VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites

Abstract

Mutations in the human VPS13 genes are responsible for neurodevelopmental and neurodegenerative disorders including chorea acanthocytosis (VPS13A) and Parkinson's disease (VPS13C). The mechanisms of these diseases are unknown. Genetic studies in yeast hinted that Vps13 may have a role in lipid exchange between organelles. In this study, we show that the N-terminal portion of VPS13 is tubular, with a hydrophobic cavity that can solubilize and transport glycerolipids between membranes. We also show that human VPS13A and VPS13C bind to the ER, tethering it to mitochondria (VPS13A), to late endosome/lysosomes (VPS13C), and to lipid droplets (both VPS13A and VPS13C). These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggest lipid transport roles for these proteins as well.

Figure 1.

VPS13A localizes at ER–mitochondria and ER–lipid droplet contact sites. (A) Cos-7 cell expressing VPS13A^Halo, GFP-Sec61β, and mito-BFP showing overlap of VPS13A fluorescence with both mitochondria and ER. Bar, 10 µm. (B) High magnifications of the region indicated by white squares in A showing precise localization of VPS13A at sites of contact between ER and mitochondria. Bar, 2 µm. (C) Fluorescence intensity for the three indicated channels along a line either tracing the long axis of a mitochondrion (top) or ER tubules (bottom). Lines used for the plots are indicated by dashed lines in the merged image in B. (D) A Cos-7 cell expressing VPS13A^Halo and GFP-Sec61β shows two patterns of VPS13A fluorescence both overlapping with the ER: dots (doughnuts; a) and elongated structures (b), shown at high magnification in the bottom panels. Bar, 10 µm; bottom panels, 3 × 3 µm. (E) Live staining of neutral lipids with BODIPY 493/503 in a Cos-7 cell expressing VPS13A^Halo showing the presence of VPS13A around lipid droplets (insets). Bar, 5 µm; insets, 1.5 × 1.5 µm. (F) Correlative light EM of a Cos-7 cell expressing VPS13A^mChweey and GFP-Sec61β. Fluorescent images of three lipid droplets (right) and corresponding cell region shown by EM (left) demonstrate abundant presence of ER around the lipid droplets. Bars: 0.5 µm (left); 1 µm (right).

Figure 2.

VPS13C localizes at ER–endosome contacts. (A) HeLa cells expressing VPS13A^mCherry and VPS13C^mClover3 show minimal overlap between the VPS13A and VPS13C signals specifically found on small round structures likely to be lipid droplets (arrowheads in insets). Bar, 10 µm; insets, 10 × 10 µm. (B) Cos-7 cells expressing VPS13C^mClover3 or RFP-Sec61β and loaded overnight with dextran Alexa Fluor 647 show VPS13C signal surrounding dextran-positive puncta, which are often surrounded by ER. Arrowheads point to a dextran-negative vesicle enwrapped by ER, likely to be a lipid droplet. Insets, 2.73 × 2.73 µm. (C) The majority of full-length VPS13C^mClover3 localizes on Rab7a-RFP–positive structures in Cos7 cells. (D) Cos-7 cells expressing VPS13C^mClover3 loaded overnight with 100 µM oleate and then fixed and stained with LipidTox. The figure shows VPS13C signal surrounding LipidTox-labeled spots (lipid droplets at high magnification in the inset) and more intense VPS13C signal around larger vesicular structures likely to be endolysosomal vesicles. Insets, 2.4 × 2.4 µm. (E–H) CRISPR/Cas9-edited HeLa cells expressing VPS13A or VPS13C tagged with 2×HA epitopes (endo-HA-VPS13A and endo-HA-VPS13C) at their genomic loci. Cells were transfected with either mito-BFP or EGFP-Rab7a, fixed, and immunostained with anti-HA antibodies (magenta). A large fraction of endo-HA-VPS13A–immunoreactive puncta are localized on mitochondria (E) and not on Rab7-positive vesicles (F). Conversely, a large fraction of endo-HA-VPS13C–immunoreactive puncta are localized on Rab7-positive vesicles (G) and not on mitochondria (H). Bars, 2 µM (B–H).

Figure 3.

The predicted FFAT motif in VPS13A and VPS13C tethers them to the ER. (A) Schematic cartoon of the putative domain architecture of human VPS13A and VPS13C. The striped region in VPS13C represent an ∼500-residue insertion likely arisen from the internal duplication of the region just upstream of it. The dashed line in the DH_L domain defines the start of the ATG homology region (see also Fig. 5 A). (B) Cos-7 cells expressing VPS13A_1–1,372-EGFP or VPS13C_1–1,390-EGFP and mCherry-VAPB show robust enrichment of VPS13A and VPS13C N-terminal fragments on the ER. (C) Corresponding constructs bearing a mutant FFAT motif (VPS13A_1–1,372^FFLI and VPS13C_1–1,390^YFSL) expressed in Cos-7 cells are instead cytosolic. Bar, 5 µm. (D) In VAPB-overexpressing cells, VPS13C populates VAP-enriched ER domains that completely surround endocytic vesicles filled with dextran. Bar, 2 µm. All amino acid numbers refer to human proteins.

Figure 4.

