperating in the opposite direction to clathrin-mediated endocytosis. Consequently,\nthis recycling pathway would not be inhibited by dynasore. The authors must verify\ntheir claims against the published literature on SNX27-retromer function.\nResponse:\nAs described above (Response to Question 7), our use of dynasore was not intended\nto block SNX27-retromer function, but rather to reduce clathrin/dynamin-dependent\nendocytic retrieval of ΔF508-CFTR. To avoid potential miscommunications, we have\nrevised the sentence by removing the last phrase. Now, it reads: “Silencing VPS26A,\ndynasore-treated conditions, further suggesting that these proteins primarily function\nin anterograde trafficking to the plasma membrane.” (Lines 464–467)\nQuestion 11: Line 444: This aligns with a report stating that Ypt6, a yeast Rab GTPase\nimplicated in the recycling of endosomal components, is involved in the UPS of Acb1\nin yeast51, collectively suggesting that retromer-mediated endosomal recycling is an\nevolutionarily-conserved pathway for stress-induced UPS.\nThis statement requires substantial clarification. The authors cite yeast Rab6\ninvolvement in starvation-induced secretion of Acb1; however, it is unclear how this\nrelates to retromer-mediated endosomal recycling or supports the claim that retromer-\nmediated endosomal recycling represents an evolutionarily conserved pathway for\nstress-induced unconventional protein secretion. The mechanistic connection\nbetween these observations needs to be explicitly established.\nResponse:\nWe acknowledge the reviewer’s point that, although Ypt6 has been shown to function\ntogether with the retromer complex in the endocytic recycling of a yeast ion\ntransporter12, the involvement of Ypt6 in yeast Acb1 UPS does not necessarily indicate\nthe involvement of retromer-mediated endosomal recycling in this process. To avoid\npotential overstatement, we have therefore removed the relevant text.\n1. Hashiguchi, Y. et al. Loss-of-function mutations of retromer large subunit genes\nsuppress the phenotype of an Arabidopsis zig mutant that lacks Qb-SNARE VTI11.\nPlant Cell 22, 159-172 (2010).\n2. Jha, S. G. & Larson, E. R. Diversity of retromer-mediated vesicular trafficking\npathways in plants. Front Plant Sci 14, 1184047 (2023).\n3. Simonetti, B. et al. SNX27-Retromer directly binds ESCPE-1 to transfer cargo\nproteins during endosomal recycling. PLoS Biol 20, e3001601 (2022).\n4. Dong, R., Saheki, Y., Swarup, S., Lucast, L., Harper, J. W. & De Camilli, P.\nEndosome-ER contacts control actin nucleation and retromer function through\nVAP-dependent regulation of PI P. Cell 166, 408-423 (2016).\n4\n5. Ghai, R. et al. Structural basis for endosomal trafficking of diverse transmembrane\ncargos by PX-FERM proteins. Proc Natl Acad Sci U S A 110, E643-652 (2013).\n6. Steinberg, F., Heesom, K. J., Bass, M. D. & Cullen, P. J. SNX17 protects integrins\nfrom degradation by sorting between lysosomal and recycling pathways. J Cell\nBiol 197, 219-230 (2012).\n7. Tseng, H. Y., Thorausch, N., Ziegler, T., Meves, A., Fassler, R. & Bottcher, R. T.\nSorting nexin 31 binds multiple beta integrin cytoplasmic domains and regulates\nbeta1 integrin surface levels and stability. J Mol Biol 426, 3180-3194 (2014).\n8. Okiyoneda, T. et al. Peripheral protein quality control removes unfolded CFTR\nfrom the plasma membrane. Science 329, 805-810 (2010).\n9. Sharma, M. et al. Misfolding diverts CFTR from recycling to degradation: quality\ncontrol at early endosomes. J Cell Biol 164, 923-933 (2004).\n10. Loo, L. S., Tang, N., Al-Haddawi, M., Dawe, G. S. & Hong, W. A role for sorting\nnexin 27 in AMPA receptor trafficking. Nat Commun 5, 3176 (2014).\n11. Hayashi, H. et al. Sorting nexin 27 interacts with multidrug resistance-associated\nprotein 4 (MRP4) and mediates internalization of MRP4. J Biol Chem 287, 15054-\n15065 (2012).\n12. Strochlic, T. I., Setty, T. G., Sitaram, A. & Burd, C. G. Grd19/Snx3p functions as a\ncargo-specific adapter for retromer-dependent endocytic recycling. J Cell Biol 177,\n115-125 (2007).\n10"}, {"round": 2, "reviewer_comments": {"reviewer_1": "SNX27 mediates the recycling of transmembrane proteins from endosomes to the plasma membrane by linking PDZ-motif cargo to retromer-mediated membrane trafficking. This is well established role of the SNX27, and the authors propose that this pathway is repurposed to facilitate stress-induced Golgi-bypass trafficking of the folding-unstable F508del CFTR to the cell surface as part of an unconventional protein secretion pathway.\nUnfortunately, despite the authors' inclusion of several additional experiments, the manuscript remains somewhat disjointed and challenging to follow. A significant weakness is that most experiments lack clearly stated hypotheses and rationale for why they were conducted. The observations are largely limited to descriptive statements without rigorous mechanistic interrogation. I would urge the authors to use established terminology throughout, rather than vague descriptions of processes that force readers to interpret the intended meaning. Additionally, all colocalization experiments must include graphical presentation of the selected regions used for quantification (e.g., histograms of fluorescence intensities in both channels along selected line profiles, or scatterplots correlating pixel intensities between channels). More fundamentally, I am not convinced that the SNX27-retromer recycling pathway in the case of CFTR should be referred to as an unconventional protein secretion pathway.\nLine 145: Retromer mediates the sorting of cargo proteins within endosomes, directing\nthem either to be recycled back to the trans-Golgi network (TGN) or transported to\nalternative intracellular destinations, rather than being targeted for degradation in\nlysosomes 22,24.\nRetromer was indeed first discovered as a protein complex responsible for the retrograde (back to the Golgi) pathway for protein recycling, hence the name retromer. However, once SNX27 was confirmed as a retromer adaptor, it was also established that the retromer-SNX27 complex is responsible for recycling to the plasma membrane (PM). It would be helpful if the authors clarify what 'alternative intracellular destinations' they are referring to and provide appropriate citations to support this statement.\nLine 155: … silencing of VPS26B reduced the ΔF508-CFTR UPS (Supplementary Fig. 2a, b); however, it also significantly\ndecreased the cytosolic protein levels of ΔF508-CFTR.\nGiven that CFTR is a transmembrane protein, additional explanation regarding the expected presence of a soluble cytosolic pool would strengthen the interpretation. The authors should provide a more precise definition of 'cytosolic levels' in this experimental context.\nLine 171: Notably, knockdowns of VPS26A, VPS29, and VPS35 significantly inhibited the UPS of wild-type CFTR (Fig. 2e–\nj). These findings indicate that the retromer complex is more crucial for the Golgi-independent UPS of CFTR rather than conventional Golgi-\nmediated transport.\nIf there is no difference between WT and mutant CFTR in the case of stress-induced Golgi bypass, could the authors clarify the rationale for focusing primarily on the mutant? The mechanism of interaction between SNX27 and CFTR is not expected to discriminate between WT and mutant forms, as it recognizes a PDZ-motif (an unstructured sequence of amino acids at the very C-terminus), and the ΔF508 mutation is not part of this interaction interface. Please clarify: is the focus on the mutant primarily driven by therapeutic relevance? If so, how does this Golgi bypass mechanism specifically inform therapeutic strategies?\nThe SNX27 recruits CFTR to the retromer complex through PDZ-based interaction\" The section presents information in a somewhat counterintuitive order.\nLine 182: \"We focused primarily on SNX27, which has been shown to recruit transmembrane protein cargos, such as the β2 adrenergic receptor29.\"\nThe rationale could be strengthened by first explaining how CFTR can be compared to the β2 adrenergic receptor. The section would be easier to follow if the authors first introduced the functional domains of SNX27 and explained the SNX27-Vps26 connection. Only then should they introduce GRASP55 (GORASP2), SNX17, and SNX2 and their potential involvement. It would also help if the authors provided a clearer rationale for why SNX2 was selected from the SNX-BAR subfamily (rather than, for example, SNX1, SNX5/6/32 – which all possess BAR-domains and can be linked to the retromer pathway) and why SNX17 was chosen over SNX31.\nLine 327: The VPS26A-SNX27 complex mediates CFTR UPS through endosomal recyclin.\nThis section requires a proper rationale for the experimental work and clear description of observations and conclusions.\nThe internalization of the CFTR channel from the plasma membrane (PM) is a constitutive process that occurs\npredominantly via clathrin-mediated endocytosis; however, this process is coupled with highly efficient recycling of CFTR.\nAccording to the literature, approximately 50% of CFTR is rapidly recycled back to the plasma membrane within a few\nminutes, while the remaining internalized pool is also redirected to the cell surface via a slower recycling route. This\ncompartment can be labeled with Rab11A and is generally referred to as the endosomal recycling compartment (ERC). Thus, both SNX27 and Rab11A are normally responsible for protein recycling to the plasma membrane through pathways that typically do not transit back through the Golgi. Therefore, in the context of CFTR trafficking, Rab11A and retromer-SNX27 are performing exactly what is expected of them: recycling protein to the PM once it arrives at the endosome.\nIt appears that by inhibiting endocytosis, the authors were attempting to distinguish two protein pools: 1) newly synthesized protein arriving at the plasma membrane and 2) the one that has been recycled from the cell surface. However, the experimental design and interpretation would benefit from clearer explanation of how these two pools are definitively separated in the context of Golgi-bypass.\nAn additional important point: SNX27 and Rab11A normally perform protein recycling independently of each other.\nTherefore, the statement that 'Rab11A is commonly involved in SNX27-mediated UPS of ΔF508-CFTR under various stimuli\nthat induce ER stress-associated UPS' requires further elaboration and supporting evidence. The observed increase in partial colocalization at current resolution does not appear sufficient to support this conclusion.\nLine 362: VPS26A and SNX27 participate in the UPS of SARS-CoV-2 spike protein\nAs with the previous section, the authors' justification and logic are extremely difficult to follow. The section begins with artificial expression of individual viral protein, omits several critical explanatory steps, and concludes with findings about SNX27 and Vps26A involvement in viral particle exocytosis rather than internalization.\nLine 438: However, silencingVPS26A, VPS29, VPS35, and SNX27 significantly reduced ΔF508-CFTR UPS even under\ndynasore-treated conditions, suggesting that these proteins primarily function in anterograde trafficking to the plasma\nmembrane rather than in retrograde retrieval from the plasma membrane.\nThis statement is inconsistent with established literature. The SNX27-retromer complex mediates protein recycling from endosomes back to the plasma membrane, operating in the opposite direction to clathrin-mediated endocytosis. Consequently, this recycling pathway would not be inhibited by dynasore. The authors must verify their claims against the published literature on SNX27-retromer function.\nLine 444: This aligns with a report stating that Ypt6, a yeast Rab GTPase implicated in the recycling of endosomal\ncomponents, is involved in the UPS of Acb1 in yeast51, collectively suggesting that retromer-mediated endosomal recycling\nis an evolutionarily-conserved pathway for stress-induced UPS.\nThis statement requires substantial clarification. The authors cite yeast Rab6 involvement in starvation-induced secretion of Acb1; however, it is unclear how this relates to retromer-mediated endosomal recycling or supports the claim that retromer-mediated endosomal recycling represents an evolutionarily conserved pathway for stress-induced unconventional protein secretion. The mechanistic connection between these observations needs to be explicitly established.", "reviewer_2": "The authors responded effectively to the reviewer comments, and the paper is now ready for publication in Nature Communications.", "reviewer_3": "The authors have thoroughly addressed all reviewer comments, and given the clear improvements and the robustness of the new data, I recommend publication of the manuscript. Congratulations to the authors for the fine work!", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter