Lee Stephanie A, Kovacs James, Stahelin Robert V, Cheever Matthew L, Overduin Michael, Setty Thanuja Gangi, Burd Christopher G, Cho Wonhwa, Kutateladze Tatiana G
Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.
J Biol Chem. 2006 Dec 1;281(48):37091-101. doi: 10.1074/jbc.M608610200. Epub 2006 Sep 19.
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.
Vam7p t-SNARE是液泡融合机制的一个重要组成部分，介导酵母中的膜运输和蛋白质分选。Vam7p通过其N端的PX结构域被招募到液泡，该结构域能特异性识别双层膜中的磷脂酰肌醇-3-磷酸（PtdIns(3)P），然而膜锚定的确切机制仍不清楚。在此，我们基于对其与脂质和胶束相互作用的结构和定量分析，描述了Vam7p PX结构域靶向和穿透膜的分子基础。我们通过核磁共振（NMR）进行的体外结合测量、单层表面张力实验和诱变得到的结果揭示了一种多价膜对接机制，该机制涉及特定的PtdIns(3)P识别，静电相互作用和伴随的疏水插入促进了这种识别。疏水成分和静电成分都增强了Vam7p PX结构域与含PtdIns(3)P膜的结合。位于PtdIns(3)P结合口袋旁边的插入缬氨酸（Val(70)）、亮氨酸（Leu(71)）和色氨酸（Trp(75)）残基被一个碱性区域包围，该区域参与与酸性脂质（如磷脂酰丝氨酸（PtdSer））的非特异性静电接触。插入残基的取代显著降低了Vam7p PX结构域的结合和穿透能力，并导致绿色荧光蛋白（EGFP）标记蛋白的细胞质重新分布。PX结构域对PtdIns(3)P和其他脂质的亲和力揭示了多价复合物内显著的协同作用，该协同作用将Vam7p稳定地锚定在液泡膜上。
