期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：29
DOI：10.1073/pnas.2203769119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Lipid transport proteins mediate lipid traffic between subcellular organelles by shielding them in hydrophobic cavities as they travel through the cytosol. Many such proteins are localized at membrane contacts, where they ferry lipids by protein modules that shuttle between the two membranes. However, a bridge-like mechanism has been suggested recently for proteins of the VPS13-ATG2 family. Here, we provide strong support for this model by visualizing a member of this family, VPS13C, in its natural context within cells, contacts between the endoplasmic reticulum (ER) and lysosomes. Its in situ architecture at these contacts is consistent with its functioning as a bridge that allows lipids to slide between closely apposed bilayers along a hydrophobic groove that runs along its entire length.
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.
