Identification of the Dictyostelium discoideum homolog of the N-ethylmaleimide-sensitive fusion protein.

The N-ethylmaleimide-sensitive fusion protein (NSF) is required for vesicular membrane fusion in multiple cellular functions. We have cloned a cDNA encoding the Dictyostelium discoideum homolog of the NSF protein. This cDNA hybridizes with a single fragment in Southern blots suggesting that NSF is encoded by a single gene in the amoeba. It is expressed constitutively during vegetative growth and throughout the differentiation cycle. The encoded gene product comprises 738 aa with a predicted molecular mass of 82 kDa. It shows the characteristic three-domain structure of NSF proteins. A more divergent amino-terminal part is followed by two highly conserved ATP-binding domains featuring Walker A and B signature sequences. The D. discoideum protein presents an overall aa sequence identity of 44% when compared to known NSF homologs. The monoclonal antibody 2E5 directed against Cricetellus griseus NSF recognizes a protein with a molecular weight of approx. 80 000 in a D. discoideum crude extract and the recombinant D. discoideum His6-NSF expressed in Escherichia coli.