Fig. 1. Vacuolar sorting of ALP and CPS is not affected in vps30 and vps38 mutant cells. (A) Wild-type (SEY6210), vps30 (JCY300), vps38 (PBY58) and vps30vps38 (PBY51) strains were grown in YNB medium to early log phase and metabolically labeled with [³⁵S] methionine/cysteine at 26°C for 10 minutes and then chased in the presence of excess non-labeled amino acids for 30 minutes. The proteins were immunoprecipitated with antibodies specific to CPY, resolved on SDS-PAGE and visualized by autoradiography. The migration positions of Golgi-modified precursor (p2) and mature (m) CPY are shown. (B) Cells were grown in YPD medium to early log phase. Total cell lysates were subjected to SDS-PAGE, followed by immunoblotting with anti-CPS and anti-ALP antibodies, respectively. CPS samples were treated with endoglycosidase H prior to electrophoresis. Precursor forms of CPS and ALP are indicated (p; 73 kDa and 76 kDa, respectively) and mature forms (m; 69 kDa and 72 kDa, respectively) are shown. (C) Fluorescence and DIC microscopy of cells expressing GFP-CPS (pGO45).