Phosphoinositides Function Asymmetrically for Membrane Fusion, Promoting Tethering and 3Q-SNARE Subcomplex Assembly

Hao Xu,William Wickner

doi:10.1074/jbc.m110.183111

Abstract

Phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are essential for rapid SNARE-dependent fusion of yeast vacuoles and other organelles. These phosphoinositides also regulate the fusion of reconstituted proteoliposomes. The reconstituted reaction allows separate analysis of phosphoinositide-responsive subreactions: fusion with SNAREs alone, with the addition of the HOPS tethering factor, and with the further addition of the SNARE complex disassembly chaperones Sec17p and Sec18p. Using assays of membrane tethering, trans-SNARE pairing, and lipid mixing, we found that PI(3)P and PI(4,5)P(2) have distinct functions that are asymmetric with respect to R-SNARE (Nyv1p) and the 3Q-SNAREs (Vam3p, Vti1p, and Vam7p). Fusion reactions with the Q-SNAREs and R-SNARE on separate membranes showed that PI(3)P has two distinct functions. PI(3)P on Q-SNARE proteoliposomes promoted Vam7p binding and association with the other two Q-SNAREs. PI(3)P on R-SNARE proteoliposomes was recognized by the PX domain of Vam7p on Q-SNARE proteoliposomes to promote tethering, although this function could be supplanted by the tethering activity of HOPS. PI(4,5)P(2) stimulated fusion when it was on R-SNARE proteoliposomes, apposed to Q-SNARE proteoliposomes bearing PI(3)P. These functions are essential for the phosphoinositide-dependent synergy between HOPS and Sec17p/Sec18p in promoting rapid fusion.

Highlights

Like other fusion events, vacuole fusion requires cognate SNARE proteins that form coiled-coil four-helix bundles either on the same membrane or on apposed membranes
With all SNAREs present on both fusion partners and in the presence of HOPS, Sec17p, and Sec18p, it is difficult to distinguish the individual functions of each phosphoinositide and whether each acts in cis or in trans to the 3Q-SNAREs and R-SNARE, which engage to form functional trans-SNARE complexes [23]
The power of the reconstitution approach, in which each relevant protein or lipid can be present or absent on each fusion partner, allows exploration of the roles and asymmetries of PI[3]P and PI[4,5]P2 functions. Are both phosphoinositides needed on each fusion partner, and is this requirement asymmetric with respect to R-SNARE and the 3Q-SNAREs that combine in trans to form SNARE complexes? What are the relationships between the HOPS and Sec17p/Sec18p chaperones and phosphoinositides? We report studies that began with SNARE liposomes in the absence of HOPS, Sec17p, or Sec18p

Summary

Introduction

Vacuole fusion requires cognate SNARE proteins that form coiled-coil four-helix bundles either on the same membrane or on apposed membranes. HOPS stimulated fusion in the absence of phosphoinositides (Fig. 4, bars 5 and 6; and supplemental Fig. 3C), suggesting that HOPS does not need PI[3]P or PI[4,5]P2 to tether SNARE-bearing proteoliposomes.

Results

Conclusion