Stringent 3Q·1R composition of the SNARE 0-layer can be bypassed for fusion by compensatory SNARE mutation or by lipid bilayer modification

Rutilio A. Fratti, Kevin M. Collins, Christopher M. Hickey, William Wickner

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

SNARE proteins form bundles of four α-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q·1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7Q283R, yet Vam7Q283R is preferred by vacuoles with Nyv1R191Q. Rotation of the position of the arginine in the 0-layer increases the Km for Vam7p but does not affect the maximal rate of fusion. Vam7Q283R forms stable 2Q·2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion.

Original languageEnglish (US)
Pages (from-to)14861-14867
Number of pages7
JournalJournal of Biological Chemistry
Volume282
Issue number20
DOIs
StatePublished - May 18 2007
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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