Binding regions for mitochondria or late endosomes lie in the C-terminal half of VPS13 proteins. (A) Cos-7 cells expressing mClover3-VPS13C_{2,415–3,309} and Rab7a-mCherry show localization of VPS13C signal on Rab7a-positive vesicles. (B) EGFP-tagged VPS13A_{1,864–2,560} has a cytosolic localization with no accumulation at the surface of mitochondria, which appear dark in the EGFP channel due to the exclusion of cytosol. (C) Cos-7 cells coexpressing mCherry-tagged VPS13A_{2,751–3,174} and mito-BFP were incubated with BODIPY 493/503 for lipid droplet (LD) labeling. VPS13A_{2,751–3,174} fluorescence clearly outlines mitochondria and lipid droplets as shown in high-magnification panels below (2.33 × 2.33 µm). (D) Cos-7 cells expressing mClover3-VPS13C_{3,310–3,753} and labeled with BODIPY 493/503 show VPS13C_{3,310–3,753} signal surrounding BODIPY-positive puncta (lipid droplets) but not mitochondria as shown in high-magnification panels below (2.33 × 2.33 µm). (E) Cos-7 cells coexpressing the mitochondrial marker mito-BFP and an mCherry-tagged VPS13A fragment (VPS13A_{2,953–3,027}) corresponding with the ATG homology region show that this fragment surrounds lipid droplets (labeled with BODIPY 493/503) but not mitochondria. Regions indicated by the white squares are shown in high magnification on the right (2.33 × 2.33 µm). (F) Representation of the predicted helical arrangement of residues 2,993–3,010 of VPS13A generated via the HeliQuest tool (heliquest.ipmc.cnrs.fr; Gautier et al., 2008). An mCherry-tagged VPS13A_{2,953–3,027} construct bearing a single amino acid substitution (V3006Q; indicated by an asterisk) in the hydrophobic face of the predicted amphipathic helix had a cytosolic localization. All micrographs were acquired by live-cell imaging, and images shown are representative of at least two independent experiments. Bars, 5 µm. All amino acid numbers refer to human proteins.

Figure 5.

N-terminal portions of Vps13 solubilize and transport lipids. (A) Schematic of VPS13 domain architecture. The crystallized fragment from C. thermophilum is indicated. (B) LC/MS/MS analysis of lipids that copurify with Vps13α showed binding to glycerolipids, with a slight preference for PC. Cellular abundance of these glycerolipids is indicated (Lees et al., 2017). Sphingomyelin represented 1% of bound lipids. No sterol, di-, or triacylglycerides were detected. (C) Vps13α was incubated with NBD-tagged lipid including NBD-ceramide (CM) and then examined by native PAGE. Phospholipids, which were visualized by their fluorescence, comigrated with protein, visualized by Coomassie staining. (D) Donor liposomes (25 µM) containing fluorescent lipids (2% NBD-PS, 2% NBD-PE, 5% DGS-NTA, 61% DOPC, and 30% PE) were mixed 1:1 with acceptor liposomes (25 µM: 65% DOPC, 30% PE, and 5% PI(4,5)P₂) in the presence or absence of Vps13α_tethered (0.125 µM). This construct tethers the Vps13α fragment between acceptor and donor liposomes via a PI(4,5)P₂-specific PH domain and a C-terminal hexahistidine sequence. The assay monitors the increase in NBD-PS fluorescence after lipid transfer from donor liposomes, where NBD fluorescence is quenched via FRET with Rhodamine-PE, to acceptor liposomes. The fluorescence increase observed is consistent with lipid transfer at a rate similar to that for the lipid transport domain of Extended-Synaptotagmin1 (0.125 µM), a previously validated glycerolipid transporter (Saheki et al., 2016; Yu et al., 2016; Bian et al., 2018). The Extended-Synaptotagmin1 lipid-transfer domain was tethered between liposomes analagously to Vps13α via a hexahistidine tag and a PH domain. The fluorescence increase is much smaller when only donor but not acceptor liposomes are present. The small but still significant fluorescence increase under these conditions is due to lipid extraction by Vps13α_tethered and would not be expected in the case of Vps13α-mediated fusion of hemifusion between donor liposomes. The dithionate and turbidity assays (Fig. S5, A and B) rule out Vps13α-mediated fusion and/or hemifusion between liposomes. The experiments were performed in triplicate. SD is indicated. (E) Ribbons diagram for Vps13_crystal colored from blue (N terminus) to red (C terminus). Predicted helix α2 (not depicted) connects residue 94 to residue 133 (stars). (F) Surface representation with carbon atoms shown in white, oxygens in red, and nitrogens in blue. View as in E (left) and rotated (right). The concave face of the scoop is entirely hydrophobic. (G) Vps13_crystal comigrates with NBD-labeled glycerolipids. (H) Vps13_crystal, Vps13_1–729, and Vps13_1–1,390 were quantitated against BSA standards using SDS-PAGE. Equimolar quantities of these proteins were incubated with fluorescent glycerolipids (NBD-PS and NBD-PE) and then run on a native gel. Vps13_1–729 and Vps13_1–1,390, which are about twice and four times as large as Vps13_crystal, bound approximately twice and 2.5× as many fluorescent lipids/protein molecule, respectively. Each experiment was performed in triplicate. SD is indicated.

Figure 6.

Diagram depicting sites of action of VPS13A and VPS13C. Schematic of VPS13A and VPS13C localization and domains responsible for such localization. LD, lipid droplet; LE, late endosome; Lys, lysosome.