Cox2p is one of three mitochondrially-encoded subunits of
cytochrome c oxidase. The first 15 codons of COX2 encode a
leader peptide that is cleaved from pre-Cox2p after translocation of its
N-terminal region through the inner-membrane, and may initiate membrane
insertion (Mol. Biol. Cell 1997, 8: 1449-60). While mammalian Cox2p
lacks a leader peptide, deletion of the yeast leader peptide coding
sequence causes a tight respiratory deficient phenotype and prevents the
accumulation of Cox2p (Genetics 1997, 145: 903-0). We have
translationally fused a reporter gene ( ARG8 m ) to
COX2 and found, surprisingly, that the leader peptide deletion
also prevents reporter protein accumulation without affecting mRNA
levels, indicating that these sequences could be required for
COX2 translation. To define the role(s) of the Cox2p leader
peptide coding sequence, we have constructed mutants with partial
deletions, point mutations, local frameshifts, or random replacements in
it. We have analyzed the effects of these mutations on both accumulation
of reporter protein, and on expression of COX2 itself, to
differentiate possible roles in translation from roles in Cox2p
function. In addition, multicopy and intragenic suppressors of these
mutations have been analyzed. Taken together, our results suggest that
the leader peptide coding sequence functions, at the mRNA level, in
COX2 translation, in addition to specifying a pre-Cox2p leader
peptide.
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