Deletion of the leader peptide of the mitochondrially encoded precursor of Saccharomyces cerevisiae cytochrome c oxidase subunit II.
Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p. The 15-amino acid leader peptide is processed after export to the intermembrane space. Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide. We generated two deletions in the S. cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site. When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype. A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a p- mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence. The resulting fusion protein was processed to yield functional Cox2p. Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide. We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded.
Torello
A T
AT
Department of Biology, Ithaca College, New York 14850, USA.
Overholtzer
M H
MH
Cameron
V L
VL
Bonnefoy
N
N
Fox
T D
TD
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics*; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D003576:Electron Transport Complex IV