Molecular mechanism of membrane docking by the Vam7p PX domain.
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.
Lee
Stephanie A
SA
Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.
Kovacs
James
J
Stahelin
Robert V
RV
Cheever
Matthew L
ML
Overduin
Michael
M
Setty
Thanuja Gangi
TG
Burd
Christopher G
CG
Cho
Wonhwa
W
Kutateladze
Tatiana G
TG
D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050767:Qc-SNARE Proteins / Q000737:chemistry / Q000502:physiology*; D050600:SNARE Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D055672:Static Electricity; D050825:Synaptosomal-Associated Protein 25; D014364:Tryptophan / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D008055:Lipids; D008823:Micelles; D050767:Qc-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; C494205:VAM7 protein, S cerevisiae; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014364:Tryptophan; D066246:ErbB Receptors; D007930:Leucine; D014633:Valine