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Figure 5


Fig. 5. Subcellular distribution of endogenous Gas1p. (A) Differential centrifugation analysis. Wild-type or arl1-mutant cells were grown in YPAD medium, subjected to velocity sedimentation and then separated into P13 and S13 fractions as described in Materials and Methods. Proteins in samples of fractions were analyzed by immunoblotting. Gas1p, Pma1p (plasma membrane marker), Pgk1p (cytosol marker) and Kex2p (Golgi marker) were identified with specific antibodies. (B) Sucrose-density-gradient centrifugation analysis. S13 was layered on top of the sucrose gradient (10-30%), which was then subjected to centrifugation as described in Materials and Methods. Twelve fractions were collected from the top and samples were analyzed by western blotting using antibodies against Gas1p, Drs2p (late Golgi marker), Emp47p (early Golgi marker), Pgk1p (cytosol marker) and Pep12p (endosome marker). The lower panel displays the quantification of the western blot results.





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