FIG. 2.

SYM1 is stress inducible. (A) A wild-type culture in mid-logarithmic-growth phase was subjected to a 1-h heat shock at 37°C, aliquots were removed at the indicated time points, and total RNA was prepared for Northern analysis as described in Materials and Methods. Radiolabeled probes were used to detect mRNA levels of the indicated genes. STI1 is a well-established heat shock gene, and PGK1 is used as a loading control. (B) Sym1 protein levels after a brief heat shock were examined in the sym1Δ mutant strain transformed with a plasmid expressing SYM1 tagged with a triple-HA epitope at the carboxyl terminus under the control of its own promoter. Equal amounts of mid-logarithmic-phase cells were harvested at 30°C or after 15 min of heat shock at 37°C, protein extracts were prepared, and the levels of Sym1-HA and the loading control PGK were analyzed by Western blotting. (C) Protein extracts were prepared from the same strain used as described for panel B from cells grown in glucose medium or after a 9-h shift to ethanol-containing medium and were analyzed by Western blotting. The polyclonal antibody used to detect the mitochondrial ATP/ADP carrier protein (AAC) also recognizes the mitochondrial outer membrane protein porin.