FIGURE 6.

Expression and phenotypes of the sss1Δ12 mutation. A, haploid yeast strains bearing genomic wild-type SSS1, Δsss1, and sss1Δ12 alleles also containing a SSS1, URA3 plasmid (FKp53) were grown on minimal medium lacking uracil for plasmid selection and on 5-FOA medium for plasmid counterselection for 3 days. All strains grew normally on minimal medium with plasmid selection. After 3 days on 5-FOA medium, the wild-type strain grew as expected, and the Δsss1 strain was lethal upon plasmid counterselection as expected. The sss1Δ12 strain was also unable to grow on this medium, indicating the lethality of removing the C-terminal 12 residues of Sss1p. B, whole cell extracts prepared from the MET3-SSS1 strains BWY875 (Δsss1) and BWY886 (sss1Δ12) grown in minimal medium without methionine or in the presence of 2 mm methionine for 8 h (to deplete Sss1p) were subjected to 16% SDS-PAGE and immunoblot analysis with Sss1p antiserum. C, strains BWY875 and BWY886 grown as above were labeled with ¹⁴C-amino acids for 5 min. The sample in lane 1 was provided by BWY875 cells grown in the absence of methionine and treated with tunicamycin (Tu). Whole cell extracts were then immunoprecipitated with ppCPY- or DPAP B-specific antiserum, and the precipitates were resolved by 10 and 7.5% SDS-PAGE, respectively. For a description of ppCPY and DPAP B ER processing, see Fig. 5 legend. The data in B and C are representative of two independent experiments.