Fig. 4. Vid22p is a glycoslyated integral membrane protein. (A) The predicted amino acid sequence of Vid22p based upon the DNA sequence of YLR373C. Vid22p is a 901 amino acid protein with a predicted molecular weight of 102 kDa and pI of 5.15. Vid22p contains 12 potential N-linked glycosylation sites (underlined regions) and one potential transmembrane domain (bold underlined region). (B) The VID22 gene was fused with a V5 coding sequence and integrated into a wild-type yeast strain. Vid22p-V5 was detected using SDS-PAGE and western blot analysis with antibodies directed against V5. Vid22p migrated as a doublet representative of glycosylated and unglycosylated forms of Vid22p (lane 1). Lysates were treated with or without endoH to determine whether Vid22p was glycosylated. (C) Cells were lysed in Con A binding buffer and incubated in the presence of Con A beads. The total, bound and unbound materials were examined for the presence of Vid22p, CPY or FBPase via western blot analysis. (D) Cells expressing Vid22p-V5 were grown in low glucose media for 2 days and then shifted to glucose rich media for 0, 60 and 120 minutes. Cells were lysed and examined for the levels of Vid22p, FBPase, Vid24p and Pma1p via western blot analysis. (E) Wild-type cells expressing Vid22p-V5 were labeled in ethanol and chased in the presence of 2% ethanol or 2% glucose. Cells were harvested at 0, 1 and 2 hours and total lysates were immunoprecipitated with anti-V5 antibodies. Radiolabeled Vid22p-V5 was visualized using a phosphorimager. (F) Vid22p-V5 cells were shifted to glucose for 30 minutes. Cells were homogenized and lysates were resuspended in either TE alone, TE containing Na₂CO₃ (ph 11.5) or TE containing 2% TX-100. The distribution of Vid22p and Pma1p in the pellet and the supernatant fractions was examined by western blotting with anti-V5 or anti-Pma1p antibodies.

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