While most SNAREs are permanently anchored to membranes by their transmembrane domains, the dually lipidated SNARE Ykt6 is found both on intracellular membranes and in the cytosol. The cytosolic Ykt6 is inactive due to the autoinhibition of the SNARE core by its longin domain, although the molecular basis of this inhibition is unknown. Here, we demonstrate that unlipidated Ykt6 adopts multiple conformations, with a small population in the closed state. The structure of Ykt6 in complex with a fatty acid suggests that, upon farnesylation, the Ykt6 SNARE core forms four alpha helices that wrap around the longin domain, forming a dominantly closed conformation. The fatty acid, buried in a hydrophobic groove formed between the longin domain and its SNARE core, is essential for maintaining the autoinhibited conformation of Ykt6. Our study reveals that the posttranslationally attached farnesyl group can actively regulate Ykt6 fusion activity in addition to its anticipated membrane-anchoring role.

VAMP4 is enriched in the trans-Golgi network (TGN) and functions in traffic from the early and recycling endosomes to the TGN, but its trafficking itinerary is unknown. Cells stably expressing TGN-enriched VAMP4 C-terminally-tagged with EGFP (VAMP4-EGFP) are able to internalize and transport EGFP antibody efficiently to the TGN, suggesting that VAMP4-EGFP cycles between the cell surface and the TGN. The N-terminal extension of VAMP4 endows a chimeric VAMP5 with the ability to cycle from the surface to the TGN. Detailed time-course analysis of EGFP antibody transport to the TGN as well as pharmacological and thermal perturbation experiments suggest that VAMP4-EGFP is endocytosed by clathrin-dependent pathways and is delivered to the sorting and then recycling endosomes. This is followed by a direct transport to the TGN, without going through the late endosome. The di-Leu motif of the TGN-targeting signal is important for internalization, whereas the acidic cluster is crucial for efficient delivery of internalized antibody from the endosome to the TGN. These results suggest that the TGN-targeting signal of VAMP4 mediates the efficient recycling of VAMP4 from the cell surface to the TGN via the sorting and recycling endosomes, thus conferring steady-state enrichment of VAMP4 at the TGN.

Searching and evaluating the Human Protein Atlas for transmembrane proteins enabled us to identify an integral membrane protein, TMEM115, that is enriched in the Golgi complex. Biochemical and cell biological analysis suggested that TMEM115 has four candidate transmembrane domains located in the N-terminal region. Both the N- and C-terminal domains are oriented towards the cytoplasm. Immunofluorescence analysis supports that TMEM115 is enriched in the Golgi cisternae. Functionally, TMEM115 knockdown or overexpression delays Brefeldin-A-induced Golgi-to-ER retrograde transport, phenocopying cells with mutations or silencing of the conserved oligomeric Golgi (COG) complex. Co-immunoprecipitation and in vitro binding experiments reveals that TMEM115 interacts with the COG complex, and might self-interact to form dimers or oligomers. A short region (residues 206–229) immediately to the C-terminal side of the fourth transmembrane domain is both necessary and sufficient for Golgi targeting. Knockdown of TMEM115 also reduces the binding of the lectins peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA), suggesting an altered O-linked glycosylation profile. These results establish that TMEM115 is an integral membrane protein of the Golgi stack regulating Golgi-to-ER retrograde transport and is likely to be part of the machinery of the COG complex.