The presence of the iron-sulfur protein (subunit V) of complex III in mitochondria of heme-deficient yeast cells lacking iron-sulfur clusters detectable by electron paramagnetic resonance

The presence of subunit V, the iron-sulfur protein, of complex III has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae which lacks 5-aminolevulinic acid synthase and, hence, is devoid of heme. The mature form (24 K Da) of the iron-sulfur protein was observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against subunit V and either the sensitive immuno-transfer technique or immunoprecipitation from dodecylsulfate-solubilized mitochondria. In addition, a slight shoulder with a molecular mass 1.5 kDa larger than the mature form was present in mitochondria from the heme-deficient cells. Electron paramagnetic resonance spectroscopy revealed the absence of iron-sulfur signals due to clusters S-1, S-2 and S-3 of succinate dehydrogenase or to Rieske's iron-sulfur cluster of complex III in mitochondria from the heme-deficient cells. The lack of iron-sulfur centers in these cells may be a consequence of the absence of sulfite reductase in the cells without heme.     

The DAN1 gene of S. cerevisiae is regulated in parallel with the hypoxic genes,but by a different mechanism

The DAN1 gene is expressed under anaerobic conditions in yeast and completely repressed during aerobic growth. The function of the gene is unknown, and genetic disruption had no effect on fitness which could be detected, even upon prolonged anaerobic growth. Expression of DAN1 was constitutive in a heme-deficient strain, indicating that heme participates in repression. Expression was blocked by heme in anaerobic medium, suggesting that heme acts as a negative co-effector rather than through its metabolic functions, i.e., in the production of a co-effector. Expression of DAN1 was regulated in parallel with the hypoxic gene ANB1, showing identical kinetics of induction and dose response to heme. However, unlike ANB1, DAN1 is not regulated by the repressor of the hypoxic regulon, ROX1, as shown by observation of normal aerobic repression of DAN1 in a strain carrying a deletion of ROX1. These results indicate the existence of a parallel regulatory system which produces an identical response to oxygen by a different mechanism than that controlling the hypoxic regulon.     

Synthesis of the proteins of complex III of the mitochondrial respiratory chain in heme-deficient cells

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5-aminolevulinic acid synthase and, hence, devoid of heme. The two 'core' proteins, apocytochrome b and the iron-sulfur protein, were observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against complex III and the sensitive immuno-transfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1. By contrast, when mitochondria from the heme-deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.