Role of two nucleotide-binding regions in an N-ethylmaleimide-sensitive factor involved in vesicle-mediated protein transport.
N-Ethylmaleimide-sensitive factor (NSF) was originally characterized as the protein that restores in vitro protein transport activity of the Golgi membranes inactivated by N-ethylmaleimide. This protein has two homologous regions, each containing the consensus sequence for nucleotide binding, and possesses ATPase activity. To investigate the role of the two nucleotide-binding regions in NSF, we have replaced two lysyl residues (Lys-274 and Lys-557) located in the consensus sequences with glutamine or methionine via site-directed mutagenesis. The mutant NSF proteins in which Lys-274 was replaced had no ability to restore protein transport between N-ethylmaleimide-treated Golgi membranes and, in addition, inhibited the protein transport assay using normal Golgi membranes. This inhibition, which was eliminated by N-ethylmaleimide treatment, was caused by the impairment of the function of donor Golgi membranes. Although wild-type NSF showed a protective effect against inhibition by the Lys-274 mutant NSF protein when added at the start of the protein transport assay, its protective effect diminished after the time for the formation of transport vesicles had passed. These results support the idea that NSF incorporated into transport vesicles is nonexchangeable for exogenously added NSF. On the other hand, the mutant proteins in which Lys-557 was replaced had slight but significant protein transport activity. They did not inhibit the protein transport assay using normal Golgi membranes. The mutant NSF proteins in which Lys-274 and Lys-557 were replaced had about 20 and 25% of the ATPase activities of wild-type NSF, respectively. Their ATPase activities were sensitive to N-ethylmaleimide and dependent on their protein concentrations, as observed in wild-type NSF.
Sumida
M
M
Institute of Scientific and Industrial Research, Osaka University, Japan.
Hong
R M
RM
Tagaya
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D052066:N-Ethylmaleimide-Sensitive Proteins; D009711:Nucleotides / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins; D008239:Lysine; D005033:Ethylmaleimide