Mechanism of Induction of Cytochrome Oxidase in Yeast: I. Kinetics of Induction and Evidence for Accumulation of Cytoplasmic and Mitochondrial Precursors

Abstract

Abstract The induction of cytochrome oxidase in Saccharomyces carlsbergensis was studied by following the time course of elaboration of (a) hemeprotein (or hemeproteins) having heme a as prosthetic group, measured spectrophotometrically at 605 mµ, and (b) the cytochrome oxidase activity, measured in cell-free extracts. The kinetics of induction was investigated after removal of inducer (air) and after the separate addition of cycloheximide, chloramphenicol, and acriflavine at different times during respiratory adaptation. The following results were obtained. 1. Anaerobically grown cells harvested from early stationary phase form hemeprotein a and cytochrome oxidase activity at a constant rate from the onset of aeration. Hemeprotein a reaches its maximal inducible level after 2 hours, whereas cytochrome oxidase activity is at a maximum after 3 hours of aeration. These levels are 33 to 37% of those of aerobically grown cells. The enzyme of induced or aerobically grown cells exhibits the same Km with respect to ferrocytochrome c. 2. Cells induced for periods up to 25 min possess the capacity to form additional cytochrome oxidase activity upon further incubation in the absence of air, whereas cells induced for longer periods are unable to do so. Cycloheximide added prior to aeration prevents the expression of this capacity, but chloramphenicol and acriflavine are without effect. 3. Separate addition of cycloheximide, chloramphenicol, and acriflavine prior to aeration allows the formation of different amounts of hemeprotein a and cytochrome oxidase activity. The amount of inhibitor-resistant synthesis of hemeprotein a is greater than that of cytochrome oxidase activity, and the kinetics of these syntheses varies with the inhibitor used. 4. Cells undergoing respiratory adaptation exhibit the capacity to form additional cytochrome oxidase activity upon further incubation with cycloheximide. This capacity is seen only during the 1st hour of induction, and its expression is inhibited by chloramphenicol. Conversely, cells induced for periods longer than 80 min can form additional cytochrome oxidase when further incubated with chloramphenicol, and the expression of this capacity is blocked by cycloheximide. During the 1st 80 min of induction, the cells can synthesize additional amounts of hemeprotein a when further incubated with either cycloheximide or chloramphenicol. 5. Cells induced for periods longer than 40 min form additional cytochrome oxidase when further incubated with acriflavine, and the expression of this capacity is inhibited by cycloheximide (between 60 and 100 min) and by chloramphenicol (between 40 and 80 min). 6. Cycloheximide inhibits the induction of cytochrome c, anaerobic growth, and incorporation of 14C-leucine into the proteins of adapting yeast, whereas chloramphenicol and acriflavine are not inhibitory.