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

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 α/β and γ subfamilies, SNAPs act as adapters between N-etbylmaleimide-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 α- and γ-SNAP, respectively. The α-/γ-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of γ-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the γ subtype. Altogether, these results suggest a specific role for γ-SNAP, distinct from that of α-SNAP.