Functional Reconstitution of Intracellular Vesicle Fusion Using Purified SNAREs and Sec1/Munc18 (SM) Proteins

Methods Mol Biol. 2019:1860:237-249. doi: 10.1007/978-1-4939-8760-3_15.

Abstract

The fusion of intracellular vesicles with target membranes is mediated by two classes of conserved molecules-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAP receptors or SNAREs) and Sec1/Munc18 (SM) proteins. A conserved function of SM proteins is to recognize their cognate trans-SNARE complexes and accelerate fusion kinetics. Here, we describe a physiologically relevant reconstitution system in which macromolecular crowding agents are included to recapitulate the crowded intracellular environment. Through this system, we elucidate the molecular mechanisms by which SNAREs and SM proteins drive vesicle fusion.

Keywords: Content mixing; Lipid mixing; Macromolecular crowding; Membrane fusion; Reconstitution; SM protein; SNARE; Vesicle fusion.

Publication types

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

MeSH terms

  • Cytoplasmic Vesicles / metabolism*
  • Exocytosis
  • Glucose Transporter Type 4 / metabolism
  • Kinetics
  • Membrane Fusion*
  • Munc18 Proteins / isolation & purification
  • Munc18 Proteins / metabolism*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • SNARE Proteins / isolation & purification
  • SNARE Proteins / metabolism*

Substances

  • Glucose Transporter Type 4
  • Munc18 Proteins
  • Recombinant Proteins
  • SNARE Proteins