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 important for assembly and/or activity of complex II of the electron transport system in Saccharomyces cerevisiae. These genes were expressed in a respiration-deficient yeast mutant in which the endogenous IP gene had been disrupted. Substitutions at the N-terminus were tolerable. Substituting the region covering the first iron-sulfur center [2Fe-2S] had no effect on assembly, while activity decreased 2-5-fold. The addition of seven amino acids from the human peptide, including four charged residues, at the C-terminus did not perturb either assembly or activity. A region between the first and second cysteine clusters was identified which when substituted caused a complete failure in the assembly of complex II. It includes a 15 amino acid stretch which shows the greatest variability between species. Larger substitutions including this segment failed as well. Exchanging the region between the second and third cysteine clusters making up the [4Fe-4S] and [3Fe-4S] centers enabled transformants to grow on nonfermentable carbon sources, yet no SDH activity was observed in vitro. The IP and FP proteins accumulate to wild-type levels in these mutants. We speculate that the lack of observed activity is due to the lability of iron-sulfur centers in isolated, broken mitochondria.
Saghbini
M
M
Department of Biology, University of California at San Diego, La Jolla 92093-0322.
Broomfield
P L
PL
Scheffler
I E
IE
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D003001:Cloning, Molecular; D004579:Electron Transport; D042963:Electron Transport Complex II; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000378:metabolism; D010101:Oxygen Consumption; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013385:Succinate Dehydrogenase / Q000096:biosynthesis* / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase