Molecular mechanism of membrane docking by the Vam7p PX domain

Stephanie A. Lee, James Kovacs, Robert V. Stahelin, Matthew L. Cheever, Michael Overduin, Thanuja Gangi Setty, Christopher G. Burd, Wonhwa Cho, Tatiana G. Kutateladze

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

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 andquantitativeanalysisofitsinteractionswithlipidsandmicelles. 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 Val70, Leu 71, and Trp75 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.

Original languageEnglish (US)
Pages (from-to)37091-37101
Number of pages11
JournalJournal of Biological Chemistry
Volume281
Issue number48
DOIs
StatePublished - Dec 1 2006

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Lee, S. A., Kovacs, J., Stahelin, R. V., Cheever, M. L., Overduin, M., Setty, T. G., Burd, C. G., Cho, W., & Kutateladze, T. G. (2006). Molecular mechanism of membrane docking by the Vam7p PX domain. Journal of Biological Chemistry, 281(48), 37091-37101. https://doi.org/10.1074/jbc.M608610200