The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway.
SAR1, the yeast gene which encodes a novel type of small GTP-binding protein, has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To further the understanding of the function of its product, a lacZ-SAR1 hybrid gene was constructed and a polyclonal antibody was raised against the hybrid protein. This antibody specifically recognizes the SAR1 gene product (Sar1p) as a 23-kDa protein in the yeast cell lysate. We examined the subcellular localization of Sar1p using this antibody. In wild-type cells, Sar1p was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER. The soluble form of Sar1p was also detected when the protein was overproduced. Immunofluorescence microscopy with the anti-Sar1p antibody showed perinuclear staining that was exaggerated in the ER-accumulating sec18 mutant. Membrane association of Sar1p was shown to be very light. Sar1p was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sar1p completely. From these results, we suggest that Sar1p is firmly located on the ER membrane where it regulates the ER-Golgi traffic.
Nishikawa
S
S
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Nakano
A
A
D000906:Antibodies / Q000276:immunology; D001692:Biological Transport; D002458:Cell Fractionation; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010957:Plasmids; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D005656:Fungal Proteins; D008565:Membrane Proteins; D019204:GTP-Binding Proteins