The VPS13A VAB domain interacts with SNX5

To identify potential human adaptors that bind the VAB domain of VPS13A, we performed a two-hybrid screen of a human cDNA library using a LexA DNA-binding domain fusion protein to the VAB domain of VPS13A as bait. We screened 1.5 × 10⁶ transformants and obtained several positive clones, two of which contained identical plasmids encoding a C-terminal splice variant of the SNX5 sorting nexin lacking the last 45 amino acid residues (Fig 1A). Notably, one of the three yeast Vps13 adaptors is also a sorting nexin, Ypt35 (Bean et al, 2018). The sorting nexins are a diverse family of proteins that contain a Phox domain and are involved in protein sorting and trafficking within the endolysosomal system (Yong et al, 2022). The endosomal protein SNX5 also contains a BAR domain and is part of the retromer complex that regulates the retrograde transport of proteins from endosomes to Golgi (Trousdale & Kim, 2015). We confirmed the specificity of the two-hybrid interaction in β-galactosidase filter assays and analyzed the determinants of this interaction using truncated and mutant fusion proteins (Fig 1B). Our results indicate that the Phox domain of SNX5 is sufficient to interact with the VAB domain of VPS13A (Fig 1B). Studies in yeast have shown that the binding of Vps13 to adaptors is mediated by repeat 6 in the VAB domain and a consensus PXP motif in the adaptors (Bean et al, 2018; Dziurdzik et al, 2020). In addition, a conserved asparagine residue in repeat 6 of the VAB domain (N2428) plays a key role in this interaction, as alanine substitution of this residue has a strong negative impact on adaptor binding (Dziurdzik et al, 2020). Serine substitution of the respective asparagine residue in VPS13D (N3521S) results in spastic ataxia, suggesting that the molecular mechanisms involved in the interaction between Vps13 and adaptors are evolutionarily conserved. We analyzed the effect of the same substitution (N2418S) in VPS13A and observed a strong reduction of the two-hybrid interaction between the VAB domain and SNX5 (Fig 1B and C). Interestingly, SNX5 contains a PXP motif located in the Phox domain (Fig 1A). Consistent with what was observed in yeast, alanine substitution of the two proline residues of this motif fully prevented interaction between the VAB domain of VPS13A and SNX5 (Fig 1B and C). Taken together, these results support the idea that SNX5 is a novel adaptor of VPS13A and that the molecular basis of the interaction between the VAB domain of Vps13 proteins and adaptors is conserved in yeast and humans. However, we cannot rule out the possibility that the PXP motif of SNX5 is not directly involved in binding, as it is part of a proline-rich sequence with a possible structural role in the Phox domain (Koharudin et al, 2009). Alternatively, the substitution of proline residues may have an indirect effect on binding through the destabilization of the Phox domain.

Different fragments of VPS13A encoding the VAB domain were fused to the GFP gene to validate the interaction of respective protein fragments with SNX5 and to study their subcellular localization in HeLa cells. We used a mCherry fusion to full length SNX5, which includes the C-terminal end missing in the two-hybrid library clone. Protein extracts from HeLa cells co-transfected with plasmids encoding the different VPS13A fragments fused to GFP (Fig 2A) and mCherry-SNX5 were subjected to pulldown. All fragments containing the VAB domain were able to interact with mCherry-SNX5 (Fig 2B), confirming the interaction previously identified in two-hybrid assays in yeast. Sorting nexins are a large family of proteins with up to 33 members in mammalian cells (Hanley & Cooper, 2020). Interestingly, a high-throughput proteomic study has shown that VPS13B coprecipitated with the sorting nexins SNX20 and SNX21 (Huttlin et al, 2015), suggesting that interaction with sorting nexins may be a general property of mammalian VPS13 proteins.

