Abstract
Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.
Highlights
N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) family members drive membrane fusion by the formation of a trans-SNARE complex consisting of specific v- and t-SNAREs present at vesicle (v) and target (t) membranes
Within the hydrophobic core of the bundle X-ray crystallography [2,3] revealed an unusual central hydrophilic layer composed of three glutamines (Q) and one arginine (R) residue, which led to the classification of Q- and R-SNAREs, respectively [4]
This interaction is exemplified in yeast, where the release of Ykt6 from endosomal membranes into the cytoplasm depends on a functional Longin domain and an intramolecular interaction with its SNARE domain to fold into a soluble, closed conformation [6]
Summary
N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) family members drive membrane fusion by the formation of a trans-SNARE complex consisting of specific v- and t-SNAREs present at vesicle (v) and target (t) membranes. The SNARE motifs are 60–70 amino acid residues long [1] and contain repeated heptad patterns of hydrophobic residues They assemble into parallel four-helix bundles stabilized by a hydrophobic helix that faces the bundle’s core. Membrane localization depends on the intramolecular interaction of the N-terminal Longin and C-terminal SNARE domains and the presence of a farnesylation and reversible palmitoylation within a CCAIM/CAAX motif at the C-terminus [5]. This interaction is exemplified in yeast, where the release of Ykt from endosomal membranes into the cytoplasm depends on a functional Longin domain and an intramolecular interaction with its SNARE domain to fold into a soluble, closed conformation [6]. A recent study identified a new geranylgeranyl transferase that plays an essential role for membrane-anchored Ykt in proper Golgi function [7]
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