Abstract
The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices. One helix contains a polybasic region (PBR) composed of Arg-164, Arg-168, Lys-172, Lys-175, Arg-179, and Lys-186. Polybasic regions are often associated with nonspecific binding to acidic phospholipids including phosphoinositides. Although the PX (phox homology) domain alone binds PI3P, we theorized that the Vam7 PBR could bind to additional acidic phospholipids enriched at fusion sites. Mutating each of the basic residues in the PBR to an alanine (Vam7-6A) led to attenuated vacuole fusion. The defective fusion of Vam7-6A was due in part to inefficient association with its cognate SNAREs and HOPS, yet the overall vacuole association of Vam7-6A was similar to wild type. Experiments testing the binding of Vam7 to specific signaling lipids showed that mutating the PBR to alanines augmented binding to PI3P. The increased binding to PI3P by Vam7-6A likely contributed to the observed wild type levels of vacuole association, whereas protein-protein interactions were diminished. PI3P binding was inhibited when the PX domain mutant Y42A was introduced into Vam7-6A to make Vam7-7A. Thus the Vam7 PBR affects PI3P binding by the PX domain and in turn affects binding to SNAREs and HOPS to support efficient fusion.
Highlights
The study of membrane lipid composition is vital to gaining a complete understanding of membrane trafficking and fusion
We report that the polybasic region (PBR) affects phosphatidylinositol 3-phosphate (PI3P) binding and protein interactions with SNAREs and HOPS needed for efficient vacuole fusion
We found that wild type Vam7 robustly stimulated fusion as observed previously, whereas Vam7-6A was impaired (Fig. 1B)
Summary
The study of membrane lipid composition is vital to gaining a complete understanding of membrane trafficking and fusion. Vam7 PBR and Vacuole Fusion tase Pah1 to facilitate the transfer of Sec18 from PA-enriched membrane domains to cis-SNARE complexes4 [19, 20]. We report that the PBR affects PI3P binding and protein interactions with SNAREs and HOPS needed for efficient vacuole fusion.
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