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.
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.
Weidenhaupt
M
M
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR314 CNRS) and Laboratoire de Chimie des Protéines, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Bruckert
F
F
Louwagie
M
M
Garin
J
J
Satre
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids; D017433:Protein Structure, Secondary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins