Analysis of the Role of Sec3 in SNARE Assembly and Membrane Fusion

Methods Mol Biol. 2019:1860:175-189. doi: 10.1007/978-1-4939-8760-3_10.

Abstract

Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and tightly regulated by a variety of factors. The exocyst complex is one of the multi-subunit tethering complexes and functions in the tethering of the secretory vesicles to the plasma membrane. We have found that the yeast Sec3, a subunit of the exocyst, binds to the t-SNARE protein Sso2 and promotes its interaction with another t-SNARE protein, Sec9. Here, we describe the structural analysis and in vitro membrane fusion assays, by which we found that Sec3 binding leads to a conformational change within Sso2, and facilitates SNARE assembly and the membrane fusion.

Keywords: Liposome; Membrane fusion; Sec3; Sec9; Soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Sso2; Structure analysis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Motifs / genetics
  • Liposomes / chemistry
  • Liposomes / metabolism
  • Membrane Fusion*
  • Models, Molecular*
  • Mutagenesis
  • Protein Binding
  • Qa-SNARE Proteins / chemistry
  • Qa-SNARE Proteins / genetics
  • Qa-SNARE Proteins / isolation & purification
  • Qa-SNARE Proteins / metabolism*
  • Qc-SNARE Proteins / chemistry
  • Qc-SNARE Proteins / isolation & purification
  • Qc-SNARE Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Liposomes
  • Qa-SNARE Proteins
  • Qc-SNARE Proteins
  • Recombinant Proteins
  • SEC3 protein, S cerevisiae
  • SEC9 protein, S cerevisiae
  • SSO2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins