THE C-TERMINAL DOMAIN OF YEAST CYTOCHROME-B IS ESSENTIAL FOR A CORRECT ASSEMBLY OF THE MITOCHONDRIAL CYTOCHROME BC1 COMPLEX

Yeast mutants modifying the C-terminal region of mitochondrial cytochr ome b were isolated and characterized. A nonsense mutation of the leuc ine codon 335 (TTA --> TAA), 50 residues before the normal C-terminus, blocks incorporation of heme into the apocytochrome b and prevents gr owth on non-fermentable substrates. The same defects were observed in a frameshift mutant (after codon 348, TAT --> TATT) in which the last 37 C-terminal residues are predicted to be replaced by a novel sequenc e of 33 amino acids. Function was regained in the nonsense mutant only by true back mutations restoring a protein of the wild-type sequence. The respiratory capacity was restored to wild-type levels in the fram eshift mutant by a variety of single base subtractions located within a window of 24 bases before or after the original +T addition, these p seudo-reversions resulted in single or multiple (up to five) consecuti ve amino acid replacements between positions 346 and 354 and restored the wild-type sequence from position 355 to 385. These data, combined with hydropathy calculations and sequence comparisons, suggest that th e C-terminal domain of cytochrome b forms a transmembrane segment esse ntial for the correct assembly of the cytochrome bc1 complex.