In situ architecture of the lipid transport protein VPS13C at ER–lysosome membrane contacts

Abstract

VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson’s disease–linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER–endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo–electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.

In silico modelling human VPS13 proteins associated with donor and target membranes suggests lipid transfer mechanisms

“…In their recent paper, Cai et al produced a more tightly wound model which spanned 29.3 nm and left both ends of the protein free of membrane. However, their measurements in cells expressing full-length VPS13C showed the distribution of distances between ER and lysosome spanned by VPS13C centred around 32.5 nm (Cai et al, 2022), the exact length of VPS13C in our models ( Video 1 ). Intriguingly, the density mapping of Cai et al (2022) showed that there was a substantial density on the C-terminal lysosomal side of the contact and little density where the protein was in contact via the chorein domain with the ER, suggesting a more stable conformation of the C terminus in their association with membranes than the N terminus.…”

“…The ER is relatively enriched in those lipid species compared to endo-lysosomes (Escribá et al, 2015;Guo et al, 2009;Horvath and Daum, 2013;Pogozheva et al, 2022;Reglinski et al, 2020;Tauchi-Sato et al, 2002;van Meer and de Kroon, 2011;van Meer et al, 2008). Despite this, the levels of phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and sphingomyelin increase in the lysosome in VPS13C knockout clones (Hancock-Cerutti et al, 2022). Intriguingly, this suggests that VPS13C-mediated lipid transfer can be compensated for by other mechanisms.…”

“…This is despite in vitro experiments which dispense entirely with the VPS13C terminal domains to accomplish lipid transfer between model membranes. A stronger association of the VPS13 C terminus to target membranes compared to the N terminal Chorein domain to donor membranes is borne out by recent studies which measured the average density of the intramembrane bridges formed by VPS13C at contacts between the ER and lysosomes (Cai et al, 2022). In their recent paper, Cai et al produced a more tightly wound model which spanned 29.3 nm and left both ends of the protein free of membrane.…”

“…VPS13C also localises at ER-lipid droplet, at ER-endo-lysosome jucntions (Kumar et al, 2018). Here, it is thought to interact with the GTPase Rab7 (McCray et al, 2010, Hancock-Cerutti et al 2022. Tethered at Golgiendosome contact sites are VPS13B proteins (Seifert et al, 2011), which were shown to bind Rab6 (Seifert et al, 2015).…”

“…An N-terminal hydrophobic groove was previously found to solubilise tens of lipid fatty acid moieties (Adlakha et al, 2022; Kolakowski et al, 2021; Kumar et al, 2018) and extends across the bulk length of the protein in the form of β-strand repeats and their laterally connected α-helices or unstructured peptides, giving rise to the recently named repeating β-groove (RBG) domain (Neuman et al, 2022). Recognition motifs and protein-binding domains decorate VPS13s along their lipid transferring channels, which span across intermembrane contact sites that may vary between ∼10-40 nm in distance (Cai et al, 2022; Neuman et al, 2022). At the C-terminus, a VPS13 Adaptor Binding (VAB) domain interacts with a Pro-x-Pro motif to mediate protein interactions (Bean et al, 2018), in proximity to a pleckstrin homology (PH)-like domain (Fidler et al, 2016) which faces the cytosol.…”