Alteration of the Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader by mitochondrial gene replacement and functional interaction with the translational activator protein PET111.

Abstract

The ability to replace wild-type mitochondrial DNA sequences in yeast with in vitro-generated mutations has been exploited to study the mechanism by which the nuclearly encoded PET111 protein specifically activates translation of the mitochondrially coded COX2 mRNA. We have generated three mutations in vitro that alter the COX2 mRNA 5'-untranslated leader (UTL) and introduced them into the mitochondrial genome, replacing the wild-type sequence. None of the mutations significantly affected the steady-state level of COX2 mRNA. Deletion of a single base at position -24 (relative to the translation initiation codon) in the 5'-UTL (cox2-11) reduced COX2 mRNA translation and respiratory growth, whereas insertion of four bases in place of the deleted base (cox2-12) and deletion of bases -30 to -2 (cox2-13) completely blocked both. Six spontaneous nuclear mutations were selected as suppressors of the single-base 5'-UTL deletion, cox2-11. One of these mapped to PET111 and was shown to be a missense mutation that changed residue 652 from Ala to Thr. This suppressor, PET111-20, failed to suppress the 29-base deletion, cox2-13, but very weakly suppressed the insertion mutation, cox2-12. PET111-20 also enhanced translation of a partially functional COX2 mRNA with a wild-type 5'-UTL but a mutant initiation codon. Although overexpression of the wild-type PET111 protein caused weak suppression of the single-base deletion, cox2-11, the PET111-20 suppressor mutation did not function simply by increasing the level of the protein. These results demonstrate an intimate functional interaction between the translational activator protein and the mRNA 5'-UTL and suggest that they may interact directly.

Evidence for a Pro-oxidant Intermediate in the Assembly of Cytochrome Oxidase

“…Whereas the lack of Cox2 may be expected to result in accumulation of the pro-oxidant Cox1 intermediate, only modest peroxide sensitivity is observed in cox2⌬ cells, and pet111⌬ cells are peroxide-resistant. Cells containing mutant alleles of COX2 with mutations in the 5Ј-untranslated region showed the expected diminution in Cox2 nascent chains but also reduced Cox1 nascent chains (36). Likewise, cells lacking Pet111 show the expected loss of Cox2 translation and also diminution in Cox1 translation (37).…”

Protein synthesis in mitochondria

“…The respiratory deficiency resulting from a mutation in PET111 is suppressed by an exact substitution of the COX2 for the COX3 51 untranslated leader, thus demonstrating that PET111 also acts via sequences upstream of the initiation codon [68]. A translational defect resulting from a point mutation in the COX2 leader can be efficiently suppressed by a second-site mutation in the PET111 gene [88]. Interestingly, the same PET111 missense mutation partially suppressed the leaky respiratory-deficient growth phenotype of a mutant in which the COX2 initiation codon has been changed to AUA [51 ].…”

“…Interestingly, the same PET111 missense mutation partially suppressed the leaky respiratory-deficient growth phenotype of a mutant in which the COX2 initiation codon has been changed to AUA [51 ]. Moreover, both the leader and the start site mutation are slightly alleviated by overproduction of PETlll [51,88], Like PET122 and PET494, overproduced PET111 has been detected in mitochondria as a tightly bound inner membrane protein [68]. No functional link between PET111 and the mitochondrial ribosome has been demonstrated so far.…”

Pet111p, an Inner Membrane-bound Translational Activator That Limits Expression of the Saccharomyces cerevisiaeMitochondrial Gene COX2

“…Mitochondria-Pet111p is expressed at very low levels (9), and we have previously not been able to detect the protein in wild-type cells (21). Yeast cells overproducing Pet111p accumulate it in mitochondria, associated with membranes.…”

“…Pet111p is known to be mitochondrially located (20) and to interact functionally with the COX2 mRNA 5Ј-UTL (21). Although the amino acid sequence of Pet111p is not highly conserved among budding yeasts, its function in specifically activating translation of the COX2 mRNA has been conserved, indicating an important role in the production of cytochrome c oxidase (22).…”