STUDIES ON THE ASSEMBLY OF COMPLEX-II IN YEAST MITOCHONDRIA USING CHIMERIC HUMAN YEAST GENES FOR THE IRON-SULFUR PROTEIN SUBUNIT

A series of chimeric human/yeast IP genes were constructed in order to investigate domains of the iron-sulfur protein (IP) that are importan t for assembly and/or activity of complex II of the electron transport system in Saccharomyces cerevisiae. These genes were expressed in a r espiration-deficient yeast mutant in which the endogenous IP gene had been disrupted. Substitutions at the N-terminus were tolerable. Substi tuting the region covering the first iron-sulfur center [2Fe-2S] had n o effect on assembly, while activity decreased 2-5-fold. The addition of seven amino acids from the human peptide, including four charged re sidues, at the C-terminus did not perturb either assembly or activity. A region between the first and second cysteine clusters was identifie d which when substituted caused a complete failure in the assembly of complex II. It includes a 15 amino acid stretch which shows the greate st variability between species. Larger substitutions including this se gment failed as well. Exchanging the region between the second and thi rd cysteine clusters making up the [4Fe-4S] and [3Fe-4S] centers enabl ed transformants to grow on nonfermentable carbon sources, yet no SDH activity was observed in vitro. The IP and FP proteins accumulate to w ild-type levels in these mutants. We speculate that the lack of observ ed activity is due to the lability of iron-sulfur centers in isolated, broken mitochondria.