Fig. 4. (A) Wild-type and sec61-2 yeast strains were transformed with high-copy plasmids coding for Ubc6p or Ubc6p^ser or with an empty vector as indicated. Plates were incubated at 30 and 36.5°C as indicated. (B) A yeast strain coding for the mutant carboxypeptidase Y (CPY^*) was transformed with high-copy plasmids coding for scUbc6p () or scUbc6p^ser () or with an empty () vector. Equal amounts of cells were labeled with ³⁵S-cysteine/methionine and chased with non-radioactive medium. Aliquots were taken after 0, 30, 60 and 90 minutes chase-time and immunoprecipitated for CPY^*, subjected to SDS-PAGE and analyzed by autoradiography. A typical experiment is shown. The average and standard deviation of four (scUbc6p^ser) or six (empty plasmid, scUbc6p) independent experiments was calculated and is displayed as a graph. (C) Yeast strain K700 strain was co-transformed with pUB23P, which encodes for the N-end rule substrate Pro-ß-Galactosidase, and with high-copy plasmids coding for scUbc6p or scUbc6p^ser, or with an empty vector. Equal amounts of cells were labeled with ³⁵S-cysteine/methionine and chased with non-radioactive medium. Aliquots were taken after 0, 20 and 40 minutes chase-time and immunoprecipitated for ß-galactosidase, subjected to SDS-PAGE and analyzed by autoradiography. A typical experiment is shown in the upper part. The average of three independent experiments was calculated and is displayed as a graph.