Figure 3.
Grd20p is required for efficient sorting of the vacuolar hydrolase CPY. (A) Strains SNY17 (GRD20), SBY4-10A (grd20-Δ1), SNY97-14D (grd20::Tn3), and SBY4-10A/pSB10 (grd20-Δ1 + pSB10) were pulsed with [35S]methionine and cysteine for 10 min and chased with unlabeled amino acids for 45 min. The strains were incubated at 30°C throughout the experiment. CPY was immunoprecipitated from the intracellular (I) and extracellular (E) fractions and analyzed by SDS-PAGE and fluorography. The left panel was overexposed to allow detection of CPY in the grd20-Δ1 strain that incorporated label poorly. (B) The following strain/plasmid combinations were propagated for several doublings at 22°C: SBY4-10A/pSB1 (GRD20), SBY4-10A/pSB1-125 (grd20-1), and SBY4-10A/pSB1-1212 (grd20-2). The strains were then shifted to 36°C for 5 min or incubated at 22°C as indicated. After a 10-min pulse and 0- and 45-min chase, CPY was immunoprecipitated and analyzed as in A. (C) Strain SBY4-10A transformed with plasmids pSB1 + pSB18 (GRD20 + GRD20), pSB1 + pSN335 (GRD20 + grd20-1), pSB1 + pSN336 (GRD20 + grd20-2), and pSB1-125 + pSN336 (grd20-1 + grd20-2) was subjected to CPY immunoprecipitation as described in B after a 45-min chase. For both A and B the percentage of CPY secreted from each strain after a 45-min chase as determined by phosphorimager analysis is indicated below each E lane. The CPY secretion values in B represent the average of two experiments, and the values from each of those experiments are shown in Table 3 for strains incubated at 36°C. The positions of ER (p1) and Golgi (p2) precursor forms and vacuole localized mature form (m) of CPY indicated were assigned based on the migration of CPY expressed in wild-type strains.