Figure 3

SEC61 - CPLV and SSH1 - CPLV do not affect ER import in heterozygous diploids. A) Bait quantitation. Left: Wildtype (1) or SEC61/SEC61-CPLV yeast (2) were grown to early exponential phase, equal amounts lysed, proteins separated by SDS-PAGE, transferred to nitrocellulose, and detected with polyclonal anti-Sec61p-N- antiserum and ¹²⁵I-Protein A. Right: Wildtype (1) or SEC61/SEC61-CPLV (2) or SSH1/SSH1-CPLV (3) cells were analyzed as above. Fusion proteins were detected with polyclonal anti-PLV-C-terminus, Pdi1p was a loading control. B) SEC61-CPLV does not affect posttranslational import into the ER, although associated with the Sec complex. Top: Import reactions containing microsomes from wildtype or SEC61/SEC61-CPLV diploid yeast, ³⁵S-labelled preproalpha factor (ppaF), and ATP were incubated at 24°C for the indicated times, reactions stopped, and proteins detected by SDS-PAGE and autoradiography. ER-imported ppaF is signal-cleaved and glycosylated (3gpaF). Bottom: Import reactions as above were incubated at 20°C for 10 min, DMSO (-) or 500 μM DSP (+) was added for crosslinking. After quenching, membranes were lysed and Sec complex-associated proteins precipitated with polyclonal anti-Sec63p-serum. Crosslinks were reduced, proteins separated by SDS-PAGE and Sec61-CPLV and Sec61p detected with anti-Sec61p-N-terminus. C) ER import of CPY and Pdi1p is not affected in bait strains. Diploid cells expressing indicated fusion proteins and a sec61-2 haploid with an ER protein import defect were grown to early log phase at 30°C, pulsed for 2 min with ³⁵S-methionine/cysteine, and chased for indicated times. Cells were lysed, CPY (top) or Pdi1p (bottom) immunoprecipitated with polyclonal antisera, separated by SDS-PAGE and detected by autoradiography. Note that cytoplasmic precursor pCPY and mature vacuolar mCPY migrate at the same position. p1: ER CPY; p2: Golgi CPY. Black bar right of Pdi1p blot indicates hypoglycosylated forms of Pdi1p which occur when translocation is inefficient (in sec61-2 cells).