The Carboxyl-terminal End of Cox1 Is Required for Feedback Assembly Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

Abstract

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced. We deleted the C-terminal 11 or 15 residues of Cox1 by site-directed mutagenesis of mtDNA. Although these deletions did not prevent respiratory growth of yeast, they eliminated the assembly-feedback control of Cox1 synthesis. Furthermore, these deletions reduced the strength of the Mss51-Cox14 interaction as detected by co-immunoprecipitation, confirming the importance of the Cox1 C-terminal residues for Mss51 sequestration. We surveyed a panel of mutations that block CcO assembly for the strength of their effect on Cox1 synthesis, both by pulse labeling and expression of the ARG8 m reporter fused to COX1. Deletion of the nuclear gene encoding Cox6, one of the first subunits to be added to assembling CcO, caused the most severe reduction in Cox1 synthesis. Deletion of the C-terminal 15 amino acids of Cox1 increased Cox1 synthesis in the presence of each of these mutations, except pet54. Our data suggest a novel activity of Pet54 required for normal synthesis of Cox1 that is independent of the Cox1 C-terminal end.

A Novel Function of Pet54 in Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

“…The reduced labeling of Cox1 and Cox1⌬C15 in the double mutants (⌬pet54/⌬cox14 and ⌬pet54/⌬coa3) showed a bypass of Cox14 and Coa3 synthesis control (Fig. 2, A and B), in contrast to what has been observed for a ⌬cox6 mutant (16) or other mutants affecting Cox1 assembly (15,18,19). A similar result was observed with strains containing introns in COX1 (data not shown), strongly suggesting that the role of Pet54 in translation of the COX1 mRNA is independent of the role in COX1 intron splicing.…”

“…This was the only mutant analyzed whose Cox1 labeling was not increased by C-terminal truncation of Cox1, suggesting that this effect might not be due simply to the lack of CcO assembly. Indeed, deletion of Pet122 or Pet494, the other COX3 mRNA translational activators, resulted in a Cox1 labeling pattern similar to that observed for general CcO assembly factor mutants reported previously (16). In CcO assembly mutants, synthesis of Cox1 is down-regulated; however, additional elimination of COX14 or COA3 recovers Cox1 synthesis, even if CcO assembly is impaired (15,18,19).…”

“…Deletion of PET54 downregulated Cox1 synthesis; however, in contrast to what was observed for most assembly mutants, deletion of the Cox1 C-terminal end did not recover Cox1 synthesis (16), indicating that Pet54 plays an additional role in this process.…”

“…In mutants that block CcO assembly, synthesis of Cox1 is down-regulated; however, deletion of the last 15 residues of Cox1 (Cox1⌬C15) recovered normal levels of Cox1 synthesis (16). This pattern was observed in many different mutants on either CcO subunits or chaperones that participate in enzyme assembly.…”

“…The only exception was a deletion of pet54. In this mutant, synthesis of Cox1 is downregulated, but deletion of the C-terminal end of Cox1 does not recover synthesis (16).…”