Person:

Klemm, Robin

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER–ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.