The VPS13A VAB domain co-localizes with SNX5

Previous studies from our group using the full-length VPS13A bearing an internal GFP tag suggested a major localization in the mitochondria, but also a minor overlapping localization with ER and endosomes (Muñoz-Braceras et al, 2019). We now use different fragments of the VPS13A to more clearly dissect the determinants of its localization and analyze possible colocalization with its interactor SNX5 using confocal microscopy. Interestingly, we found that cells transfected with the constructs encoding the VAB, VAB+, and VAB++ fragments resulted in a punctate pattern over a general cytosolic background, with the smallest fragment, GFP-VAB, diffusing also into the nucleus (Fig 3A). Furthermore, as previously described, additional inclusion of the C-terminal part of the protein containing the ATG_C and PH domains (VAB-Ct) directs the fusion protein to the mitochondria (Kumar et al, 2018) (Fig 3A), although some distinct puncta, often associated to the mitochondria, can also be observed.

The punctate pattern observed with the different fragments of VPS13A (VAB, VAB+, VAB++) would be consistent with localization in endosome-like structures. Strikingly, a complete co-localization was observed when these fragments were produced together with mCherry-SNX5 in HeLa cells (Figs 3B and S1), which further supports the interaction between these two proteins and the endosomal localization of the VAB-containing fragments. Full-length endogenous VPS13A also co-localizes with mCherry-SNX5 in HeLa cells, although only 10% of SNX5 and 10% of VPS13A signal overlap with each other in these cells (Fig S2). This is consistent with previous analysis that revealed a similar level of co-localization of VPS13A with RAB7 (Muñoz-Braceras et al, 2019). These results indicate that the interaction between full-length VPS13A and SNX5 is restricted to a subpopulation of SNX5-containing structures and involves a relatively small fraction of VPS13A. The fact that co-localization is much higher with the isolated VPS13A fragment containing the VAB domain suggests that the other domains of VPS13A that mediate its association with other organelles limit its availability to interact with SNX5.

The importance of the VAB domain defects in pathology is highlighted by a VPS13D mutation causing substitution of a conserved asparagine residue in the repeat 6 of the VAB domain, which results in spastic ataxia (Dziurdzik et al, 2020). Notably, introduction of the corresponding mutation into the DNA fragment encoding the VAB+ fragment of VPS13A reduced its level of co-localization with SNX5, in agreement with the results obtained in two-hybrid assays (Fig 3C). These results, together with those obtained for VPS13D (Guillén-Samander et al, 2021), suggest that the VAB domains of mammalian VPS13 proteins act as a critical determinant of the localization of these proteins, as previously reported for the yeast Vps13 protein (Bean et al, 2018).

Although the fragment containing only the VAB domain is able to interact with SNX5 in yeast two-hybrid and pull-down assays, and also co-localize with SNX5 in HeLa cells, we cannot rule out that co-localization of VPS13A and SNX5 in vivo at endogenous levels requires additional determinants, such as the APT1 domain that has been shown to bind PI3P and PI5P (Kolakowski et al, 2020). The synergistic action of both determinants may be critical for the stabilization of the association of VPS13A with specific SNX5-containing endosomes in vivo. This model has been proposed for the yeast-sorting nexin Ypt35 and the PI3P-binding APT1 domain of Vps13, which together appear to be required for Vps13 recruitment to endosomal and vacuolar membranes (Kolakowski et al, 2020).

SNX5-VPS13A VAB+ domain puncta localize in endosome subdomains associated with recycling tubular–vesicular structures in proximity to mitochondria and ER

Next, we characterized the nature of SNX5-containing endosomes that co-localize with the VAB domain of VPS13A and the possible MCSs involved. Previous studies in HeLa and HEK293 cells have shown that SNX5 is present in PI3P-enriched domains that are associated (but not completely overlapping) with other endosomal markers, suggesting that SNX5 label-specific endosomal subdomains (Merino-Trigo et al, 2004; Kerr et al, 2006). Despite this endosomal localization, the Phox domain of SNX5 (and that of SNX6 and SNX32) appears unable to interact with PI3P or any other phosphoinositide (Chandra et al, 2019). In contrast, these domains can mediate protein–protein interactions (Paul et al, 2017; Sun et al, 2017; Simonetti et al, 2019). The localization of SNX5 to PI3P enriched-domains could be mediated by its interaction with SNX1 or SNX2 through the BAR domains (Hong, 2019). In addition, SNX5 can also be recruited to the plasma membrane under stimulation of the EGF signaling pathway (Merino-Trigo et al, 2004; Lim et al, 2008). Consistent with these previous data, co-localization analysis with different markers showed that puncta containing SNX5 or the VPS13A VAB+ fragment associate with the early and late endosome markers RAB5 and RAB7, respectively (Fig 4A), although this association is limited to a fraction of VAB+ puncta (17% and 22% of puncta associate with RAB5 and RAB7, respectively; at least 10 cells for each marker were used for quantification). Furthermore, SNX5 puncta were found closely associated with abnormal tubulo-vesicular profiles induced by overexpression of a chimeric form of the cation-independent mannose 6-phosphate receptor (CI-MPR) denoted as CI-MPRtail (Fig 4B) (Waguri et al, 2003). These results are consistent with the described role of SNX5 in retromer-independent retrograde transport of CI-MPR between endosomes and the Golgi (Hara et al, 2008; Kvainickas et al, 2017; Simonetti et al, 2017). The BAR domain of SNX5 and SNX6 is thought to be required to induce the membrane bending necessary for the biogenesis of tubulo-vesicular transport carriers. Notably, our previous studies in VPS13A-depleted HeLa cells have shown defects in cathepsin maturation and reduced lysosomal degradative capacity affecting the efficiency of degradation by autophagy (Muñoz-Braceras et al, 2015, 2019), these phenotypes being compatible with defects in lytic enzymes trafficking to lysosomes. Interestingly, inactivation of VPS13 in yeast also prevents vacuolar trafficking of carboxypeptidase Y (Bankaitis et al, 1986), most likely because of the defect in endosome-Golgi cycling of Vps10, which plays the same role as CI-MPR in humans (Brickner & Fuller, 1997).

Previous studies described the main localization of VPS13A at the interface between ER and the mitochondria (Kumar et al, 2018; Muñoz-Braceras et al, 2019), although localization between mitochondria and endosomes was also reported by our group (Muñoz-Braceras et al, 2019). To further support the later possibility, we analyzed by video-lapse confocal microscopy the possible association of SNX5 and VAB+-containing vesicles with the mitochondria. Fig 4C and Video 1 and Video 2 show events of co-localization or close association overtime between SNX5 or the VAB+ fragment with the mitochondria, indicating a transient tethering of the two organelles.

Recent structural studies suggest that the elongated shape of the VPS13 proteins fits into the gap between organelles and acts as a bridge allowing lipid flow between the two organelles in MCSs. Consistent with this model, the N-terminal region of VPS13A and VPS13C contains an FFAT motif that interacts with the ER VAPA/B proteins, whereas their C-terminal region contains the determinants for interaction with mitochondria (in both VPS13A and C) or endosomes (in VPS13C) (Kumar et al, 2018). Our results now suggest that the C-terminal region of VPS13A can also interact with SNX5-containing endosomes in addition to mitochondria. The VAB domain of VPS13A mediates the interaction with endosomes through SNX5, whereas the ATG_C and PH domains are responsible for the association with mitochondria. Because both determinants are topologically located at the C-terminal end of the protein but at different sites, the possibility of a three-way junction between the ER, mitochondria, and SNX5-containing endosomes is possible. Consistent with this hypothesis, we were able to identify triple co-localization events between the VAB+ fragment or GFP-SNX5, mitochondria, and ER by confocal microscopy (Figs 5A, S3, and S4). The VAB+ fragment cannot be responsible for the establishment of three-way junctions as it lacks the ER and mitochondrial-binding determinants. However, we believe that SNX5 mediates its recruitment to endosomal domains that in some cases are associated with preexisting three-way contacts, probably mediated by additional proteins. Three-way interactions among ER, mitochondria, and lysosomes have been previously documented by cell imaging (Valm et al, 2017), and a four-way contact among ER, mitochondria, Golgi, and lysosomes is required for mitochondrial division (Nagashima et al, 2020), suggesting that the interplay between multiple organelles may be more important that initially thought.

