Abstract
Immunodetection of protein carbonyl groups demonstrates that growth arrest elicited by carbon or nitrogen starvation causes an increased oxidation of proteins in Saccharomyces cerevisiae. Mutant analysis suggests that the response regulator Pos9p is involved in mitigating self-inflicted oxidative damages in G(0) cells, whereas Yap1p is primarily required in growing cells. The data also suggest that oxidation of target proteins is not a priori an effect of arrest of cell division or nutrient depletion and cannot be explained by the respiratory activity alone nor a high ratio of catabolic/anabolic activity in G(0) cells. Instead, we observed that starvation elicits a transition in the respiratory state (from phosphorylating to nonphosphorylating respiration) and that this transition is associated with a stepwise increase in protein oxidation. During carbon starvation, this transition and increase in oxidation occurs immediately as the carbon source is depleted, growth is arrested, and the respiratory rate falls drastically. In contrast, during nitrogen starvation and excess carbon the respiratory state transition and stepwise increase in protein oxidation are markedly delayed and occur long after the nitrogen source has been depleted and division and growth-arrested. Oxidation in G(0) cells could be enhanced by treating cells with low concentrations of antimycin A and attenuated with myxothiazol, indicating that protein oxidation is intimately linked to reactive oxygen species generated by semiquinones of the Q-cycle. Thus, the work presented suggests that the degree of coupling in the mitochondrial respiratory apparatus rather then the overall rate of respiration affects the degree of protein oxidation in nondividing yeast cells.
Highlights
It has been proposed that aging results from random deleterious events, and oxidative damage has been suggested to be one major contributor to such stochastic degeneration of organisms and their cells
In prokaryotic model systems, such as E. coli, it is known that the levels of both primary and secondary oxidative defense proteins increase markedly during growth arrest and that the population becomes increasingly resistant to external oxidative stresses [10, 11]
Protein Oxidation Increases in G0 Cells and Is Enhanced in pos9 Mutants—The carbonyl content of total proteins was measured immunochemically, and densitometric quantification of the blots demonstrated a severalfold increase in carbonyl content in wild-type yeast cells as a result of arrest of proliferation during starvation (Fig. 1)
Summary
Growth medium of auxotrophic strains (see Table I) was similar to the carbon starvation medium but contained 1 g/l of glucose (so that cell density never constituted a limitation for oxygen diffusion), leucine (25 mg/l), uracil (9 mg/l), histidine (9 mg/l), and methionine (6 mg/l) and was buffered with 100 mM potassium hydrogen phthalate and NaOH at pH ϭ 5. To obtain respiration rates for pseudostates 4 (PS4) and 3 (PS3), the cells were treated with (see “Treatments with Drugs”) TET and CCCP added directly into the cuvette used for measurement of oxygen consumption. From these values and the natural respiratory rate, the respiratory state value (RSV) was calculated.
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