Identification of functionally important regions of the Saccharomyces cerevisiae mitochondrial translational activator Cbs1p.
Translation of cytochrome b mRNA in yeast mitochondria requires activation by the nuclear-encoded Cbs1p. According to the current model, Cbs1p tethers cytochrome b mRNA to the inner mitochondrial membrane via interaction with the 5'-untranslated leader. Cbs1p is predicted to be a hydrophilic protein with two hydrophobic segments near the carboxyl-terminal end, which are both too short to span the membrane. Nevertheless Cbs1p is tightly associated with the mitochondrial membrane, as shown by its behaviour in extraction experiments with taurodeoxycholate. In an attempt to define functionally important regions of Cbs1p, we created a number of mutant alleles by random and directed mutagenesis. We report that a Cbs1p mutant protein lacking the mitochondrial presequence is still able to complement a Deltacbs1 strain, suggesting that the presequence does not contain essential mitochondrial targeting information. Mutations in a cluster of positively charged amino acids at the extremeC-terminus have no effect on Cbs1p function, but removal of this segment severely impairs Cbs1p function. Truncation of 12 or more amino acids from the C-terminus results in a completely defective protein. We further show that both short hydrophobic regions are essential for Cbs1p function, although membrane association is observed even in the absence of these regions.
Krause-Buchholz
U
U
Institute of Genetics, Dresden University of Technology, D-01062 Dresden, Germany.
Tzschoppe
K
K
Paret
C
C
Ostermann
K
K
Rödel
G
G
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003573:Cytochrome b Group / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
C060724:CBS1 protein, S cerevisiae; D003573:Cytochrome b Group; D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators