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Fig. 3. (A) Gyp5p and Gyl1p are present in three main cellular pools: yeast cells expressing Gyp5p-Myc, Gyl1p-HA and Sec4p-GFP were submitted to subcellular fractionation to generate P13, P100 and S100 fractions. The volumes of fractions loaded on each lane of SDS-PAGE correspond to 2x107cells. The nitrocellulose membranes were incubated with either anti-Myc, anti-HA or anti-GFP antibodies, then stripped from the antibodies and incubated again with anti-Pma1p antibodies (from R. Serrano) and anti-actin antibodies. (B) Gyp5p and Gyl1p co-fractionate with plasma membrane and vesicles markers: the P13 and P100 fractions shown in A were loaded on linear 20-60% sucrose gradients. Fractions were probed by immunoblotting with different antibodies as described in A. Dpm1p is an ER membrane dolichol-phosphate mannose synthase. (C) Gyp5p and Gyl1p behave as peripheral membrane proteins: yeast cells expressing Gyp5p-Myc and Gyl1p-HA were submitted to membrane extraction, as described in Materials and Methods. S, supernatant; P, membrane pellet. (D) Gyp5p and Gyl1p are phosphorylated proteins: total protein extracts of yeast cells expressing Gyp5p-Myc and Gyl1p-HA were submitted to calf intestine alkaline phosphatase (CIP) treatment, as described in Material and Methods, resolved on SDS-PAGE and revealed with either anti-Myc or anti-HA antibodies.





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