Figure 4.
The measured proximity between Sec62p and Sec63p is due to both proteins being in one complex. (A) Cells bearing Sec63CRUp and Nug-Sec62p were transformed with a plasmid containing either Sec62p, Sec62Dha, Ste14Dha, Tpi1ha, or an empty plasmid, all under the control of the PGAL1-promoter (lanes a–e). Approximately 105, 104, 103, and 102 cells were spotted on selective media lacking uracil and containing either glucose to repress or galactose to induce the PGAL1 promoter. (B) S. cerevisiae cells (104) were plated as described in panel A on selective media containing galactose and lacking uracil, and colonies were counted after 4 d. The average of seven independent experiments is shown. Approximately 800 colonies were recovered upon overexpression of Sec62p. This number was arbitrarily set as 100. (C) Overexpression of the ha epitope-bearing proteins was confirmed by immunoblot analysis of extracts of S. cerevisiae cells coexpressing Sec63CRUp, Nug-Sec62p, and the following constructs: Tpi1ha (lanes a and f), Ste14Dha (lanes b and g), Sec62Dha (lanes c and h), Sec62p (lanes d and i), and empty vector (lanes e and j). Cells were grown in glucose (lanes a–e) to repress and grown in galactose (lanes f–j) to induce the expression of the proteins.