Lipid-Induced Conformational Switch Controls Fusion Activity of Longin Domain SNARE Ykt6

Abstract

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.

Structure and function of longin SNAREs

“…In fact, even in the presence of Varp, VAMP7 is still able to engage in SNAREcomplex formation in vitro (Schäfer et al, 2012). The strong autoinhibition of Ykt6 fits well with its ubiquitous cellular distribution, including a cytosolic pool, as this will prevent the interaction of cytosolic (closed) Ykt6 with non-cognate t-SNARE proteins (Wen et al, 2010).…”

“…Farnesylation is irreversible and occurs after Ykt6 translation. Surprisingly, this addition does not anchor Ykt6 into membranes, and X-ray crystallography experiments have demonstrated that, following farnesylation, Ykt6 switches from a semi-closed to a predominantly closed and fusion-inactive conformation (Pylypenko et al, 2008;Wen et al, 2010). This traps the lipid molecule inside a hydrophobic pocket that is formed by the interface between the longin domain and the coiled-coil domain, and thus inhibits membrane insertion of Ykt6.…”

“…This traps the lipid molecule inside a hydrophobic pocket that is formed by the interface between the longin domain and the coiled-coil domain, and thus inhibits membrane insertion of Ykt6. In this configuration, the coiled-coil domain folds as five separated domains (four α-helices and one β-sheet) that wrap around the longin domain (without modifying its structure) and interact with it through hydrophobic interactions and hydrogen bonds (Wen et al, 2010) (Fig. 4A).…”

“…In that study, the longin domain was required for the incorporation of Sec22 into coat protein complex II (COPII)-coated vesicles and for its export from the ER to the Golgi, further pointing to an important role of the longin domain in SNARE protein sorting. COPII assembles on the ER from three (Wen et al, 2010)] bound to a dodecylphosphocholine (DPC) molecule (cyan), which mimics the C-terminal farnesylation of the protein. The entire SNARE coiled-coil domain is visible in the structure.…”

Distinct Palmitoylation Events at the Amino-terminal Conserved Cysteines of Env7 Direct Its Stability, Localization, and Vacuolar Fusion Regulation in S. cerevisiae

“…Vac8, a phosphorylated and palmitoylated vacuolar membrane protein, is involved in efficient vacuole fusion, inheritance, and cytosol-to-vacuole trafficking (12,13,20). Ykt6 is a vacuolar SNARE that mediates its own protein palmitoylation during an early stage of homotypic vacuole fusion (21)(22)(23)(24)(25). The t-SNARE Vam3 is palmitoylated and functions with Vam7p in vacuolar protein trafficking and mediates docking/fusion of late transport intermediates with the vacuole (26,27), and Meh1/Ego1 is a palmitoylated vacuolar protein involved in regulating autophagy (28,29).…”