Activities of mutant Sar1 proteins in guanine nucleotide binding, GTP hydrolysis, and cell-free transport from the endoplasmic reticulum to the Golgi apparatus.
Sar1p belongs to a unique subfamily of the small GTPase superfamily and is essential for the formation of vesicles that transport proteins from the endoplasmic reticulum to the Golgi apparatus. We have obtained mutants of the yeast SAR1 gene, which show several different phenotypes in cell growth and protein transport [Nakano, A. , Otsuka, H., Yamagishi, M., Yamamoto, E., Kimura, K., Nishikawa, S., and Oka, T. (1994) J. Biochem. 116, 243-247; Yamanushi, T., Hirata, A., Oka, T., and Nakano, A. (1996) ibid. 120, 452-458]. In this study, we have purified five mutant Sar1 proteins using an Escherichia coli expression system and characterized their biochemical properties in detail. Three of them prefer GDP binding to GTP binding and are thus regarded as GDP-form mutants, and one is insensitive to the GTPase-activating protein and is almost fixed in the GTP-bound state. The GDP mutants are defective in vesicle formation in vitro, whereas the GTP mutant can drive vesicle formation but not the overall transport to the Golgi. These mutants will be useful for further understanding of the regulation of the GTPase cycle of Sar1p.
Saito
Y
Y
Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama, 351-0198, Japan.
Kimura
K
K
Oka
T
T
Nakano
A
A
D019943:Amino Acid Substitution; D001665:Binding Sites; D002474:Cell-Free System; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006056:Golgi Apparatus / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D020559:Monomeric GTP-Binding Proteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae