Fig. 5. Yeast cells carrying alanine replacements of the phosphorylatable threonines in Sec63p display protein translocation defects. (A) Pulse analysis of CPY translocation. Wild-type cells (WT), cells carrying the alanine replacements in position 652 (T652A) or 654 (T654A) of Sec63p, or sec62-1 ts cells (sec62-1) were labeled with [³⁵S]-methionine for 5 minutes and subjected to a CPY immunoprecipitation. P1 and P2 indicate the positions of the translocated and preproCPY the position of the nontranslocated fraction of CPY on the autoradiogram of the gel (Pilon et al., 1997). The shift in the apparent molecular weight after EndoH treatment (+) confirmed the localization of P1 and P2 in the secretory pathway. (B) Steady-state analysis. Yeast cells carrying the indicated alleles of SEC63 or SEC62 and expressing signal-sequence-bearing C_ub-Ura3p constructs were spotted on plates without histidine and with or without uracil (+Uracil or –Uracil, respectively) to select for the presence of the plasmids. Growth of cells on SD-ura plates indicates a translocation defect for the respective signal sequence. (C) Synthetic lethality. Yeast cells containing SEC63 or sec63T654A and expressing Sec62p from a URA3 plasmid were transformed with the different TRP1 plasmids containing the indicated alleles of SEC62 carrying the Dha module at their C-termini (Wittke et al., 2000). Cells were plated on 5-FOA-containing media. Cells that contain the sec63T654A and the sec62C35-DHA allele do not growth on this media, indicating a synthetic lethality between the two alleles.