ACTIVITIES OF MUTANT SAR1 PROTEINS IN GUANINE-NUCLEOTIDE-BINDING, GTPHYDROLYSIS, AND CELL-FREE TRANSPORT FROM THE ENDOPLASMIC-RETICULUM TOTHE GOLGI-APPARATUS

Sar1p belongs to a unique subfamily of the small GTPase superfamily an d is essential for the formation of vesicles that transport proteins f rom the endoplasmic reticulum to the Golgi apparatus. We have obtained mutants of the yeast SARI gene, which show several different phenotyp es in cell growth and protein transport [Nakano, A., Otsuka, H., Yamag ishi, M,, Yamamoto, E,, Kimura, K,, Nishikawa, S,, and Oka, T, (1994) J, Biochem. 116, 243-247; Yamanushi, T., Hirata, A., Oka, T,, and Naka no, A. (1996) ibid. 120, 452-458], In this study, we have purified fiv e mutant Sari proteins using an Escherichia coli expression system and characterized their biochemical properties in detail. Three of them p refer GDP binding to GTP binding and are thus regarded as GDP-form mut ants, and one is insensitive to the GTPase-activating protein and is a lmost fixed in the GTP-bound state, The GDP mutants are defective in v esicle formation in vitro, whereas the GTP mutant can drive vesicle fo rmation but not the overall transport to the Golgi. These mutants will be useful for further understanding of the regulation of the GTPase c ycle of Sar1p.