TMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis

Domain: neuroscience
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
The bidirectional transport of Ca2+ into and out of mitochondria regulates metabolism, signaling, and cell fate. While influx is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, efflux mechanisms are more diversified, involving Na+ or H+ exchange pathways. We here demonstrate that TMEM65 is a fundamental component of the Ca2+ efflux machinery of mitochondria. Its overexpression specifically enhances Na+- and Li+-dependent mitochondrial Ca2+ extrusion. This effect is inhibited by CGP-37157 and does not depend on NCLX, currently considered the bona fide mitochondrial Na+/Ca2+ exchanger. Its downregulation chronically elevates basal [Ca2+]mt and impairs efflux upon stimulation. In Caenorhabditis elegans, deletion of TMEM65 homologs compromises embryonic development under mild thermal stress, causing necrotic lesions that are suppressed by genetic inhibition of MCU-1. These findings highlight a molecular component that may be relevant in pathological settings in which excessive mitochondrial Ca2+ accumulation critically contribute to degenerative pathways.

Introduction
Calcium (Ca2+) acts as a universal and ubiquitous intracellular messenger, exerting control over every facet of cellular pathophysiology1. Such a remarkable versatility relies on the one hand on a large array of dedicated Ca2+ binding and/or transporting proteins, and on the other hand on the strictly controlled compartmentalization of [Ca2+] changes. In such a sophisticated framework, mitochondria significantly shape intracellular Ca2+ dynamics and downstream Ca2+-dependent pathways. In healthy cells at rest, intramitochondrial [Ca2+] ([Ca2+]mt) matches cytoplasmic [Ca2+] ([Ca2+]cyt) at approximately 100 nM. Upon cellular stimulation, mitochondria undergo a rapid transient rise in [Ca2+]mt that largely exceed bulk [Ca2+]cyt, up to 1 mM in a few cases2,3. This marked accumulation of Ca2+ is in part due to the highly negative organelle membrane potential (−180 mV), but also to the strategic positioning of mitochondria near Ca2+-releasing channels of the endoplasmic reticulum or the plasma membrane, where local micro/nano-domains of high [Ca2+] are generated4. Inside mitochondria, Ca2+ acts as a positive modulator of three dehydrogenases (PDH, IDH3 and OGDH), which stimulating oxidative metabolism and eventually ATP synthesis5. In pathological conditions, excessive Ca2+ accumulation within the organelle matrix (commonly referred to as Ca2+ overload) can potentially trigger permeability transition, ultimately leading to membrane permeabilization that may promote the release of pro-death factors6–8. Functioning as auxiliary cellular Ca2+ reservoirs, mitochondria contribute to buffering intracellular [Ca2+] transients, thereby influencing the spatio-temporal patterns of [Ca2+] oscillations, either within micro/nano-domains or on a bulk scale (e.g., throughout the entire cell)9. Consequently, impairment of mitochondrial Ca2+ homeostasis has been implicated in the pathogenesis of a variety of human diseases, ranging from neuromuscular diseases to brain injuries10,11.

From a biophysical perspective, [Ca2+]mt is dynamically determined by the rates of Ca2+ influx versus efflux across the IMM. Pioneering investigations conducted in the 1960s and 1970s elucidated the mechanisms underlying this regulation: Ca2+ entry into mitochondria is facilitated by an electrogenic uniporter, while Ca2+ extrusion occurs through two distinct mechanisms - one dependent on sodium ions (i.e. mediated by a Na+/Ca2+ exchanger) and the other Na+-independent (i.e. mediated by a H+/Ca2+ exchanger)12,13. At the molecular level, mitochondrial Ca2+ influx is orchestrated by the MCU complex, whose precise composition varies across tissues and may encompass components such as MCU, MCUb, EMRE, MICU1, MICU2, and MICU314. As to the efflux, the protein NCLX, encoded by the SLC8B1 gene, has been identified as the mediator of Na+-dependent Ca2+ extrusion15, whereas two distinct proteins, LETM116,17 and TMBIM518,19, have been proposed to function as mitochondrial H+/Ca2+ exchangers. Given the breakthrough discoveries of the last years, it is not possible to rule out that other molecular components may contribute to mitochondrial Ca2+ transport. Here, we identify TMEM65 as an unexpected, broadly conserved protein that enables Na+-dependent mitochondrial Ca2+ efflux. We found that genetic manipulation of TMEM65 expression causes striking effects on mitochondrial Ca2+ homeostasis in cultured cells. Specifically, TMEM65 overexpression effectively suppresses mitochondrial [Ca2+]mt transients independently of NCLX, while its silencing prevents the decay of the organellar Ca2+ rises, resulting in markedly elevated resting [Ca2+]mt levels. Consistent with the prominent contribution of TMEM65 to organelle Ca2+ clearance, we show that genetic deletion of TMEM65 homologues in Caenorhabditis elegans causes an overt necrotic phenotype that could be prevented by inhibiting mitochondrial Ca2+ influx, thereby pointing out that mitochondrial Ca2+ overload is a crucial determinant of cell demise. Since a human subject harboring a homozygous TMEM65 loss-of-function mutation exhibits a neuromuscular phenotype reminiscent of mitochondrial disease20, it is reasonable to consider MCU inhibition as a potential therapeutic strategy for conditions characterized by excessive mitochondrial Ca2+ accumulation.

Results
TMEM65 is a conserved protein of the IMM
We previously performed a bioinformatic analysis and identified candidate mitochondrial proteins of the inner membrane with a putative function in Ca2+ signaling21. Besides MCU, the list contains the recently described mitochondrial H+/Ca2+ exchanger TMBIM5 (also known as GHITM/MICS1). Thus, we sought to further inspect the list of presumed Ca2+ transporters using an evolutionary analysis to look for proteins with a conservation profile similar to MCU, and focused on a protein named TMEM65 (Fig. 1a). Among these candidates, TMEM65 is a 25 kDa protein with 3 transmembrane domains and a glycine zipper motif that are well conserved in kinetoplastids, green algae (but not in other plants) and metazoan (Fig. 1b and Supplementary Fig 1a-b). Mammalian TMEM65 also shows an apparent poly-acidic tail near the C terminus. Although two works proposed TMEM65 as a protein positioned to cardiac sarcolemma22,23, multiple lines of evidence indicate that TMEM65 is a bona fide mitochondrial protein. In this regard, i) TMEM65 expression is not limited to the heart but rather is ubiquitous (highest in the cerebellum, lowest in the liver, Supplementary Fig 1c), ii) sequence analysis of TMEM65 reveals a conserved, prototypical mitochondrial targeting signal at the N-terminal (Fig. 1b, predicted MPP-cleaved peptide indicated in cyan), and iii) TMEM65 is present in all high quality mitochondrial datasets based on robust proteomic evidence, including MitoCarta24, MitoCOP25 and MitoPhos26. To confirm these predictions and assess TMEM65 localization, we cloned, tagged (3xFlag) and expressed human TMEM65 in HeLa cells. Immunoblot analyses show a major band running with high specificity at 20 kDa, in line with the predicted molecular weight of the mature protein after processing by the Mitochondrial Processing Peptidase (MPP). The unprocessed 25 kDa precursor is also detectable upon overexpression (Fig. 1c). Immunofluorescence analyses confirm co-localization with the mitochondrial marker SDHA (Fig. 1d). To test protein topology across the IMM, we performed Proteinase K (PK) protection assay in isolated mitoplasts. Digestion of intact (but not permeabilized) mitoplasts causes the loss of full-length TMEM65 and the appearance of a smaller fragment, indicating that a significant portion of TMEM65 resides within the matrix (Fig. 1e). Of note, the Flag peptide fused at the C-terminus of TMEM65 is lost after PK treatment (Fig. 1e), thereby demonstrating that TMEM65 is inserted in the IMM as depicted in Fig. 1b. This membrane orientation is also supported by other studies27, including an independent spatially-resolved proteomic screening that mapped Tyrosine 77 within the matrix (Y77, highlighted in grey in Fig. 1b)28.

TMEM65 expression modulates mitochondrial Ca2+ homeostasis
We next tested the role of TMEM65 on mitochondrial Ca2+ handling by monitoring [Ca2+]mt in living intact HeLa cells using mitochondrial-targeted genetically-encoded Ca2+ indicators (GECI) based on the Ca2+-sensitive bioluminescent protein Aequorin29. Control and TMEM65-overexpressing cells were continuously perfused with modified Krebs-Ringer buffer and challenged with 100 μM histamine, an inositol 1,4,5-trisphosphate (IP3)-generating agonist that elicits Ca2+ release from the endoplasmic reticulum, leading to a transient increase of [Ca2+] in both cytoplasm and mitochondria. Interestingly, TMEM65 overexpression robustly inhibits (approximately 80%) the peak of [Ca2+]mt upon cellular stimulation (Fig. 2a). Importantly, this effect on mitochondrial Ca2+ handling is specific, since no major changes were detected in terms of cytosolic Ca2+ dynamics (Supplementary Fig 2a-b) and mitochondrial membrane potential (Supplementary Fig 2c), hence ruling out possible confounding factors. To obtain additional insights into the structure-to-function relationship of TMEM65, we generated a set of mutants by introducing individual substitutions in the most conserved amino acids or by disrupting relevant motifs (i.e. glycine zipper and poly-acidic tail, Supplementary Fig 2d). All TMEM65 variants are expressed at a levels similar or greater compared to wild-type, with the exception of the mutant lacking the transmembrane glycine zipper motif (GZ dead), that completely fail to express at protein level, probably due to an impairment in membrane packing (Supplementary Fig 2e-f)30. We evaluated histamine-induced [Ca2+]mt peaks in cells expressing wild-type (wt) or mutant TMEM65. Our data indicate that D132, D167, G170 and E178 are required for TMEM65 activity, while G140L and Q195E mutants are very similar to wild type (Supplementary Fig 2g-h). The TMEM65 mutant lacking the poly-acidic tail still retain some effect, although to a lower extent when compared to wt. Surprisingly, the E144Q mutant shows even stronger effect than the wt. None of these mutants affects the expression of the primary mitochondrial Ca2+ influx and efflux transporters (Supplementary Fig 2f). Overall, these data suggest a clear, specific involvement of TMEM65 in the regulation of mitochondrial Ca2+ handling.

We next evaluated the effect of TMEM65 downregulation on organelle Ca2+ dynamics by testing different synthetic unmodified siRNAs. When agonist-induced peak [Ca2+]mt values were compared, we obtained inconsistent results among siRNA sequences (Supplementary Fig 3a-b) despite comparable efficiency in reducing TMEM65 protein level (Supplementary Fig 3e), an outcome likely due to off-target effects. To enhance specificity, we used endoribonuclease-prepared siRNAs (esiRNA)31. This approach reveals increased [Ca2+]mt peak upon specific downregulation of TMEM65 (Fig. 2a, b). More importantly, in addition to the effect on maximal [Ca2+]mt values, TMEM65 silencing alters the kinetic profile of [Ca2+]mt transients. In particular, a delayed Ca2+ clearance after the peak is noticeable, an indication of impaired mitochondrial Ca2+ extrusion. To better appreciate this effect, we re-analyzed all Ca2+ traces by normalizing [Ca2+]mt on peak [Ca2+]mt (Supplementary Fig 3c). The slope of the resulting trace is thus proportional to mitochondrial Ca2+ efflux rate (Supplementary Fig 3d). This analysis clearly reveals that TMEM65 overexpression enhances mitochondrial Ca2+ extrusion (notwithstanding lower [Ca2+]mt peak), while its silencing elicits the opposite effect. Remarkably, all tested siRNAs consistently decreased mitochondrial Ca2+ efflux, irrespective of their wavering effect on peak [Ca2+]mt (Supplementary Fig 3a-d). Furthermore, expression analyses of the two main mitochondrial Ca2+ transporters (MCU for the influx, NCLX for the efflux) revealed no significant changes (Supplementary Fig 3f). Due to the lack of specificity of the commercial antibodies tested, NCLX protein levels could not be reliably assessed (Supplementary Fig 4). Altogether, these data suggest an involvement of TMEM65 in mitochondrial Ca2+ efflux rather than in influx. To further substantiate our data, we evaluated mitochondrial Ca2+ dynamics using a GFP-based GECI, the mitoGCaMP6 probe, that also allows the more precise quantification of baseline [Ca2+]mt and extrusion kinetics32. Compared to controls, both overexpression and downregulation of TMEM65 alter mitochondrial Ca2+ dynamics, further supporting that TMEM65 is a key regulator of mitochondrial Ca2+ efflux (Fig. 2c). Specifically, TMEM65 overexpression causes a negligible decrease of resting Ca2+, a histamine induced upstroke similar to control, at least in the initial phase, but most evidently, a steep return to baseline after the peak (Fig. 2c). Conversely, TMEM65-silenced cells show chronically elevated resting matrix Ca2+ levels, and histamine-induced [Ca2+]mt elevations that last over several minutes (Fig. 2c, d). Altogether, our detailed analyses of mitochondrial Ca2+ dynamics uncover a prominent role of TMEM65 in controlling mitochondrial Ca2+ efflux.

TMEM65 mediates Na+-dependent mitochondrial Ca2+ efflux
We next explore the mechanism underlying TMEM65 effect on mitochondrial Ca2+. It is known that experiments carried out in intact cells have the benefit of examining mitochondria in their native environment. However, these experimental settings suffer from two main drawbacks. Firstly, mitochondrial Ca2+ influx and efflux constantly coexist, thus preventing the genuine segregation of the two phenomena. Secondly, efflux relies on two concurrent but separate mechanisms, one Na+-dependent and the other Na+-independent. To overcome these limitations, we performed [Ca2+]mt measurements in digitonin-permeabilized cells, where mitochondria can be exposed to buffers with defined ionic composition. In this experimental setup, mitochondrial Ca2+ uptake was initiated by perfusing mitochondria of plasma-membrane permeabilized cells with an intracellular-like buffer containing fixed [Ca2+], until a plateau [Ca2+]mt is reached (i.e. when influx rate equals efflux rate). Then, MCU inhibitor Ruthenium Red (RuR) was added to block the influx, resulting in a progressive decrease of [Ca2+]mt. The rate of this decrease represents a reliable measurement of mitochondrial Ca2+ efflux. Perfusion with Na+-free buffer allows the evaluation of organelle Na+-independent efflux, while incorporation of 10 mM NaCl in the RuR-containing buffer enhances mitochondrial Ca2+ extrusion, thereby enabling the quantification of Na+-dependent efflux. Li+ can also be used as Na+ surrogate, although less effective33. Using this approach, we found that both TMEM65 overexpression and downregulation showed no effects on mitochondrial Ca2+ influx or Na+-independent efflux. Rather, TMEM65 overexpression greatly enhanced Na+- and Li+-dependent mitochondrial Ca2+ efflux. Indeed, its silencing blunted the Na+-dependent component of organellar Ca2+ extrusion (Fig. 3a-d, Supplementary Fig 5a-b). To further support this finding, we took advantage of the specific pharmacological inhibitor of mitochondria Na+/Ca2+ exchange, the benzothiazepine CGP-3715734. This compound causes an increase in agonist-induced [Ca2+]mt peaks in control cells due to the inhibition of organelle Ca2+ efflux35. Strikingly, CGP-37157 was able to abrogate the effect elicited by TMEM65 overexpression, restoring normal mitochondrial Ca2+ dynamics (Fig. 3e-g). Overall, these results indicate that i) mitochondrial Ca2+ efflux strongly counteracts both in resting conditions and in all phases of agonist-evoked responses the action of MCU and ii) TMEM65 is a key contributor to the process of Na+-dependent mitochondrial Ca2+ efflux.

TMEM65 acts independently of NCLX
The potential relationship between TMEM65 and the mitochondrial Na+/Ca2+ exchanger NCLX warrants investigation. Although the TMEM65 sequence shows no similarity to that of known exchangers—suggesting a possible modulatory role—functional analyses indicate that its overexpression and silencing strongly enhance and suppress, respectively, Na+-dependent mitochondrial Ca2+ extrusion. This positions TMEM65 as the molecular rate-limiting factor for the process. Conflicting reports in the literature36,37 prompted us to test whether TMEM65 activity depends on the sole characterized mitochondrial Na+/Ca2+ exchanger. In cells acutely silenced for NCLX (Supplementary Fig 3f), TMEM65 overexpression markedly reduced [Ca2+]mt peaks (Fig. 4a, b), indicating functional independence. To rule out compensation from residual NCLX, we generated both polyclonal and monoclonal NCLX knockout (KO) cell lines. In a consistent manner, TMEM65 overexpression lowered agonist-induced [Ca2+]mt peaks (Fig. 4c) and increased Ca2+ extrusion rates in intact (Fig. 4e) and permeabilized cells (Fig. 4f, g), confirming that NCLX is dispensable for TMEM65 function. In the same models, the Na+/Ca2+ exchange inhibitor CGP-37157 not only abrogated the stimulatory effect of TMEM65 overexpression (Fig. 4b) but also remained active in NCLX-silenced and NCLX-KO cells (Fig. 4b; Supplementary Fig 6). Together, these results demonstrate that TMEM65 operates independently of NCLX, and indicate TMEM65 as one of the molecular targets of CGP-37157.

TMEM65 deficiency causes necrotic lesions in vivo in C. elegans
To better understand the physiological relevance of TMEM65 in vivo, we decided to employ the nematode Caenorhabditis elegans. Based on protein alignment (BLAST) using human TMEM65 as a query, we found that C33H5.19/tag-321 and C03B8.3 genes encode two homologs (here referred to also as CeTMEM65-1 and CeTMEM65-2, respectively) (Fig. 5a, Supplementary Fig 7a-c), one of which (TAG-321) with a higher percent identity and a predicted AlphaFold 3D structure relatively similar to the human protein variant (Fig. 5b). First, we tested the expression and functionality of CeTMEM65 in mammalian cells. Western blot shows that CeTMEM65-1 is efficiently expressed also in HeLa cells, while CeTMEM65-2 fails to be detected, probably due to instability of the protein at 37°C (Supplementary Fig 7d). In terms of activity, the effect of CeTMEM65-1 on mitochondrial Ca2+ handling is comparable to its human counterpart, indicating that their biochemical function is maintained through evolution (Supplementary Fig 7e). Next, we performed CRISPR/Cas9 gene editing and generated deletions of the promoters and exons within tag-321 and C03B8.3 genes. Although both tag-321 (bon103) and C03B8.3 (bon104) were (null) alleles (Supplementary Fig 7f), homozygous mutants did not display any obvious difference compared to wt (N2) nematodes at normal growth conditions (20°C, E. coli B-type strain as a bacterial food). When we generated C03B8.3;tag-321 double (null) mutants, we observed a significant reduction of hatched larvae compared to wt, tag-321 (bon103) and C03B8.3 (bon104) mutants at 20°C (Fig. 5c). Lifespan assays at 20°C showed no differences between wt and mutant nematodes, whereas there was a trend toward a minor decreased median lifespan for CeTMEM65 single and double mutants compared to wt at 25°C (Fig. 5d-e and Supplementary tables ST1-3). Intriguingly, while at 20°C we could find larvae in plates from wt and CeTMEM65 single mutants, at 25°C the large majority of C03B8.3;tag-321 (null) mutant animals laid eggs that did not hatch. Motivated by this intriguing observation, we decided to assess the number of larvae that could develop and survive at 25°C (Fig. 5f). Compared to wild type animals, only ~5% of all C03B8.3 (bon104);tag-321 (bon103) mutants hatched (Fig. 5g), with most of the degenerating embryos at gastrula/comma stages showing large vacuoles (Fig. 5h). Of note, C03B8.3 (null) mutants had a hatching rate similar to wt, whereas tag-321 (null) animals showed a modest, although significant increased number of larvae that could hatch at 25°C (Fig. 5g). Given the resemblance of the large vacuoles to necrotic structures, we reasoned that mcu-1 (lof) and consequent inhibition of mitochondrial Ca2+ influx would restore normal hatching of CeTMEM65 (null) mutants at 25°C (Fig. 5i). We first attempted to generate C03B8.3;mcu-1;tag-321 triple mutants using the already existing alleles. However, since mcu-1 and tag-321 genes are both on chromosome IV at a very closed distance (less than 1 cM), we could not introduce the mcu-1 (ju1154) allele into the existing C03B8.3 (null);tag-321 (null) background. To overcome this problem, we performed another gene editing in mcu-1 (ju1154) animals and generated an equivalent lesion within tag-321 gene. DNA sequencing confirmed that the newly generated tag-321 (bon105) allele was matching the previous tag-321 (bon103) mutation (Supplementary Fig 7g-h). We backcrossed the new strain, generated C03B8.3(bon104);mcu-1(ju1154);tag-321(bon105) triple mutants and assessed animal hatching. Remarkably, we found that mcu-1 (lof) completely suppressed the degeneration of C03B8.3;tag-321 double mutant embryos at 25°C (Fig 5j, k), indicating a genetic interaction between mcu-1 and CeTMEM65 and further supporting the prominent role of TMEM65 as a key regulator of mitochondrial Ca2+ efflux. To confirm our findings and rule out any eventual egg-laying defects of individual hermaphrodites, we performed a population-based hatching assay in which eggs were extracted from hundreds of gravid young adult hermaphrodites and plated on bacteria-seeded NGM plates (Fig. 5l). Consistent with our previous findings, while the large majority of C03B8.3;tag-321 double mutant embryos did not hatch at 25°C, C03B8.3(bon104);mcu-1(ju1154);tag-321(bon105) triple mutants hatched and developed at a comparable rate of wt animals (Fig. 5m). Using this experimental paradigm, CeTMEM65 single mutants did not show any difference compared to wt (Fig. 5n).). Of note, the hatching rate of mcu-1 (ju1154) was comparable to wt (Supplementary Fig 7i). Together, our in vivo evidence demonstrates that TMEM65 deficiency may facilitate mitochondrial Ca2+ overload when cells are exposed to environmental challenges (e.g., restrictive temperatures) that may induce degenerative processes (necrosis-like death). Finally, we tested whether NCLX loss of function could recapitulate the same phenotype observed in C03B8.3;tag-321 double mutants (Fig. 5o). It was previously suggested that NCX-9 is the putative NCLX-type Na+/Ca2+ exchanger in C. elegans38. Using two ncx-9 (lof) alleles, we carried out our population-based hatching assay and found that NCX-9 loss did not alter C. elegans hatching at 25°C (Fig. 5p). Although we cannot rule out that other NCLX-type exchangers may compensate for NCX-9 deficiency and/or contribute to mitochondrial Ca2+ extrusion, our data report unique developmental defects associated with CeTMEM65 dysfunction that causes death in an MCU-dependent manner.

In conclusion, we confirmed the critical function of TMEM65 in regulating mitochondrial Ca2+ fluxes. TMEM65 is both sufficient to enhance Na+-dependent mitochondrial Ca2+ extrusion, as shown by overexpression experiments, and necessary to prevent organellar Ca2+ overload, as evidenced by siRNA-mediated knockdown in vitro and in vivo in in C. elegans mutants. Importantly, TMEM65 activity does not require the presence of NCLX, which has long been considered the bona fide mitochondrial Na+/Ca2+ exchanger. This unexpected result aligns with recent data showing that TMEM65 alone, when reconstituted in liposomes, exhibits intrinsic Ca2+ transport activity. These findings establish TMEM65 as an autonomous molecular entity with unique Na+/Ca2+ exchange capacity and prompt a critical reassessment of the genuine contribution of NCLX to mitochondrial Ca2+ homeostasis. More importantly, our findings also shed light on the pathogenic mechanism underlying a reported human neuromuscular syndrome associated with TMEM65 deficiency, where mitochondrial dysfunction emerges as a central feature. Based on our in vivo experimental paradigm, MCU inhibition may represent a viable strategy to mitigate the deleterious effects of TMEM65 loss. Considering that excessive mitochondrial Ca2+ accumulation is implicated in numerous diseases, our work lays the foundation for therapeutic approaches aimed at restoring mitochondrial Ca2+ balance.

Methods
Chemicals, cell culture and transfection
All chemicals were purchased from Sigma-Aldrich, unless otherwise specified. All the experiments were performed in HeLa cells (ATCC Number: CCL-2) cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco#41966052, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), containing penicillin (100 U/ml) and streptomycin (100 μg/ml) (Euroclone). NCLX-KO cells were purchased from SyntheGo (now EditCo). Knockout cells were generated using Crispr-Cas9 technology by targeting exon 2 of the SLC8B1 gene using the following guide RNA sequence: ACACCUGAAGCUGGAAACUG. When needed, cells were seeded on either glass coverslips (onto 13- or 24-mm in diameter) or 96-well plates and allowed grow to 50-80% confluence before transfection. Transfection of siRNA was performed using RNAiMAX (Thermo Fisher Scientific) according to manufacturer’s instructions. Transfection of plasmid DNA was performed using polyethylenimine (PEIMAX, Polysciences).

Plasmids and siRNAs
Plasmids encoding C-term 3xFlag tagged human TMEM65 (NM_194291.2), C. elegans TAG-321 (NM_068883.5), and C. elegans C03B8.3 (NM_066278.5) were purchased from VectorBuilder. Untagged human TMEM65 was amplified using the following primer: fw 5’- ATTAGCTAGCACCACCATGTCCCGGCTG-3’, rv 5’- ATTAGAATTCTTAACTTTTCGTTTCCAGTTTTTCATCTTCTTC-3’. The PCR fragment was cloned into pcDNA3.1 (Thermo Fisher Scientific) using NheI and BamHI sites.
Mutagenesis was performed using In-Fusion cloning (Takara) using the following primer pairs:

D132N fw: 5’-CTTTTTGAATAATGCAATTATGATTGTTGCTGG
rv: 5’-GCATTATTCAAAAAGCCAAACCCTATGAAAGG
G140L fw: 5’-TGTTGCTTTAACCCATATTGAAATGTCTATTGG
rv: 5’-TGGGTTAAAGCAACAATCATAATTGCATTATCC
E144Q fw: 5’-CCATATTCAAATGTCTATTGGAATTATTTTGGGAA
rv: 5’-GACATTTGAATATGGGTTCCAGCAACAATCA
D167N fw: 5’-TGTGTCAAACCTAGCTGGACTTGGACTTGC
rv: 5’-GCTAGGTTTGACACAAGATTTCCCAAAGCAGC
G170L fw: 5’-TCTAGCTCTTCTTGGACTTGCAGGCTACG
rv: 5’-CCAAGAAGAGCTAGATCTGACACAAGATTTCCC
E178Q fw: 5’-CTACGTTCAAGCATTGGCTTCCAGGTTAGG
rv: 5’-AATGCTTGAACGTAGCCTGCAAGTCC
Q195E fw: 5’-ACCAAAGGAAGTTGACATGTGGCAAACACGTC
rv: 5’-TCAACTTCCTTTGGTGTGAGATCAGGAATTGAC
ΔPolyAc fw: 5’-GAGGAGGTAAACTGGAAACGAAAAGTGACTAC
rv: 5’-CCAGTTTACCTCCTCCAAAGAAAATTAAAGG
GZ dead fw: 5’-TGCTGTGACTATTGCATGCATTCTAGCTATGT TTCCTTTAATTTTCTTTGGAG
rv: 5’-GCAATAGTCACAGCAACAGCTTTTGCCAAATG TGTACTAAGACGTGTTTG

All constructs were verified by Sanger sequencing.
The following siRNAs were purchased from Sigma-Aldrich: esiRNA-RLuc (cat no EHURLUC), esiRNA-TMEM65 (cat no EHU042431), esiRNA-SLC8B1 (cat no EHU154091), siRNA Universal Negative Control #2 (cat no SIC002), siRNA-TMEM65#1 (cat no SASI_Hs01_00147974), siRNA-TMEM65#2 (cat no SASI_Hs02_00371420), siRNA-TMEM65#3 (cat no SASI_Hs01_00147977), siRNA-TMEM65#4 (SASI_Hs01_00147978).

Antibodies, SDS-PAGE and western blot
Cells were lysed in RIPA buffer (150 mM NaCl, 25 mM Tris-Cl pH 8, 1 mM EGTA-Tris, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with Complete EDTA-free protease inhibitor mixture (Roche Applied Science) and PhosStop (Roche Applied Science) for 30 minutes on ice. Crude extracts were centrifuged 15000g for 10 minutes to remove debris, and proteins in the supernatant were quantified using the BCA Protein Assay Kit (Pierce). Approximately 30 μg of proteins were dissolved in LDS sample buffer (Life Technologies) supplemented with 10 mM dithiothreitol, heated for 5 minutes at 95°C and loaded on 4-12% Bis-Tris NuPage gels (Thermo Fisher Scientific). After electrophoretic separation, proteins were transferred onto nitrocellulose membranes by semi dry transfer (BioRad). Membranes were blocked for 1 hour at RT with 5% non-fat dry milk in TBS-T (50 mM Trizma, 150 mM NaCl and 0.1% Tween) and probed with the indicated primary antibodies overnight at 4°C. Isotype matched, horseradish peroxidase-conjugated secondary antibodies (1:10000, BioRad) were used followed by detection by chemiluminescence (SuperSignal Pico, Pierce). The following primary antibodies were used: anti-GRP75 (1:1000, SCBT sc-133137), anti-TMEM65 (1:1000, Thermo Fisher Scientific PA5-112762), anti-Flag (1:1000, Cell Signaling #2368), anti SDHA (1:1000, Cell Signaling #5839), anti-PDHA1 (1:1000, Cell Signaling #2784), and anti-MITOK (1:10000, Sigma HPA010980), anti-NCLX (Proteintech 21430-1-AP, Thermo PA5-114330 and Abcam ab83551). Western blots are representative of at least three independent preparations. For Western blot analyses of C. elegans samples, adult animals were collected in water and snap-frozen in liquid nitrogen. C. elegans pellets were then sonicated in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). Lysates were centrifuged as described above and protein concentrations were quantified via Bradford assay (Sigma-Aldrich). Approximately 20-30 μg of protein was resolved on 8% polyacrylamide gels and transferred onto nitrocellulose membranes using semi-dry Trans-Blot TurboTM (Bio-Rad). Following antibody incubation (anti-GFP, 1:1000, Roche #118144600001), membranes were developed in ECL and imaged using the Chemidoc imaging system (Bio-Rad). Uncropped images of Western blots used for the assembly of final figures are provided as Supplementary Figs.

RNA extraction, reverse transcription, and qPCR
Total RNA was extracted from HeLa cells using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). For cDNA synthesis, 1 μg of RNA from each sample was reverse transcribed using the SuperScript II cDNA synthesis kit (Thermo Fisher Scientific), following the manufacturer’s protocol. Oligo(dT)12–18 primer (Thermo Fisher Scientific) was used for first-strand cDNA synthesis, along with dNTPs (deoxynucleotide triphosphates, Thermo Fisher Scientific) and reverse transcriptase.
The resulting cDNA was analyzed by quantitative PCR (qPCR) using TaqMan probe-based chemistry to assess relative gene expression levels. qPCR reactions were carried out in 384-well plates using TaqMan Gene Expression Master Mix (Applied Biosystems) and gene-specific TaqMan probes. Amplification was performed on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific) under standard cycling conditions. Relative gene expression was calculated using the ΔΔCt method, with TOMM20 or ACTIN serving as the endogenous control, as indicated in figure legend. The following probes were used: TOMM20-VIC (cat no Hs00740685_sH), ACTB-VIC (cat no Hs01060665_g1), MCU-FAM (cat no Hs00293548_m1), MCU-FAM (cat no Hs00293548_m1), SLC8B1-FAM (cat no Hs00905331_m1).

Bioinformatic analyses
To evaluate conservation profiles of MCU and TMEM65, reference proteomes of eukaryotic species were retrieved from Uniprot as fasta files (Release 2022_02, 25-May-2022). Only species containing at least 3000 entries in main (canonical) set were considered for further analysis. Homology searches were performed using phmmer (HMMER version 3.3.2)39. Human MCU and TMEM65 protein sequences were used as input against reference proteomes using BLOSUM62 as scoring matrix. Hits with an E-value below 0.05 were considered as homologs. Multiple sequence alignments (ClustalW) and phylogenetic trees (RAXML) were performed using MegAlign Pro (DNASTAR version 17.3.1). Visualization of protein topology was generated using Protter40. Prediction of transmembrane domains was performed using TMHMM41. The presence of mitochondrial targeting peptides was evaluated using TPP342, MitoFates43 and TargetP44. Highly conserved amino acids were identified by performing a multiple sequence alignment across selected species and selecting residues with a sequence logo score ≥ 3.5 bits (log2-scaled; maximum score = 4.3 bits for 20 amino acids). To evaluate tissue expression of TMEM65, RNA-seq expression values (TPM) were retrieved from GTEx portal45. To normalize to mitochondrial abundance, we calculated the average expression of a large subset of broadly expressed mitochondrial proteins, and normalized TMEM65 expression to these values.

Mitochondrial isolation and proteinase K protection assay
Mitochondria were isolated from HeLa cells through differential centrifugation as previously described46. Mitoplasts were obtained through osmotic swelling by incubating mitochondrial fraction in 20 mM Tris-Cl pH 7.4 for 20 minutes on ice. An equal amount of mitoplasts was treated with proteinase K (100 μg/ml) at room temperature for the indicated time and the proteolytic reaction was quenched by PMSF addition. Samples were then loaded on SDS-PAGE and processed for Western blot as described above. Results are representative of at least three different proteolytic reactions.

Immunofluorescence and confocal imaging
HeLa cells expressing TMEM65-3xFlag were grown on 24 mm coverslips until 50% confluence. Cells were then washed with PBS, fixed in 4% formaldehyde for 10 minutes and quenched with 50 mM NH4Cl in PBS. Cells were permeabilized for 10 minutes with 0.1% Triton X-100 in PBS and blocked in PBS containing 2% BSA for 1 hour. Cells were then incubated with primary antibodies for 3 hours at room temperature and washed 3 times with 0.1% Triton X-100 in PBS. The following primary antibodies were used: anti-SDHA (1:100, Cell Signaling #5839), anti-Flag (1:100, Cell Signaling #2368), The appropriate isotype matched AlexaFluor-conjugated secondary antibodies (1:500, Thermo Fisher Scientific) were incubated for 1 hour at RT and coverslips were mounted with ProLong Diamond Antifade reagent (Thermo Fisher Scientific). Images were acquired on a Leica TCS-SP5-II-RS-WLL equipped with a 100x, 1.4 N.A. Plan-apochromat objective. AlexaFluor 488 was excited by the 488 nm laser line and images were collected in the 495-535 nm range. AlexaFluor 555 was sequentially excited with the 543 nm laser line and signal was collected in the 555-600 nm range. Pixel size was set below 100 nm to meet the Nyquist criterion. For each image, a z-stack of the whole cell was acquired, with a step size of 130 nm. Images are presented as maximum projections of the whole stack and were generated using the Fiji image processing package based on ImageJ47. Images are representative of at least three independent transfections.

[Ca2+] measurements
For experiments in intact cells using bioluminescent probes, HeLa cells were grown on 96-well plates and cotransfected with an Aequorin based probe48,49 together with the indicated constructs. siRNAs were delivered through RNAiMAX-mediated reverse transfection 72 hours before experiments. Plasmids were delivered through PEI-mediated forward transfection 36/48 hours before experiments. Untargeted siRNA (esiRNA-RLuc or universal negative siRNA) and/or pcDNA3.1 were used as controls. For aequorin reconstitution, cells were incubated for 1 hour at 37°C with 5 μM coelenterazine in KRB (Krebs-Ringer modified buffer: 125 mM NaCl, 5 mM KCl, 1 mM Na3PO4, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, pH 7.4) supplemented with 1 mM CaCl2. After reconstitution, cells were washed and placed in 70 μl of KRB and luminescence was recorded on a PerkinElmer Envision plate reader equipped with a two-injector unit. Each well was monitored for 50 seconds during which histamine was first injected (final concentration is 100 μM) to activate calcium transients, and then a hypotonic, Ca2+-rich, digitonin-containing solution was added to discharge the remaining aequorin pool. Output data were analyzed and calibrated with a custom made macro-enabled Excel workbook. Where indicated, cells were pre-treated with either vehicle (DMSO) or 20 μM CGP-37157 (Tocris Bioscience).
For experiments in permeabilized cells using bioluminescent probes, HeLa cells were grown on 13 mm round glass coverslips at 50-80% confluence and cotransfected with a low-affinity mitochondrial-targeted aequorin-based probe together with the indicated constructs. For aequorin reconstitution, cells were incubated for 1-2 hours at 37°C with 5 μM coelenterazine in KRB supplemented with 1 mM CaCl2 and then transferred to the perfusion chamber of a custom-built luminometer49,50. Experiments in permeabilized cells were performed in an intracellular-like buffer (IB: 130 mM KCl, 2 mM K2HPO4, 5 mM glutamic acid, 2 mM malic acid, 1 mM MgCl2, 20 mM HEPES, pH 7.0), supplemented with additional 10 mM KCl, 10 mM NaCl or 10 mM LiCl as specified. Plasma membrane was permeabilized by perfusing cells for 1 min with 100 μM digitonin dissolved in IB containing 500 μM EGTA. Mitochondrial Ca2+ uptake in permeabilized cells was initiated by perfusing IB containing 5 μM Ca2+, and influx was blocked by adding 10 μM ruthenium red (RuR). The experiments were terminated by lysing the cells with 100 μM digitonin in a hypotonic Ca2+-rich solution (10 mM CaCl2 in H2O), thus discharging the remaining aequorin pool. The light signal was collected and calibrated into [Ca2+] values by an algorithm based on the Ca2+ response curve of aequorin at physiological conditions of pH, [Mg2+] and ionic strength51,52.
For experiments in intact cells using fluorescent probes, HeLa cells were grown on 24 mm round glass coverslips at 30-50% confluence and cotransfected with mitoGCaMP6 together with the indicated constructs. Cells were transferred to an imaging chamber and incubated in KRB supplemented with 1 mM CaCl2. Images were acquired on an Olympus IX-73 microscope equipped with a 40x/1.3 N.A. oil-immersion semiapochromat objective (UPLFLN40XO). Excitation light was selected with a Deltaram V high speed monochromator (Photon Technology International) equipped with a 75W Xenon Arc lamp. MitoGCaMP6 emission was collected through a 525/50 filter (Chroma) mounted on an OptoSpin 25 wheel (Cairn research). Images were captured with a Kinetix 22 sCMOS camera (Photometrics). The system is controlled by Metamorph 7.10 (Molecular devices) and was assembled by Crisel Instruments. MitoGCaMP6 was used in excitation ratiometric mode, where cells are alternatively illuminated every second at 485 and 410 nm32. Typical exposure time was 100 milliseconds. After background correction, 485/410 fluorescence ratio was calculated for each cell. Where indicated, 100 μM histamine was added, followed by addition of 3 μM ionomycin and 5 mM CaCl2 to check for homogenous probe saturation. Analysis was performed with the Fiji distribution of ImageJ47. Data are presented as fluorescence ratio (R, 485/410 nm), and each data point represents one coverslip (containing approximately 1 to 5 individual cells).

ΔΨm measurements
The measurement of mitochondrial membrane potential is based on the distribution of the mitochondrion-selective lipophilic cation dye tetramethylrhodamine, methyl ester (TMRM, Thermo Fisher Scientific). Cells were loaded with 20 nM TMRM for 30 minutes at 37°C and then transferred to the imaging system. Images were acquired on a Zeiss Axiovert 200 microscope equipped with a 40x/1.3 N.A. Plan Fluor objective. Excitation was performed with a Deltaram V high speed monochromator (Photon Technology International) equipped with a 75W Xenon Arc lamp. Images were captured with a Evolve 512 Delta EMCCD (Photometrics). The system is controlled by Metamorph 7.5 and was assembled by Crisel Instruments. TMRM excitation was performed at 560 nm and emission was collected through a 590–650 nm bandpass filter. On each coverslips images of 5 random fields were acquired with a fixed 200 milliseconds exposure time. At the end of each experiment, 10 μM CCCP was added to collapse ΔΨm. data are presented as average background-corrected fluorescence in resting conditions. Data were obtained from at least three independent preparations. All analyses were performed with the Fiji distribution of ImageJ.

Caenorhabditis elegans strains and maintenance
Nematode cultures were maintained at 20°C unless differently stated. C. elegans was grown on nematode growth media (NGM) plates seeded with a bacterial lawn of OP50 E. coli following standard culture methods. The following strains were used in this study: wild type N2 (Bristol), BAN544 tag-321(bon103)IV, BAN545 C03B8.3(bon104)III, BAN547 C03B8.3(bon104)III;tag-321(bon103)IV, BAN580 C03B8.3(bon104)III;mcu-1(ju1154)IV;tag-321(bon105)IV, CZ19982 mcu-1(ju1154)IV. We are grateful to Dr. Damien M. O’Halloran (Institute for Neuroscience, George Washington University, Washington, D. C, USA) for kindly providing ncx-9(gk234237) and ncx-9(gk773708). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40OD010440).

CRISPR/Cas9 gene editing
Young adult C. elegans hermaphrodites were injected with customized injection mix of Cas9 protein (pre-complexed with sgRNAs), target-specific sgRNAs, target donor homology repair template (ssODN) (InVivo Biosystem, Eugene, Oregon, USA) and a plasmid encoding [myo-2p::GFP]. F1 offspring were screened for GFP expression in the pharynx, while F2 were genotyped using specific oligonucleotides upstream and downstream the expected deletions. DNA sequencing was used to confirm the mutation. Selected positive mutants were backcrossed at least five times with wt (N2) males.

DIC microscopy
Eggs from adult nematodes were extracted via bleaching, washed twice with water and then transferred onto glass slides. Images were acquired with an EPI-SCOPE 1 Apotome microscope equipped with a 63x oil immersion objective (Zeiss).

Hatching assays
Two independent experimental paradigms were used to assess hatching of larvae. In the first one, animals were grown at 20°C, collected at adulthood and bleached with a hypochlorite solution. The extracted eggs were transferred onto bacteria-seeded NGM plates and incubated at either 20°C (for maintenance) or 25°C. Individual young adults were then transferred on freshly bacteria-seeded plates and the hatched progeny was quantified over time (up to 48 hours). For each experiment, between 8 and 15 young adult nematodes per genotype were used. The second hatching assay was a population-based analysis. Animals were grown at 20°C, collected at adulthood and bleached with a hypochlorite solution. Eggs were plated in freshly prepared bacteria-seed plates and transferred at 25°C until hatched animals reached adulthood. Then, synchronized gravid hermaphrodites were collected and bleached with a hypochlorite solution. The extracted eggs were seeded on plates, counted and incubated at 25°C. Hatched larvae were quantified using a stereomicroscope.

Lifespan assay
Gravid adult animals grown at 20°C were collected and bleached using a NaClO/NaOH solution to extract eggs. Synchronized populations were then placed in an incubator at either 20°C or at 25°C, according to the experimental paradigm. Animals were transferred every 2 days and scored at least every other day for touch-provoked movement until death. Animals that died abnormally (e.g., internal hatching, vulva protrusions) were scored as censored. Survival curves were generated using GraphPad Prism software (GraphPad Software Inc., San Diego, USA).

Statistics and reproducibility
No statistical method was used to predetermine sample size. All experiments were repeated independently at least 3 times with similar results. For the analysis of [Ca2+] dynamics, representative traces are shown together with bar graphs. No data were excluded from the analyses. Experiments were not randomized, and investigators were not blinded to allocation during experiments or outcome assessment. Bar graphs display mean ± standard deviation (s.d.), with individual data points represented by dots unless otherwise indicated. Variance was assessed using one-way, two-way, or three-way ANOVA, as specified in the figure legends. Comparisons between two groups were performed using unpaired two-sample t-tests. For multiple comparisons, Holm–Šidák post hoc tests were applied. Where applicable, raw data and exact p-values are provided in the source data tables. All statistical analyses were conducted using OriginPro (OriginLab), Prism (GraphPad), or Excel (Microsoft).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Vetralla et al. characterize TMEM65 as a transmembrane mitochondrial protein involved in regulating Na+-dependent efflux of mitochondrial Ca2+ and shed light on the neurodegenerative mechanism of TMEM65 deficiency. The authors use flag tagged protein to identify TMEM65 as a transmembrane mitochondrial protein and genetically encoded calcium indicators to characterize the effect of overexpression, genetic silencing, and mutation of TMEM65 on mitochondrial Ca2+ homeostasis. With the C. elegans model, they shed light on the pathological mechanism of TMEM65 deficiency. The regulation of Ca2+ homeostasis, specifically molecular players that may be involved in protecting against Ca2+ overload, is of great interest to the field of mitochondrial biology. The authors make several claims that are not fully supported by their data and importantly do not consider potential compensatory regulation of NCLX when changing TMEM65 expression. Addressing these discrepancies either by changing the claims or generating data that does support their existing claims will make the manuscript stronger.", "reviewer_2": "The authors demonstrate the function of TMEM65 as a pivotal member of the Ca2+-efflux machinery in mitochondria. TMEM65 overexpression enhances Na+- and Li+-dependent mitochondrial Ca2+ extrusion, while its downregulation elevates resting mitochondrial Ca2+ and inhibits efficient Ca2+ efflux upon cellular activation, culminating in mitochondrial calcium overload and mitochondrial dysfunction. In C. elegans, deletion of TMEM65 orthologues results in defects of eggs upon exposure to mild temperature-stress. Here are points that need to be addressed.\n\n1) In Figure 1, HSP60 is used as a mitochondria-specific marker. Panel 1D shows abundance of unmerged HSP60 and TMEM65 signals. HSP60 is known to reside in the cytoplasm and even the extracellular space, therefore a more specific mitochondrial marker should be used (e.g. CoxIV) to argue mitochondrial expression of TMEM65.\n\n2) Figure 1E- a low molecular weight marker should be added to the WB (below 25 kDa, to better recognize the TMEM65 bands after proteinase K treatment).\n\n3) This study does not show if TMEM65 forms a complex with NCLX. A simple immunoprecipitation experiment (or proximity ligation assay (PLA)) would address this issue.\n\n4) Results from C. elegans suggest that TMEM65 ortholog/orthologs are part of a protein complex (see point 3, about NCLX co-IP), since the C03B8.3 deletion alone fails to produce any phenotype. Only one ortholog out of 2 was tested for ability to regulate Ca2+ efflux. Given that both TMEM65 C. elegans orthologs show low protein sequence similarity (52 and 35%) to mammalian TMEM65, both genes must first be proven to code for mitochondrial proteins involved in regulation of calcium efflux.\n\n5) Even a double KO of the TMEM65 orthologues in C. elegans produces a relatively minor phenotype, therefore perhaps a different in vivo model would better represent the function of TMEM65 or better model its loss of function effects.", "reviewer_3": "In this study, Vetralla and colleagues work to identify the functional significance of TMEM65 in calcium metabolism and the impact to organismal homeostasis. The authors clearly demonstrate the importance of C. elegans tag-321 and C03B8.3 (TMEM65 homologs) to embryonic development. The authors show that combining null mutations of tag-321 and C03B8.3 resulted in severely compromised hatching at elevated temperature of 25 °C. This finding is in contrast to the single mutants that show little defects alone, supporting their role as paralogs. This defect with loss of both tag-321 and C03B8.3 was suppressed by addition of a third mutation in mcu-1, the well-established mitochondrial calcium importer. By alleviating the developmental defect, most embryos were able to hatch. In the context of a fully penetrant hatching defect, suppression in this case represents a powerful readout for epistasis and confirms an antagonistic relationship between loss of the calcium influx and efflux machinery. Loss of the efflux machinery results in toxicity and this is compensated by disruption of import to alleviate accumulation of toxic levels of calcium. The findings for calcium homeostasis are exciting. Further, this work demonstrates the utility of model organisms to understand disease relevant pathways."}, "author_response": "We would like to thank the reviewer for appreciating our efforts and highlighting the important contribution of our study in the field of mitochondrial biology. Below, we outlined the experiments that we propose to perform in order to address the reviewer’s concerns.\n\n1. Much of the results in this paper are not properly dissociated from NCLX regulation. While the authors acknowledge that NCLX has been identified as a mediator of Na+-dependent Ca2+ extrusion, they claim modulating TMEM65 expression has effects on Na+-dependent Ca2+ extrusion due to the function of TMEM65 without considering NCLX activity. Please address this through investigation of NCLX expression in cells with altered TMEM65 expression.\nFollowing the reviewer’s comment, we will perform immunoblot analyses of NCLX expression in TMEM65 overexpressing and deficient cells. Because there are no reliable NCLX antibodies available (a well-known problem in the field), we will also analyze NCLX transcripts using qPCR.\n\n2. The authors claim TMEM65 variants were expressed at a similar level, but by eye one can see at least a two-fold increase in abundance between the expression of the WT protein and E178Q protein among other differences. It may be best to write that the mutants expressed at WT levels, or greater, except GZ dead. Would TMEM65 overexpression affect NCLX expression? It is possible that the changes observed with these mutants is not due to TMEM65 function per se, but due to the effects of TMEM65 on NCLX stability. Please check of NCLX levels cell cells that express TMEM65 variants.\nWe agree with the reviewer and will adjust the text accordingly. Having that said, we are confident that the effect on Ca2+ signaling is not influenced by the expression, since those are population-based recording of co-transfected cells with TMEM65 variants and aequorin. In support of our arguments, Western blots show that some TMEM65 mutants (e.g., D132N; E144Q) are highly expressed, nevertheless they do not behave as wt TMEM65, possibly indicating that they are probably loss of function. As suggested by the reviewer, we will analyze the expression levels of NCLX in cells expressing TMEM65 mutants by WB and qPCR (see previous comment).\n\n3. The authors claim that TMEM65 is an essential component and/or the strongest contributor to the process of mitochondrial Ca2+ efflux. This is a misinterpretation of the data, as the process of mitochondrial Ca2+ efflux still happens with TMEM65 genetically silenced, therefore the claim of essential component is not supported, and no other contributors to the process of mitochondrial Ca2+ efflux are analyzed, therefore the comparative word “strongest” is not appropriate. Please compare the effects that TMEM65 knockdown and NCLX knockdown have on Na+-dependent Ca2+ efflux.\nFollowing the reviewer’s comment, we will adjust the text and tune down our conclusions. Having that said, it is not possible to rule out that the transient silencing of TMEM65 may not be sufficient to completely abrogate Ca2+ efflux. To address this issue, we will try to measure Na+-dependent mitochondrial Ca2+ efflux in mitochondria isolated from wt and TMEM65-KO HAP1 cells, that are commercially available (although preliminary experiments show HAP1 are not the best model to study cellular calcium dynamics). In addition, we will compare the relative effect of NCLX and TMEM65 on mitochondrial calcium transients through overexpression and silencing experiments in HeLa cells. We also recently obtained NCLX-KO HeLa cells, a cellular model that will be useful to test the whether the effect of TMEM65 is dependent on the presence of NCLX.\n\n4. The authors claim that the double mutant nematodes had a median lifespan that was slightly less than wt and single mutant nematodes when assessed at 25 °C. However, statistical analysis of the median lifespans is not shown in the source data table or supplementary table.\nWe may have overlooked at this important detail. Although all the data summary and statistics (compared to wt) were reported in the extended lifespan table, we agree to add the statistics also in the summary table as requested by the reviewer.\n\na. Please add p values (compared to wt) in the Statistical analysis of lifespan assays at 25 °C.\nWe will follow the advice of the reviewer.\n\nb. In assessing the individual lifespan assays, a p value (compared to wt) is reported that does not match the medians. It is unclear what this p value is reporting and whether that is a better representation of lifespan than comparing medians. Please clarify what this p value represents and address why median lifespan was used instead of mean lifespan or another measurement of lifespan.\nTo report the lifespan assay data, we follow the guidelines of the scientific community. In the extended lifespan table, the p value is calculated by comparing the survival curves and by using Log-rank Mantel-Cox test. It is generally requested that for each lifespan assay the median and max survival must be reported. As in our study, the mean lifespan can be included when several biological replicates are available.\n\nc. The authors claim that the preliminary evidence showed no major survival difference between wt and CeTMEM65 single and double mutant nematodes. However, in supplementary table 1 the authors report a significant p value (compared to wt) for C03B8.3(bon104). Please address this discrepancy.\nWe do not feel confident to make any claim about the lifespans at 20 °C because we performed the experiment only once. If the reviewer considers this aspect of relevance, we will provide three additional biological replicates as we did for the experiments at 25 °C. We believe that the most relevant data are those obtained at 25 °C, however we are carrying out further lifespan also at 20°C.\n\n5. The authors claim that CGP-37157 restores normal mitochondrial Ca2+-dynamics, but there is a significant difference between control and TMEM65 overexpressing cells with regards to normalized Ca2+ efflux (Fig. 3g). Thus, this claim is not supported. Please address this discrepancy.\nWe may have exaggerated the conclusions of the experiment, as there is indeed a small but statistically significant difference in TMEM65-overexpressing cells treated with CGP-37157. However, the difference compared to untreated cells is substantial. We think that the residual effect is attributed to the overexpression of TMEM65, causing a rightward shift in the CGP-37157 inhibition curve, indicative of a direct effect of CGP-37157 on TMEM65. To verify this hypothesis, we will measure the dose/response curve of GCP in control and TMEM65-overexpressing cells. In addition, we will also repeat the experiment using fluorescent genetically-encoded calcium indicators, that are better suited to monitor efflux kinetics. We will change the text accordingly.\n\n6. It is unclear what criteria is used to identify TMEM65 when looking at proteins with a conservation profile similar to MCU. Please describe how the evolutionary analysis led to TMEM65 out of the fourteen proteins identified in Sharma et al 2015.\nThe list of 14 proteins, which might play a role in mitochondrial Ca2+ dynamics, was documented in De Stefani et al., Nature 2011. Our original analysis was limited to few selected species. Here we performed a broader evolutionary analysis and spotted TMEM65 as a protein with the closest conservation profile to MCU. We will expand the methods section to better explain our in silico analysis. Conversely, the paper “Sharma et al 2015” refers to a manuscript from Gramolini lab claiming that TMEM65 is localized to the plasma membrane of cardiomyocytes. We will include in the rebuttal some comments on that paper on its limitations and potential explanation of their results. However, we do not believe that such comments can be accommodated in the final text, except for some brief references in the discussion section.\n\n7. Figure 1b – Highly conserved amino acids criteria are not provided. How conserved is “highly” and in what species is this conservation? Please provide details on the conserved amino acids. How many species were used in this analysis?\nWe apologize for the lack of methodological details in defining “highly conserved” amino acids in TMEM65 sequence. We performed a multiple sequence analysis of a manually curated list of protein sequences from different species (approximately 200 species from multiple clades). We considered amino acids as 'highly conserved' if they had a score of at least 3.5 bits in the sequence logo (log2-scale, with a maximum of 4.3 bits for 20 amino acids). Roughly, this corresponds to amino acids conserved in at least 80% of analyzed species. We added these details in the “Methods” section.\n\n8. The authors claim that failure to detect expression of CeTMEM65-2 is probably due to the instability of the protein at 37 °C, but they give no reason for this claim. Please discuss the reasoning behind this claim.\nThe exogenous expression of native C. elegans proteins (an organism that grows at room temperature and is heat-sensitive) in mammalian cells is often unsuccessful because worm proteins are unstable at 37°C, especially membrane proteins. We were surprised to be able to successfully express CeTMEM65-1 (Flag-tagged TAG-321) in HeLa cells at 37°C, which represents the exception rather than the rule. In contrast, we were unable to detect CeTMEM65-2, despite the transfection being carried out under the same conditions as the other plasmids. We also attempted to clone a codon-optimized CeTMEM65-2 into a different expression vector, but obtained the same results (no expression of the worm homologue). We will also try to express CeTMEM65 isoforms in a different cellular model that usually guarantees better expression of transgene such as HEK293 cells.\n\n9. The authors describe a slight developmental delay in the null mutants, but do not describe this effect quantitatively or qualitatively. Please describe the effect and the significance of it.\nFollowing the reviewer’s comment, we will provide the data in the revised version of the manuscript.\n\n10. Extended Data Figure 5d includes DmeTMEM65, which the reader must assume refers to Drosophila melanogaster since this is not explained in the text or the figure legend. The expression of this protein shows how a different non-human ortholog protein may be expressed, but without explanation this detracts from the rest of this figure. Please address the inclusion of this Western lane.\nWe included the Drosophila melanogaster lane in the final figure by mistake. For the record, TMEM65 from flies can be successfully expressed in HeLa cells and causes the same effect as its human counterpart. We will adjust the figure according to the reviewer’s suggestion.\n\n11. Fig. 1c: pcDNA3.1 is not explained in the figure legend. Please either replace pcDNA3.1 with “empty vector” or explain the label of pcDNA3.1 in the figure legend to increase readability.\nWe agree with the reviewer and will adjust the labels accordingly.\n\n12. Fig.1E: Typo - “(full lenght)” should be “(full length).” Please fix this typo.\nWe thank the reviewer for her/his comment and will adjust the text accordingly.\n\n13. Extended Data Figure 2f: WT label is not aligned with the WT bar. Please align properly.\nWe will adjust the figure accordingly.\n\na. In all other cases of the [Ca2+]mt curves the curves precede the quantification, whereas this figure has the opposite order. Please change the ordering for the sake of consistency.\nWe agree with the reviewer’s suggestion and proceed accordingly.\n\n14. In the source data table file, the tab named “Fig 3g” is out of order. Please place the tab “Fig 3g” between Fig 3f and Fig 4a.\nWe will correct the mistake.\n\n15. In the source data table file, the tab named “Extended Fig 5c” refers to data present in Extended Fig 5e. Please rename the tab.\nWe will rename the table as kindly suggested.\n\nReviewer #2:\n\n1) In Figure 1, HSP60 is used as a mitochondria-specific marker. Panel 1D shows abundance of unmerged HSP60 and TMEM65 signals. HSP60 is known to reside in the cytoplasm and even the extracellular space, therefore a more specific mitochondrial marker should be used (e.g. CoxIV) to argue mitochondrial expression of TMEM65.\nFollowing the reviewer’ s comment, we will repeat confocal imaging analysis and include additional mitochondrial markers confirming the mitochondrial localization of TMEM65.\n\n2) Figure 1E- a low molecular weight marker should be added to the WB (below 25 kDa, to better recognize the TMEM65 bands after proteinase K treatment).\nWe will add the low molecular weight marker as requested. Please also consider that uncropped western blots containing all MW markers are already included as supplementary figures.\n\n3) This study does not show if TMEM65 forms a complex with NCLX. A simple immunoprecipitation experiment (or proximity ligation assay (PLA)) would address this issue.\nFollowing reviewer’s suggestion, we will perform immunoprecipitation and/or PLA between NCLX and TMEM65. However, we can anticipate that this won't be as straightforward as the reviewer thinks. The available antibodies against NCLX, in fact, perform poorly. We will use epitope-tagged versions of NCLX, although they display poor (if not any) mitochondrial localization (we will of course provide evidence for this). Alternatively, we can test the functional interaction between TMEM65 and NCLX. We will compare the relative effect of NCLX and TMEM65 on mitochondrial calcium transients through overexpression and silencing/KO experiments in HeLa cells (see Rev#1 point 3). Finally, we will also take advantage of the C.elegans model to test the relationship between TMEM65 and NCLX. Indeed, a putative NCLX homologue has been described in nematodes (i.e. NCX-9, see PMID: 28196860). We will perform hatching assay at 25°C to test whether C. elegans NCLX loss of function phenocopies CeTMEM65(null) mutations.\n\n4) Results from C. elegans suggest that TMEM65 ortholog/orthologs are part of a protein complex (see point 3, about NCLX co-IP), since the C03B8.3 deletion alone fails to produce any phenotype. Only one ortholog out of 2 was tested for ability to regulate Ca2+ efflux. Given that both TMEM65 C. elegans orthologs show low protein sequence similarity (52 and 35%) to mammalian TMEM65, both genes must first be proven to code for mitochondrial proteins involved in regulation of calcium efflux.\nAs the referee may know, it is relatively difficult to overexpress functional C. elegans proteins in mammalian cells. This is because they are relatively unstable at 37 °C. However, we would like to draw the reviewer’s attention to the following arguments: i) both TAG-321 and C03B8.3 are predicted with high confidence to possess a mitochondrial transit peptide at the N-terminal (calculated with PredSL); ii) sequence alignment of human TMEM65 to either TAG-321 or C03B8.3 show highly significant scores (3e-35 and 9e-8, respectively). Please note that looking simply at % of sequence similarity is often misleading. For reference, human BCL2 and worm CED-9 (two well established homologues) are “only” 44% similar (using the same scoring matrix we used for our alignment); iii) another possible explanation for the data obtained in C. elegans is that the two orthologs are expressed in different cell types and/or at different times during embryonic development, and that the deletion of both is necessary to uncover the phenotype. A preliminary analysis of publicly available single-cell data in C. elegans (PMID 31488706) seems to support this hypothesis.\nIn any case, we understand the reviewer’s concern and we will repeat again the experiments with C03B8.3 in different cell types (e.g. HEK293).\n\n5) Even a double KO of the TMEM65 orthologues in C. elegans produces a relatively minor phenotype, therefore perhaps a different in vivo model would better represent the function of TMEM65 or better model its loss of function effects.\nWe are very puzzle by the reviewer’s comment and its attitude toward the data in nematodes. As outlined by the other two experts in the field, our C. elegans data show the relevance of TMEM65 in cell death during embryogenesis, which is not a minor phenotype. We show that in certain conditions TMEM65 deficiency causes necrosis, which can be prevented by MCU-1 inhibition. By rescuing an obvious phenotype (death), we provide mechanistic evidence on how TMEM65 plays a role in pathophysiology. Taken together, our data are perfectly in line with the findings in transgenic mice, demonstrating an evolutionarily conserved function of TMEM65 in mitochondrial calcium signaling. We will take further advantage of the powerful C. elegans model by i) performing proteomic profiling of C03B8.3;tag-321 double mutants and mcu-1 rescue at 25 ºC, and ii) comparing the phenotype of CeNCLX(lof) vs CeTMEM65(null) nematodes, along with mcu-1 rescue experiments.\n\nReviewer #3:\nWe thank the reviewer for her/his kind words and the time s/he dedicates in reading the submitted study. We appreciate very much that s/he finds our work of interest and supports its publication.\n\nAdditional experiments requested by Nature Communications editor during our zoom call\nAs discussed with Dr. Angela Parrish we will perform additional experiments to expand the in vivo part:\n• Proteomic profiles of C03B8.3;tag-321 double mutants and mcu-1 rescue effects at 25 ºC\n• Hatching assays at 25°C in ncx-9(lof) C.elegans, the putative homologue of human NCLX. If any phenotype is present, we will also test whether loss of mcu-1 can rescue the phenotype, as in the case of CeTMEM65(null)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revised version of the manuscript, the authors improved the manuscript by addressing my previous concerns, particularly on the potential interplay between TMEM65 and NCLX. They generated NCLX knockout cell lines to test if TMEM65 function depends on NCLX, and showed that TMEM65 over-expression behaves similarly in WT and NCLX KO cell lines, suggesting that the two proteins are likely to work independently, at least in the cell culture system used in this manuscript. This is an important finding for the field. For this revision, I have minor critiques as outlined below:\n\n(1) Based on the data presented on Fig. 3g, the authors claim that \"TMEM65 is the molecular target of CGP-37157\". I think claim specificity claims after adding 50uM of drug is not justified. This is a very high concentration of drug. Other calcium channels, including NCLX, have also claimed to be molecular targets of this compound, based on similar experiments. This compound is likely to be less specific, especially at high concentrations. So TMEM65 could be *one* of the targets, but the data shown does not support the claim that it is *the* target.\n\n(2) The authors show their data on NCLX antibody validation efforts, and I think the data should be included in supplementary/extended figures as this information on antibody quality will be useful for the community.\n\n(3)The authors also mention that they conducted TMEM65-NCLX co-immunoprecipitation experiments using different conditions, detergents etc in their rebuttal, but do not show the data in the rebuttal, or the manuscript. Thus, it is not possible for the reviewers to judge the claim that the two proteins do not interact. If proper controls are used, lack of interaction between the two proteins under the conditions the authors tried will be valuable information and should be included in the manuscript.", "reviewer_2": "The authors have demonstrated that TMEM65 is absolutely relevant to Ca2+ efflux from mitochondria. The authors show clearly that TMEM65 localizes to the IMM. Its overexpression dramatically enhances Na+-dependent Ca2+ extrusion, completely abrogated by the pharmacological inhibitor CGP-37157, demonstrating that TMEM65 is the major target of this inhibitor. TMEM65 downregulation chronically elevates basal mitochondrial Ca2+ levels and impairs Ca2+ efflux when Ca2+ is conducted across the IMM by MCU. The authors show clearly that TMEM65 acts independently of NCLX, and go further, suggesting that TMEM65 may have a more important role in mitochondrial Na+/Ca2+ exchange than NCLX, since its OE and KD effects on mitochondrial Ca2+ extrusion are dramatic, either in the absence or presence of NCLX. Deleting the TMEM65 homologues in Caenorhabditis elegans (CeTMEM65) affected embryonic development in a temperature dependent manner. The CeTMEM65 (null) alleles caused necrotic lesions that were mitochondrial Ca2+ dependent, since suppressed by inhibiting the mitochondrial calcium uniporter MCU-1. The results are completely convincing because of the OE and KD in the absence and presence of NCL The findings are clear and workmanlike. The authors describe a primary role for TMEM65 in mitochondrial Ca2+ extrusion that is nicely recapitulated in the in vivo experiments. I recommend publication without further delay."}, "author_response": "Given the large amount of data generated during the revision, we decided to reformat our manuscript and give more space to some data, while others were omitted. For example, we did not include C. elegans proteomics, since it does not add too much to the core findings of our study. By focusing on the main evidence, we believe that the manuscript is more understandable and easier to read for a broader audience.\n\nReviewer #1.\n\nWe would like to thank the reviewer for appreciating our efforts and highlighting the important contribution of our study in the field of mitochondrial biology. Please find below the point-by-point response to reviewer’s comments.\n\n1. Much of the results in this paper are not properly dissociated from NCLX regulation. While the authors acknowledge that NCLX has been identified as a mediator of Na+-dependent Ca2+ extrusion, they claim modulating TMEM65 expression has effects on Na+-dependent Ca2+ extrusion due to the function of TMEM65 without considering NCLX activity. Please address this through investigation of NCLX expression in cells with altered TMEM65 expression.\nComment 1.1. We agree with the reviewer on the fact that the original version of our manuscript lacks data on NCLX, which is currently considered the bona fide mitochondrial Na+/Ca2+ exchanger. As suggested, we tested whether altered NCLX expression could be responsible for differences in mitochondrial calcium handling caused by modulation of TMEM65 levels.\nUnfortunately, we were not able to evaluate NCLX protein levels, due to the lack of a reliable NCLX antibodies, a well-known limitation in the field (Hagenston et al., 2022; Pathak et al., 2020). During the revision, we tried one more time to validate NCLX antibodies from three different commercial providers (Proteintech, Abcam, and Thermo Fisher). We tested these antibodies by comparing the following conditions:\n1. Control: HeLa cells transfected with a mock vector (pcDNA3.1) and non-targeting esiRNA (esiRNA-RLuc)\n2. esiRNA-NCLX: HeLa cells after acute (72h) downregulation of NCLX\n3. esiRNA-TMEM65: HeLa cells after acute (72h) downregulation of TMEM65\n4. TMEM65-3xFlag: HeLa cells after acute (48h) overexpression of TMEM65\n5. NCLX-HA: HeLa cells after acute (48h) overexpression of NCLX\n6. HeLa-SGO_WT: parental cell line from a commercial provider (SyntheGo) used for genome editing\n7. HeLa-SGO_NCLX-KO(pool): pool of HeLa cells knockout for NCLX custom generated by a commercial provider. Gene editing was verified by sequencing\n8. HeLa-SGO_NCLX-KO(clone A4): single clone of NCLX-KO HeLa cells generated by a commercial provider. Gene editing was verified by sequencing\n9. HAP1_WT: control HAP1 cells (parental cell line) obtained from the largest commercially available (Horizon discovery) bank of isogenic cell lines\n10. HAP1_NCLX-KO: ready-made NCLX knockout HAP1 cells, commercially available. Gene editing was verified by sequencing\n11. HAP1_TMEM65-KO: ready-made TMEM65 knockout HAP1 cells, commercially available. Gene editing was verified by sequencing\nFigure R1 shows the results of our validation. Please consider that the expected molecular weight of NCLX is 64 kDa, while in the literature NCLX has been claimed to run at 50 kDa, with a putative SDS-resistant dimer running at 100 kDa. In any case, even if extensive signal can be detected, none of the bands is specific for NCLX.\nFigure R 1. Validation of some commercially available NCLX antibodies.\nTo overcome this limitation, we performed qPCR assays to evaluate mRNA levels of key components of mitochondrial calcium handling (NCLX, TMEM65 and MCU) using commercial TaqMan probes. Overall, neither overexpression nor silencing of TMEM65 causes significant changes in NCLX transcripts, thus supporting the specificity of the effect. These data have been included in the revised manuscript as Extended Data Figure 3f. Please also consider that we have now also included additional experimental evidence indicating that TMEM65 act independently of NCLX, on the basis of functional and genetic approaches (see comments 1.3 and 2.3). We think that altogether these results demonstrate that TMEM65 and NCLX are two molecularly distinct transport systems.\n\n2. The authors claim TMEM65 variants were expressed at a similar level, but by eye one can see at least a two-fold increase in abundance between the expression of the WT protein and E178Q protein among other differences. It may be best to write that the mutants expressed at WT levels, or greater, except GZ dead. Would TMEM65 overexpression affect NCLX expression? It is possible that the changes observed with these mutants is not due to TMEM65 function per se, but due to the effects of TMEM65 on NCLX stability. Please check of NCLX levels cell cells that express TMEM65 variants.\nComment 1.2. We agree with the reviewer and adjusted the text accordingly. Having that said, we are confident that the effect on Ca2+ signaling is not influenced by the expression, since those are population-based recording of cells co-transfected with TMEM65 variants and aequorin. In support of our arguments, Western blots show that some TMEM65 mutants (e.g., D132N; E144Q) are highly expressed, nevertheless they do not behave as wt TMEM65, possibly indicating that they are probably loss of function. As suggested by the reviewer, we analyzed the expression levels of NCLX (and MCU as well) in cells expressing TMEM65 mutants by qPCR (as specified in comment 1.1, Western blot is not possible due to the lack of reliable NCLX antibodies). Also in this case, expression of TMEM65 mutants has no significant effect on NCLX expression levels, further highlighting the specificity of our findings. These data have been included in the revised manuscript as Extended Data Figure 2f.\n\n3. The authors claim that TMEM65 is an essential component and/or the strongest contributor to the process of mitochondrial Ca2+ efflux. This is a misinterpretation of the data, as the process of mitochondrial Ca2+ efflux still happens with TMEM65 genetically silenced, therefore the claim of essential component is not supported, and no other contributors to the process of mitochondrial Ca2+ efflux are analyzed, therefore the comparative word “strongest” is not appropriate. Please compare the effects that TMEM65 knockdown and NCLX knockdown have on Na+-dependent Ca2+ efflux.\nComment 1.3. We took in consideration the reviewer’s comment and adjust the text accordingly.\nFirst, we would like to highlight that according to our results, silencing of TMEM65 is sufficient to abrogate mitochondrial Na+-dependent Ca2+ efflux, at least in the cellular model we tested. Indeed, the residual Ca2+ efflux that is still visible in TMEM65 silenced cell is due to the Na+-independent transporters, that remain active also in Na+-containing buffers (to our knowledge, there are no obvious experimental conditions where Na+-independent efflux can be fully inhibited). Accordingly, efflux rate in esiRNA-TMEM65 transfected cells is 4.8 +/- 1.26 umol/sec in the absence of Na+, and 4.69 +/- 0.76 umol/sec in the presence of Na+ (difference is not statistically significant), as originally shown in figures 3c and d.\nAs recommended by the reviewer, we conducted additional experiments to further explore the relationship between TMEM65 and NCLX, combining both genetic and pharmacological approaches.\nOf note, two recent studies published during the revision process have presented markedly different perspectives on this issue (Garbincius et al., 2025; Garbincius & Elrod, 2025; Zhang et al., 2025). Our primary objective was to assess whether the effect of TMEM65 depends on what is currently recognized as the bona fide mitochondrial Na+/Ca2+ exchanger, namely NCLX. To address this, we first examined the effect of TMEM65 overexpression following acute NCLX silencing (silencing efficiency confirmed by qPCR, see Extended Data Figure 3f). We then analyzed agonist-induced mitochondrial Ca²+ transients in intact cells, finding that TMEM65 overexpression markedly reduced peak [Ca2+]mt in both control and NCLX-silenced cells (see Fig. 4a–b, revised manuscript). These results indicate that NCLX expression levels do not limit the effect elicited by TMEM65 overexpression.\nTo further substantiate this result, we generated NCLX KO HeLa cells, with sequence validation provided by the commercial supplier (SyntheGo, now EditCo). To reinforce the robustness of our conclusions, selected experiments were performed both in‑ a “polyclonal” NCLX KO population and in a clonal line derived from single cell isolation. We then assessed the impact of TMEM65 overexpression on mitochondrial Ca² transport in control and NCLX KO cells. Analyses conduc‑ted in both intact and permeabilized cells consiste‑ntly revealed that TMEM65 overexpression rapidly stimulates Na dependent mitoc⁺hondrial Ca² efflux even in th‑e absence of NCLX (see Fig. 4 along with the paragraph “TMEM65 acts independently of NCLX” of the revised manuscript).\nTak⁺e‑n together with the genetic evi⁺dence (as outlined in comment 2.3), this set of experiments provides clear and consistent support for the view that TMEM65 and NCLX are functionally distinct molecular entities. This conclusion is in line with the recent demonstration that TMEM65 alone can mediate Ca2+ transport when reconstituted in vitro (Zhang et al., 2025). Concurrently, our data indicate that the contribution of NCLX to mitochondrial Ca² extrusion is limited, at least within the experimental models employed here. Specifically, both silencing and genetic ablation of NCLX lead to only a modest increase in peak [Ca² ] in intact cells (not statisti⁺cally significant, although the p-value approaches 0.05), an ⁺ₘₜ effect that is entirely absent in permeabilized cells. Consistent with this, Na dependent Ca² transport remains pharmacologically inhibitable in both NCLX silenced and NCLX KO cells (see also comment 1.5). Collectively, these findings suggest a marginal role for ⁺N‑CLX as a mit⁺ochondrial Na /Ca² exchanger and raise the possibility of its involve‑ment in a different sub‑cellular compartment.\nWe acknowledge that these observations carry important implications for the broader NCLX literature; how⁺ever⁺, the present study is centered on the functional characterization of TMEM65. A comprehensive evaluation of the relative contributions of NCLX and TMEM65 to sodium dependent mitochondrial calcium transport—likely to vary across experimental systems—falls beyond the scope of this work.\n\n4. The authors claim the double mutant nematodes had a median lifespan that was slightly less than wt and single mutant nematodes when assessed at 25 °C. However, statistical analysis of the median lifespans is not shown in the source data table or supplementary table.\nComment 1.4. In the revised manuscript, we have provided all the experimental details for each individual lifespan assay performed at 20ºC and 25ºC. Since we carried out additional analysis (n=4 biological replicates), we have re-calculated the mean±SEM of the median lifespan for each of the four strains (Supplementary table ST1). While at 20ºC there is no difference in the mean of the median lifespan compared to wt (N2), at 25ºC C03B8.3(bon104);tag-321(bon103) show a slight decreased survival compared to wt (N2) (p=0.0237). As we are talking of ~1 day (days: 15.00 ± 0.41 vs 13.50 ± 0.29), we wrote as follows: Lifespan assays at 20°C showed no differences between wt and mutant nematodes, whereas there was a trend toward a minor decreased median lifespan for CeTMEM65 single and double mutants compared to wt at 25°C (Figure 5d-e and Supplementary table ST1). We are open to any alternative statement.\n\na. Please add p values (compared to wt) in the Statistical analysis of lifespan assays at 25 °C.\nDue to space limitations in the figures and legends, it’s not feasible to include all p-values given the number of conditions tested. However, all raw data and corresponding statistical analyses, including exact p-values, are fully provided in the source data tables.\n\nb. In assessing the individual lifespan assays, a p value (compared to wt) is reported that does not match the medians. It is unclear what this p value is reporting and whether that is a better representation of lifespan than comparing medians. Please clarify what this p value represents and address why median lifespan was used instead of mean lifespan or another measurement of lifespan.\nTo report the lifespan assay data, we follow community guidelines by reporting median and maximum lifespan as indicative measures of survival. For each e individual lifespan assay, reported p-values are derived from log-rank (Mantel-Cox) tests, which compare the entire survival distributions while accounting censoring. For the “lifespan data summary” table, the p-values refer to the mean±SEM of the median lifespan that was calculated from the 4 individual experiments. For transparency, we have reported all this information. We are open to any alternative approach to publish our findings.\n\nc. The authors claim the preliminary evidence showed no major survival difference between wt and CeTMEM65 single and double mutant nematodes. However, in supplementary table 1 the authors report a significant p value (compared to wt) for C03B8.3(bon104). Please address this discrepancy.\nAs mentioned above, in the revised manuscript we have provided additional biological replicates that properly address the reviewers’ comment. Ou statements are based on statistical analysis for each individual experiment as well as mean±SEM of the median survival of the four biological replicates are available in the Supplementary Table ST1.\n\n5. The authors claim that CGP-37157 restores normal mitochondrial Ca2+-dynamics, but there is a significant difference between control and TMEM65 overexpressing cells with regards to normalized Ca2+ efflux (Fig. 3g). Thus, this claim is not supported. Please address this discrepancy.\nComment 1.5. We thank the reviewer for his/her comment and have revised the text to moderate our conclusions accordingly. There is indeed a small but statistically significant difference in the efflux rate of TMEM65-overexpressing cells treated with CGP-37157. We reasoned that the remaining residual effect we observed was directly attributable to the overexpression of the TMEM65 protein, which induces a robust increase in Na+-dependent mitochondrial Ca2+ efflux. To test this hypothesis, we replicated the experiment using both standard (i.e. 20 µM) and a higher (50 µM) concentration of CGP-37157. The results of this experiment (now included as figure 3g) indicated that in cells where the expression of TMEM65 was artificially increased, significantly higher concentrations of CGP-37157 were necessary to fully suppress the enhanced mitochondrial Ca2+ extrusion. These results may suggest that TMEM65 is the genuine molecular target of CGP-37157. At first glance, this indication seems to contradict the literature. However, the discrepancy is only apparent. Although it is frequently reported that CGP-37157 is an inhibitor of NCLX, it's crucial to recognize that CGP-37157 is actually an inhibitor of Na+-dependent Ca2+ efflux. As far as we currently know, it functionally inhibits the process, not a specific transporter. Accordingly, the activity of CGP-37157 has been described well before the discovery of NCLX as mitochondrial Na+/Ca2+ exchanger (Cox et al., 1993; Cox & Matlib, 1993). Accordingly, we tested the effect of CGP-37157 in cells were NCLX was acutely downregulated (esiRNA-NCLX) or genetically deleted. Surprisingly, CGP-37157 is perfectly capable of inhibiting mitochondrial Ca2+ efflux also in the absence of NCLX (Fig 4a-b, Extended Data Figure 5). Altogether, our data suggest that the primary target of CGP-37157's activity is TMEM65 rather than NCLX. However, it's important to note that only dedicated experiments using in vitro reconstituted TMEM65 and NCLX could definitely solve this issue. Accordingly, a recent publication supports this finding (Zhang et al., 2025).\n\n6. It is unclear what criteria is used to identify TMEM65 when looking at proteins with a conservation profile similar to MCU. Please describe how the evolutionary analysis led to TMEM65 out of the fourteen proteins identified in Sharma et al 2015.\nComment 1.6. The list of 14 proteins potentially involved in mitochondrial Ca² dynamics was originally documented in De Stefani et al., Nature (2011). Our initial analysis focused on a limited number of species; in the present study, we performed a broader evolutionary compariso⁺n and identified TMEM65 as the protein with the closest conservation profile to MCU. By contrast, the paper “Sharma et al., 2015” refers to a separate study published in Nature Communications by the Gramolini lab. That work reports TMEM65 localization at the plasma membrane of cardiomyocytes, particularly enriched at intercalated discs. A detailed assessment of that study falls outside the scope of the present work, which provides compelling evidence for the mitochondrial localization of TMEM65, consistent with multiple independent studies in mitochondrial proteomics (as discussed in the main text).\nSince the reviewer raised this point, we would like to highlight some limitations of the referenced manuscript. In that study, the authors employed a protocol for the putative enrichment of plasma membrane proteins originally validated only in Dictyostelium discoideum (Chaney & Jacobson, 1983). This protocol was applied to mouse neonatal and human fetal ventricular cardiomyocytes, and membrane-depleted and membrane-enriched fractions were analyzed by mass spectrometry. Upon careful examination of the supplementary data, we noted the following:\n• TMEM65 appears significantly enriched in the putative plasma membrane fraction of human samples (FDR = 0, Supplementary Table 4), but not in murine samples (FDR = 0.37692, Supplementary Table 3).\n• Supplementary table 4 contains the list of human proteins claimed to be “enriched at the plasma membrane”. This list is composed of 306 proteins when high stringency is used (FDR <0.01). Among these, 86 proteins (28%) are known to be located in mitochondria (i.e, they are listed in Mitocarta). Considering that mitochondria cover approximately 7% of the whole proteome, we can conclude that the supposed “plasma membrane fraction” is not only contaminated with mitochondria, but rather it is a subcellular fraction where mitochondrial proteins are over-represented.\n• Among the top 20 proteins with the highest enrichment scores (FoldChange), we found:\n9 proteins lacking transmembrane domains (based on UniProt and TM helix prediction tools: HIST1H2BJ, HIST1H2BK, H2AFX, PPP1CC, L2HGDH, OCIAD1, FAHD2A, ABHD11, RAP1B)\n2 common contaminants in proteomic preparations (keratins: KRT18, KRT6A)\n6 known mitochondrial proteins (MitoCarta: NDUFA11, SLC25A20, MTCH1, L2HGDH, ABHD11, GHITM)\n2 proteins localized to the endoplasmic reticulum (UniProt: LSS, SSR4)\n1 endosomal protein (UniProt: OCIAD1)\nOnly 1 bona fide plasma membrane protein (CXADR)\nThese findings underscore the limitations of the subcellular fractionation protocol used, which—as is often the case—may not yield pure compartment-specific fractions\n• Mass spec data were confirmed by Western blot. Despite the validation efforts, the antibody (now discontinued) reacts with multiple bands. In this paper (Gramolini lab Nat Comm 2015), the authors claim that TMEM65 specific bands are at 22 and 44 kDa (monomer and dimer respectively). However, the same research group published an additional paper on TMEM65 in 2022 (Gramolini lab Nat Comm 2022). Using a different antibody, they were still able to detect two specific bands, but at a different molecular weight (17 and 25 kDa), as shown in figure R2. This indicate the poor reliability of antibody, a well-known issue in every laboratory.\nFigure R 2.\nOverall, we find the experimental evidence suggesting TMEM65 localization at the plasma membrane to be unconvincing. In contrast, multiple independent lines of evidence—based on diverse methodologies—strongly support its exclusive mitochondrial localization. These include: i) broad tissue expression (not limited to the heart), ii) presence of a mitochondrial transit peptide at the N-terminus, iii) TMEM65 is present in all mitochondrial databases, including Mitocarta (where differential centrifugation + density based separation were performed, ending with a very pure mitochondrial fraction), MitoCop, IMPI, iv) proximity-labeling techniques using APEX (Rhee et al., 2013), v) classical biochemical approaches (Nishimura et al., 2014), and vi) our present work defining a clear molecular function for TMEM65 at mitochondria.\n\n7. Figure 1b – Highly conserved amino acids criteria are not provided. How conserved is “highly” and in what species is this conservation? Please provide details on the conserved amino acids. How many species were used in this analysis?\nComment 1.7. We apologize for the lack of methodological details in defining “highly conserved” amino acids in TMEM65 sequence. We performed a multiple sequence analysis of a manually curated list of protein sequences from different species (approximately 200 species from multiple clades). We considered amino acids as 'highly conserved' if they had a score of at least 3.5 bits in the sequence logo (log2-scaled, with a maximum of 4.3 bits for 20 amino acids). Roughly, this corresponds to amino acids conserved in at least 80% of analyzed species. We added these details in the “Methods” section.\n\n8. The authors claim that failure to detect expression of CeTMEM65-2 is probably due to the instability of the protein at 37 °C, but they give no reason for this claim. Please discuss the reasoning behind this claim.\nComment 1.8. The exogenous expression of native C. elegans proteins (an organism that grows at room temperature and is heat-sensitive) in mammalian cells is often unsuccessful because worm proteins are unstable at 37°C, especially membrane proteins. We were surprised to be able to successfully express CeTMEM65-1 (Flag-tagged TAG-321) in HeLa cells at 37°C, which represents the exception rather than the rule. In contrast, we were unable to detect CeTMEM65-2, despite the transfection being carried out under the same conditions as the other plasmids.. We also generated additional constructs for CeTMEM65-2 expression (e.g. by changing the expression vector, codon optimization, etc) and used other cell types (e.g. HEK293 cells that usually express high levels of exogenous proteins). Despite our efforts, we never detected CeTMEM65-2 by Western blot. We thus concluded that CeTMEM65-2, as many other proteins from nematodes, cannot be exogenously expressed in mammalian cells.\n\n9. The authors describe a slight developmental delay in the null mutants, but do not describe this effect quantitatively or qualitatively. Please describe the effect and the significance of it.\nComment 1.9. Following the reviewer’s comment, we carried out additional experiments. We herein provide a better statistical analysis of the developmental timing of our three null mutants compared to wt animals (n=7 biological replicates). We adjusted the text accordingly. To slim the manuscript, we did not include these data neither in the main nor in the supplementary figures. However, we are happy to add this panel in the manuscript, if the reviewer thinks that it is appropriate.\nFigure R 3\n\n10. Extended Data Figure 5d includes DmeTMEM65, which the reader must assume refers to Drosophila melanogaster since this is not explained in the text or the figure legend. The expression of this protein shows how a different non-human ortholog protein may be expressed, but without explanation this detracts from the rest of this figure. Please address the inclusion of this Western lane.\nComment 1.10. We included the Drosophila samples in the Western blot by mistake. Following the reviewer’s comment, we replaced the previous panel with a new figure.\n\n11. Fig. 1c: pcDNA3.1 is not explained in the figure legend. Please either replace pcDNA3.1 with “empty vector” or explain the label of pcDNA3.1 in the figure legend to increase readability.\nComment 1.11. We agree with the reviewer and adjusted the labels accordingly.\n\n12. Fig.1E: Typo - “(full lenght)” should be “(full length).” Please fix this typo.\nComment 1.12. We thank the reviewer for her/his comment. We corrected the figure accordingly.\n\n13. Extended Data Figure 2f: WT label is not aligned with the WT bar. Please align properly.\nComment 1.13. Thanks, we adjusted the figure accordingly.\n\na. In all other cases of the [Ca2+]mt curves the curves precede the quantification, whereas this figure has the opposite order. Please change the ordering for the sake of consistency.\nWe thank the reviewer for the suggestion. We changed the figure accordingly.\n\n14. In the source data table file, the tab named “Fig 3g” is out of order. Please place the tab “Fig 3g” between Fig 3f and Fig 4a.\nComment 1.14. We corrected the figure.\n\n15. In the source data table file, the tab named “Extended Fig 5c” refers to data present in Extended Fig 5e. Please rename the tab.\nComment 1.15. Thanks, we corrected the label.\n\nReviewer #2:\n\n1) In Figure 1, HSP60 is used as a mitochondria-specific marker. Panel 1D shows abundance of unmerged HSP60 and TMEM65 signals. HSP60 is known to reside in the cytoplasm and even the extracellular space, therefore a more specific mitochondrial marker should be used (e.g. CoxIV) to argue mitochondrial expression of TMEM65.\nComment 2.1. Following the reviewer’s comment, we performed TMEM65-3xFlag colocalization experiments with additional mitochondrial proteins through immunofluorescence. Although CoxIV antibodies were not available in our lab, we obtained full colocalization with other bona fide mitochondrial markers, including TOM20 and components of complex I and II. We have now included a colocalization between TMEM65-3xFlag and SDHA in revised Figure 1d.\n\n2) Figure 1E- a low molecular weight marker should be added to the WB (below 25 kDa, to better recognize the TMEM65 bands after proteinase K treatment).\nComment 2.2. We added a lower molecular weight marker as requested. Please also consider that uncropped western blots containing all MW markers are included as supplementary figures.\n\n3) This study does not show if TMEM65 forms a complex with NCLX. A simple immunoprecipitation experiment (or proximity ligation assay (PLA)) would address this issue.\nComment 2.3. We agree with the reviewer on the fact that the original version of our manuscript lacked data on NCLX, which is currently considered the bona fide mitochondrial Na+/Ca2+ exchanger.\nUnfortunately, working with NCLX is far from being “simple”. No commercial antibodies are available for WB and/or CoIP, as detailed in comment 1.1, making it virtually impossible to detect the endogenous protein (which is also rarely detected by MS). An alternative approach is to overexpress a tagged version, although some studies report (mis)localization of overexpressed NCLX in non-mitochondrial membranes. For instance, see (Palty et al., 2004, 2010), where overexpressed NCLX is reported to “spillover” to plasma membrane. Despite these limitations, as suggested by the reviewer, we attempted to verify a potential physical interaction between TMEM65 and NCLX by co-transfecting HeLa and HEK293 cells with TMEM65-3xFlag and NCLX-HA. In all the experimental conditions (detergents tested: Triton X100, DDM, and digitonin) we failed to observe an interaction between the two proteins. However, such a result cannot definitively exclude the possibility of an interaction (in this case, lack of evidence is not evidence of the lack).\nWe therefore implemented two different approaches to study a potential relationship between TMEM65 and NCLX. The first set of experiments was dedicated to verifying whether a functional relationship between the two proteins exist, by measuring the effect of TMEM65 overexpression in control and NCLX-KO cells. As detailed in comment 1.3, TMEM65 increases Na+-dependent mitochondrial Ca2+ efflux even in the absence of NCLX. This indicates that NCLX and TMEM65 are two molecularly distinct transporters.\nIn the second approach, we used the C. elegans model to genetically verify a possible relationship between the two proteins. The ncx-9 gene is reported to encode the C. elegans homolog of NCLX (Sharma et al., 2017) with a predicted subcellular localization to the mitochondria (Uniprot, O16242). We generated mutants carrying ncx-9 (lof) alleles and tested their phenotype. The results now presented in Figure 5p show that the genetic loss of ncx-9 has no impact on the percentage of hatched animals. The different phenotype of CeTMEM65(null) vs CeNCLX(lof) worms further supports the idea that these two proteins act independently. This is also in line with a recent report showing\n\n4) Results from C. elegans suggest that TMEM65 ortholog/orthologs are part of a protein complex (see point 3, about NCLX co-IP), since the C03B8.3 deletion alone fails to produce any phenotype. Only one ortholog out of 2 was tested for ability to regulate Ca2+ efflux. Given that both TMEM65 C. elegans orthologs show low protein sequence similarity (52 and 35%) to mammalian TMEM65, both genes must first be proven to code for mitochondrial proteins involved in regulation of calcium efflux.\nComment 2.4. . As the referee may know, it is relatively difficult to overexpress functional C. elegans proteins in mammalian cells. This is because they are relatively unstable at 37 °C. However, we would like to draw the reviewer’s attention to the following arguments: i) both TAG-321 and C03B8.3 are predicted with high confidence to possess a mitochondrial transit peptide at the N-terminal (calculated with PredSL); ii) sequence alignment of human TMEM65 to either TAG-321 or C03B8.3 show highly significant scores (3e-35 and 9e-8, respectively). Please note that looking simply at % of sequence similarity is often misleading. For reference, human BCL2 and worm CED-9 (two well established homologues) are “only” 44% similar (using the same scoring matrix we used for our alignment); iii) another possible explanation for the data obtained in C. elegans is that the two orthologs are expressed in different cell types and/or at different times during embryonic development, and that the deletion of both is necessary to uncover the phenotype. A preliminary analysis of publicly available single-cell data in C. elegans (Packer et al., 2019) seems to support this hypothesis.\nSince we took the reviewer’s concern seriously, we tried to overexpress C03B8.3. We generated additional constructs for CeTMEM65-2 expression (e.g. by changing the expression vector, codon optimization, etc) and used other cell types (e.g. HEK293 cells that usually express high levels of exogenous proteins), alone or in combination with CeTMEM65-1. Despite our efforts, we never detected CeTMEM65-2 by Western blot. We thus concluded that CeTMEM65-2, as many other proteins from nematodes, cannot be exogenously expressed in mammalian cells.\n\n5) Even a double KO of the TMEM65 orthologues in C. elegans produces a relatively minor phenotype, therefore perhaps a different in vivo model would better represent the function of TMEM65 or better model its loss of function effects.\nComment 2.5. While we acknowledge the limitation of C. elegans work, our data clearly provide crucial insights into the physiological role of TMEM65, as also highlighted by Reviewer 3. Through our cross-species analyses, we have demonstrated that TMEM65 deficiency can induce embryonic death in specific conditions. Mechanistically, this severe phenotype that can be rescued by inhibiting MCU-1. This rescue experiment provides strong mechanistic evidence supporting TMEM65's involvement in MCU-1 activity and, therefore, Ca2+-dependent cellular processes. For a broader audience, these data clearly demonstrate the evolutionarily conserved function of TMEM65 in modulating mitochondrial calcium signaling, further emphasizing its impact in cell physiology and death, as shown in our C. elegans assays.\n\nReviewer #3:\nWe thank the reviewer for her/his kind words and the time s/he dedicates in reading the submitted study. We appreciate very much that s/he finds our work of interest and supports its publication."}, {"round": 3, "reviewer_comments": {"reviewer_1": "In this revised version of the manuscript, the authors improved the manuscript by addressing my previous concerns, particularly on the potential interplay between TMEM65 and NCLX. They generated NCLX knockout cell lines to test if TMEM65 function depends on NCLX, and showed that TMEM65 over-expression behaves similarly in WT and NCLX KO cell lines, suggesting that the two proteins are likely to work independently, at least in the cell culture system used in this manuscript. This is an important finding for the field. For this revision, I have minor critiques as outlined below:\n\n(1) Based on the data presented on Fig. 3g, the authors claim that \"TMEM65 is the molecular target of CGP-37157\". I think claim specificity claims after adding 50uM of drug is not justified. This is a very high concentration of drug. Other calcium channels, including NCLX, have also claimed to be molecular targets of this compound, based on similar experiments. This compound is likely to be less specific, especially at high concentrations. So TMEM65 could be *one* of the targets, but the data shown does not support the claim that it is *the* target.\n\n(2) The authors show their data on NCLX antibody validation efforts, and I think the data should be included in supplementary/extended figures as this information on antibody quality will be useful for the community.\n\n(3)The authors also mention that they conducted TMEM65-NCLX co-immunoprecipitation experiments using different conditions, detergents etc in their rebuttal, but do not show the data in the rebuttal, or the manuscript. Thus, it is not possible for the reviewers to judge the claim that the two proteins do not interact. If proper controls are used, lack of interaction between the two proteins under the conditions the authors tried will be valuable information and should be included in the manuscript.", "reviewer_2": "The authors have demonstrated that TMEM65 is absolutely relevant to Ca2+ efflux from mitochondria. The authors show clearly that TMEM65 localizes to the IMM. Its overexpression dramatically enhances Na+-dependent Ca2+ extrusion, completely abrogated by the pharmacological inhibitor CGP-37157, demonstrating that TMEM65 is the major target of this inhibitor. TMEM65 downregulation chronically elevates basal mitochondrial Ca2+ levels and impairs Ca2+ efflux when Ca2+ is conducted across the IMM by MCU. The authors show clearly that TMEM65 acts independently of NCLX, and go further, suggesting that TMEM65 may have a more important role in mitochondrial Na+/Ca2+ exchange than NCLX, since its OE and KD effects on mitochondrial Ca2+ extrusion are dramatic, either in the absence or presence of NCLX. Deleting the TMEM65 homologues in Caenorhabditis elegans (CeTMEM65) affected embryonic development in a temperature dependent manner. The CeTMEM65 (null) alleles caused necrotic lesions that were mitochondrial Ca2+ dependent, since suppressed by inhibiting the mitochondrial calcium uniporter MCU-1. The results are completely convincing because of the OE and KD in the absence and presence of NCL The findings are clear and workmanlike. The authors describe a primary role for TMEM65 in mitochondrial Ca2+ extrusion that is nicely recapitulated in the in vivo experiments. I recommend publication without further delay."}, "author_response": "We are grateful to the reviewer for the positive evaluation of our study.\n\n(1) Based on the data presented on Fig. 3g, the authors claim that \"TMEM65 is the molecular target of CGP-37157\". I think claim specificity claims after adding 50uM of drug is not justified. This is a very high concentration of drug. Other calcium channels, including NCLX, have also claimed to be molecular targets of this compound, based on similar experiments. This compound is likely to be less specific, especially at high concentrations. So TMEM65 could be *one* of the targets, but the data shown does not support the claim that it is *the* target.\nWe agree with thew point raised by the reviewer and changed the text accordingly.\n\n(2) The authors show their data on NCLX antibody validation efforts, and I think the data should be included in supplementary/extended figures as this information on antibody quality will be useful for the community.\nAs suggested by the reviewer, we have now included the (lack of) validation of the NCLX antibodies, presented in Supplementary Figure 4.\n\n(3)The authors also mention that they conducted TMEM65-NCLX co-immunoprecipitation experiments using different conditions, detergents etc in their rebuttal, but do not show the data in the rebuttal, or the manuscript. Thus, it is not possible for the reviewers to judge the claim that the two proteins do not interact. If proper controls are used, lack of interaction between the two proteins under the conditions the authors tried will be valuable information and should be included in the manuscript.\nAs discussed in our previous response, we are not entirely convinced that a negative CoIP result can be taken as definitive evidence of the absence of interaction, even when appropriate controls are applied. We fully appreciate the reviewer’s perspective; however, we feel it would not be appropriate to present data that we ourselves consider inconclusive.\n\nReviewer #2:\n\nWe thank the reviewer for the appreciation of our work."}]

Decision letter