In summary, although previous results indicate a major localization of VPS13A in the ER-mitochondrial junctions (Kumar et al, 2018; Muñoz-Braceras et al, 2019), now our results support the idea that a minor fraction of VPS13A localizes in the interphase between these junctions and a subpopulation of endosomes trough the interaction with the sorting nexin SNX5. This interaction is mediated by the conserved VAB domain in VPS13A and a putative PxP motif in SNX5 (Fig 5B). Although these contacts are minor and transient, the lytic enzymes trafficking defects associated with VPS13A inactivation suggest that this localization could have an important impact on endosome–Golgi retrograde transport. The possibility that this trafficking defect is associated with the human disease chorea-acanthocytosis warrants further investigation.

Two-hybrid analysis

The two-hybrid screen for LexA-VSP13A(VAB)-interacting proteins was carried out in the Saccharomyces cerevisiae strain TAT7 (MATa, leu2-3,112, trp1-901, his3-Δ200, ade2-101, gal80Δ, LYS2:(lexAop)4-HIS3, ura3:(lexAop)8-lacZ) co-transformed with pNuLex(U)-VPS13A(VAB) and a human fetal liver cDNA library (Clontech). Selection of His+ transformants was performed in the presence of 5 mM 3-aminotriazole (3-AT) and transformants were subsequently screened for β-galactosidase activity using a filter lift assay. X-gal filter lift assays were performed as described previously (Yang et al, 1992) and developed for 2 h. For β-galactosidase quantitative assays, four independent transformants were grown to logarithmic phase in synthetic dextrose (SD) medium and enzymatic activity was assayed in permeabilized yeast cells and expressed in Miller units (Miller, 1972). Standard genetic methods were followed and yeast cultures were grown in SD medium lacking appropriate supplements to maintain the selection for plasmids (Rose et al, 1990).

Plasmids

Plasmid pNuLex(U)-VPS13A(VAB) encoding a LexA fusion to the VAB domain of VPS13A was constructed by inserting a PCR fragment containing the VPS13A coding sequence codons 1869-2481 into the BamH1 site of pNuLex(U), a URA3 derivative of pEG202-NLS obtained by replacing the 3xHA cassette of pWS93 (Song & Carlson, 1998) by a cassette containing the LexA and nuclear localization signal sequence of pEG202-NLS. pGAD-SNX5, the positive clone isolated in the two-hybrid screen, is a pACT2 (Clontech) derivative encoding a C-terminally truncated splice variant of SNX5 lacking the last 45 amino acids. Missense and additional C-terminal truncating mutations were obtained by using mutagenic PCR. pmCherry-SNX5 and pEGFP-SNX5 were generated by cloning the full-length SNX5 coding sequences in the polylinker of pmCherry (Bueno-Arribas et al, 2021) and pEGFP-C3 (GenBank Accession Number #U57607). VPS13A fragments were obtained by PCR and inserted into pEGF-C3.

HeLa cells, transfections, immunofluorescence, and confocal microscopy

HeLa cells were a gift from Dr. Alberto Muñoz. The cells were grown in complete DMEM medium (D5648; Sigma-Aldrich), supplemented with 10% FBS (1027-106; Gibco) and 1X penicillin–streptomycin (15140-122; Gibco). DNA transfection was performed using Lipofectamine 2000 (11668019; Invitrogen) according to the manufacturer’s instructions. Briefly, Lipofectamine 2000 and DNA were mixed in serum-free Opti-MEM medium (31985062; Life Technologies), after which the DNA–Lipofectamine 2000 complexes were added to the cells. For most plasmids, the medium was changed to DMEM at 6 h after transfection, with the exception of GFP-SNX5 and mCherry-SNX5, for which the medium was not replaced after transfection. The cells were visualized 24 h after transfection, with the exception of SNX5 expression plasmids, in which case, they were visualized after 16 h. For SNX5 expression, cells with low expression levels were chosen.

For in vivo confocal analysis cells were seeded in eight-well plates (μ-Slide eight Well), after which they were transfected with the expression plasmids. 24 h after transfection, the cells were directly visualized using an inverted laser confocal microscope (LSM710; Zeiss), under standard conditions, 37°C and 5% CO², (Heating System Ibidi). For MitoTracker Red (M7512; Molecular probes) staining, live cells were incubated at 1:5,000 for 15 min.

For immunofluorescence, the cells were seeded on sterile 12 × 12 mm glass coverslips, placed in 24-well plates and transfected as described above. They were fixed with 3% PFA in PBS, pH 7.4, for 30 min at RT. Washes were performed with 1X PBS (133 mM NaCl, 8 mM Na²HPO⁴, pH 7.4) and incubated with 100 mM glycine in PBS for 30 min at RT. The cells were permeabilized with cold methanol at −20°C for 10 min. The coverslips were then washed with PBS and incubated with the blocking solution, 3% BSA, 0.2% Triton X-100 in PBS, for 1 h at RT. Subsequently, they were incubated for 3 h at RT with the primary antibodies dissolved in the blocking solution, washed with PBS, and incubated with the appropriate secondary antibodies, also dissolved in the blocking solution, for 1 h at RT. Coverslips were washed with PBS and mounted on a slide using Prolong Diamond Antifade Mountant (P36970; Molecular probes). Images were acquired using an inverted laser confocal microscope (LSM710; Zeiss) and analyzed with ImageJ software. The anti-VPS13A antibody from Sigma-Aldrich (HPA021662) was used at 1:75.

Immunoprecipitation and Western blot techniques

For co-immunoprecipitation experiments, the commercial GFP-Trap kit (GFP-Trap A beads; Chromoteck, gtak-20) was used following the manufacturer’s instructions.

HeLa cells were transfected with the plasmids encoding the different GFP-fused VPS13A fragments, and with the mCherry-SNX5 plasmid. 24 h after transfection, the cells were washed with PBS and suspended in lysis solution (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40), supplemented with protease inhibitors (P8340; Sigma-Aldrich) (1:100) and phosphatases (2.5 mM NaF, 0.2 mM Na³VO⁴). Cell lysates were centrifuged at 12,000g, 15 min, 4°C and the supernatant was transferred to a new tube. 5% of this supernatant was separated from the sample to obtain total cell lysates, whereas the rest was diluted in washing solution (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2.5 mM NaF, 0.2 mM Na³VO⁴, and protease inhibitors). The GFP-Trap bead suspension was added to the samples after washing, and the mixtures were incubated for 3 h at 4°C under continuously rotating conditions. Subsequently, the mixtures were centrifuged at 2,000g, 2 min, 4°C, after which the supernatant was removed. The GFP-Trap beads were washed five times with the washing solution and the samples were prepared for analysis by WB.

NuPage Tris-acetate 3–8% polyacrylamide gradient gels (EA0378; Invitrogen) were used for the analysis of the co-immunoprecipitation samples. The separated proteins were transferred to PVDF membranes (Immobilon-P polyvinylidene difluoride; IPVH00010; Millipore) by wet transfer. Subsequently, they were blocked in 5% milk powder in TBS-T (50 mM Tris, 150 mM NaCl, 0.5% Tween), for 1 h at RT. The membranes were then incubated with the corresponding primary antibody dissolved in 5% milk powder in TBS-T, at 4°C overnight. The next day, the membranes were washed with TBS-T for 1 h and incubated with the corresponding secondary antibody dissolved in 2% milk powder in TBS-T, at RT for 1 h. Finally, the membranes were washed again with TBS-T for 1 h, before incubation with the chemiluminescent substrate (Super Signal West Pico PLUS Chemiluminescent substrate; 34580; Thermo Fisher Scientific). The chemiluminescent signal was detected using photographic films (CURIX RP2 Plus films; Agfa). The anti-GFP antibody from Sigma-Aldrich (G1544) was used at 1:4,000. The anti-DsRed antibody from Clontech (632496) was used at 1:10,000.

Yeast protein extracts were prepared by the NaOH/trichloroacetic lysis method (Volland et al, 1994) and analyzed by SDS–PAGE and immunoblotting with anti-LexA (R990-25; Invitrogen) or anti-SNX5 (sc-515215; Santa Cruz Biotechnology). The antibodies were detected by enhanced chemiluminescence with ECLPlus reagent (GE Healthcare).