Figure 8.

Newly synthesized Ste6 is not transported to the cell surface in a sec1-1 mutant at nonpermissive temperature. The intracellular Ste6 distribution in STE6 deletion strains transformed with the single-copy CUP1p-STE6 plasmid pRK158 was examined by sucrose density gradient fractionation (20–50% [wt/wt] sucrose, fraction 1: low sucrose density) and by Western blotting with anti-Ste6 antibodies. The cells were either continuously grown at 25°C (A and C) or were first grown at 25°C and then shifted to 37°C 1 h (B and D) before extract preparation. STE6 expression from the CUP1 promoter was induced 45 min before extract preparation by the addition of 0.5 mM CuSO₄ to the cultures, i.e., copper was added 15 min after the 37°C cultures had been shifted to nonpermissive temperature. (A and B) RKY1001/pRK158 (wild type). (C and D) RKY1177/pRK158 (end4 sec1-1). The putative secretory vesicle peak is boxed (D).