Fig. 3.

ret2(HARS)/dsl1-ΔE mutant cells show defects in growth and in CPY trafficking. (A) Isogenic strains bearing mutations at either the DSL1 or RET2 (δ-COP) gene loci, labeled as dsl1-ΔE and ret2(HARS), were serially diluted, spotted, and grown at the indicated temperatures for 48 h. The double mutant harboring both the ret2(HARS) and dsl1-ΔE allele was lethal at 37 °C (no colonies formed after 1 wk) but was viable at lower temperatures, although with a slight growth defect. All combinations are shown in biological duplicate. (B) Cells of the indicated strains were grown to midlog at 30 °C in yeast extract peptone dextrose (YEPD) medium and then were shifted for 2 h to either 34 °C or 37 °C. Glass bead extracts of collected cells were separated by 7.5% SDS-PAGE and probed with α-CPY antibodies (40). Positions of p1 (ER), p2 (Golgi), and mature (m, vacuolar) forms of CPY are indicated. Note that p1 CPY accumulates in ret2(HARS) dsl1-ΔE double-mutant cells at 34 °C and more strongly at 37 °C but does not accumulate in single-mutant cells.