Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (soluble N-ethylmaleimide-sensitive factor-attachment proteins) families in Dictyostelium discoideum

Eur J Biochem. 2000 Apr;267(7):2062-70. doi: 10.1046/j.1432-1327.2000.01212.x.

Abstract

The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Dictyostelium / genetics
  • Dictyostelium / metabolism*
  • Humans
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Phylogeny
  • Plasmids
  • Protein Structure, Secondary
  • Proto-Oncogene Proteins c-myc / metabolism
  • Sequence Homology, Amino Acid
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Vesicular Transport Proteins*

Substances

  • Carrier Proteins
  • Membrane Proteins
  • Proto-Oncogene Proteins c-myc
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
  • Vesicular Transport Proteins