Molecular mechanism of membrane docking by the Vam7p PX domain

被引:40
作者
Lee, Stephanie A.
Kovacs, James
Stahelin, Robert V.
Cheever, Matthew L.
Overduin, Michael
Setty, Thanuja Gangi
Burd, Christopher G.
Cho, Wonhwa
Kutateladze, Tatiana G.
机构
[1] Univ Colorado, Hlth Sci Ctr, Dept Pharmacol, Aurora, CO 80045 USA
[2] Univ Illinois, Dept Chem, Chicago, IL 60607 USA
[3] Indiana Univ, Sch Med, Dept Biochem & Mol Biol, South Bend, IN 46615 USA
[4] Univ Notre Dame, Walther Ctr Canc Res, Dept Chem & Biochem, South Bend, IN 46617 USA
[5] Univ N Carolina, Sch Med, Dept Pharmacol, Chapel Hill, NC 27599 USA
[6] Univ Birmingham, CR UK Inst Canc Studies, Birmingham B15 2T, W Midlands, England
[7] Univ Penn, Sch Med, Dept Cell & Dev Biol, Philadelphia, PA 19104 USA
基金
英国生物技术与生命科学研究理事会;
关键词
D O I
10.1074/jbc.M608610200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX domain that specifically recognizes PtdIns(3) P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns(3) P recognition that is facilitated by electrostatic interactions and accompanying hydrophobic insertion. Both the hydrophobic and electrostatic components enhance the Vam7p PX domain association with PtdIns(3) P-containing membranes. The inserting Val(70), Leu(71), and Trp(75) residues located next to the PtdIns(3) P binding pocket are surrounded by a basic patch, which is involved in nonspecific electrostatic contacts with acidic lipids, such as PtdSer. Substitution of the insertion residues significantly reduces the binding and penetrating power of the Vam7p PX domain and leads to cytoplasmic redistribution of the EGFP-tagged protein. The affinities of the PX domain for PtdIns(3) P and other lipids reveal a remarkable synergy within the multivalent complex that stably anchors Vam7p at the vacuolar membrane.
引用
收藏
页码:37091 / 37101
页数:11
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