Mutational analysis of the Sar1 protein, a small GTPase which is essential for vesicular transport from the endoplasmic reticulum.
SAR1 encodes a 21-kDa GTPase, which is required for vesicle formation from the endoplasmic reticulum in yeast. Although it belongs to the expanding small GTPase superfamily, there are interesting structural features that are unique to the Sar1 protein. We performed a site-directed mutational study to identify the amino acid residues that are essential for the Sar1p function. Among seven mutants we constructed, four are functionless by themselves, while two confer temperature sensitivity to cells. When the mutant proteins are overproduced in wild-type cells, all of these six show a dominant negative effect on cell growth. The replacement by serine of the only cysteine residue present in Sar1p caused no significant change in the growth phenotype. These findings are not only important for analyzing the mechanism of the Sar1p action in yeast, but will also be very useful for studying the function of Sar1p counterparts in higher eukaryotes.
Nakano
A
A
Department of Plant Sciences, Graduate School of Science, University of Tokyo.
Otsuka
H
H
Yamagishi
M
M
Yamamoto
E
E
Kimura
K
K
Nishikawa
S
S
Oka
T
T
D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae