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

Abstract

Cytochrome c oxidase assembly requires the synthesis of the mitochondria-encoded core subunits, Cox1, Cox2, and Cox3. In yeast, Pet54 protein is required to activate translation of the COX3 mRNA and to process the aI5␤ intron on the COX1 transcript. Here we report a third, novel function of Pet54 on Cox1 synthesis. We observed that Pet54 is necessary to achieve an efficient Cox1 synthesis. Translation of the COX1 mRNA is coupled to the assembly of cytochrome c oxidase by a mechanism that involves Mss51. This protein activates translation of the COX1 mRNA by acting on the COX1 5-UTR, and, in addition, it interacts with the newly synthesized Cox1 protein in high molecular weight complexes that include the factors Coa3 and Cox14. Deletion of Pet54 decreased Cox1 synthesis, and, in contrast to what is commonly observed for other assembly mutants, double deletion of cox14 or coa3 did not recover Cox1 synthesis. Our results show that Pet54 is a positive regulator of Cox1 synthesis that renders Mss51 competent as a translational activator of the COX1 mRNA and that this role is independent of the assembly feedback regulatory loop of Cox1 synthesis. Pet54 may play a role in Mss51 hemylation/conformational change necessary for translational activity. Moreover, Pet54 physically interacts with the COX1 mRNA, and this binding was independent of the presence of Mss51.Cytochrome c oxidase (CcO) 3 is the last electron acceptor of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this enzyme contains 12 subunits, three of which (Cox1, Cox2, and Cox3) are encoded by the mitochondrial DNA. Assembly of CcO is a complex process regulated by more than 25 factors and chaperones (for reviews, see Ref. 1). The first steps of CcO biogenesis involve the translational activation of the mitochondria-encoded mRNAs COX1, COX2, and COX3 by mRNA-specific proteins. Translational activation of the COX1 mRNA depends on Pet309 and Mss51 (2, 3), whereas COX2 translation depends on Pet111 (4, 5), and COX3 mRNA translation depends on Pet54, Pet122, and Pet494 (6 -9). These proteins act on the target mRNA 5Ј-UTRs to allow translation by the mitochondrial ribosomes. They interact with each other and with the mitochondrial inner membrane and are thought to tether translation initiation close to the assembly sites of CcO in the membrane (for a review, see Ref. 10) (11). The mitochondria-encoded Cox1, Cox2, and Cox3 subunits are proposed to assemble from three different modules, each containing a specific subset of nucleus-encoded subunits (12-14).Cox1, the largest subunit of the CcO, carries the heme aa 3 -Cu B center to reduce oxygen. Synthesis of Cox1 inside mitochondria is highly regulated. If CcO assembly is blocked by mutations on either integral subunits or accessory chaperones, Cox1 synthesis is down-regulated (15, 16). It is proposed that by this mechanism, mitochondria avoids accumulation of pro-oxidant Cox1 intermediates (17). In addition to its role as translational activator of COX1 mRNA, Mss51 also physically inter...

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules

“…Most, if not all, structural subunits have been found to be associated with Cox1p and Cox3p (11,12). In addition to the structural subunits, the Cox1p module also contains translational regulators Mss51p, Cox14p, and Coa3p as well as several other factors involved in the maturation of the protein (13)(14)(15)(16)(17)(18)(19).…”

The Cox1 C-terminal domain is a central regulator of cytochrome c oxidase biogenesis in yeast mitochondria

“…Mss51 sequestration in the COA complexes depends upon interactions with Cox14 that occur when Cox1 is newly synthesized (1,21,26). The Cox1⌬C15 mutation compromises the interaction between Mss51 and Cox14, as detected by co-immunoprecipitation (30), making free Mss51 more abundant to promote COX1 mRNA translation (27,32). We therefore investigated whether binding of Mss51 to Cox14 and other components of the COA complexes was similarly compromised in our Cox1 missense mutants.…”

“…Mss51 molecules are present in distinct complexes within mitochondria that are distinguishable by blue-native gel electrophoresis (BN-PAGE) and sedimentation velocity (24,27,29,33,34). Mss51 is detected in high-molecular-weight COA complexes that correspond to Cox1 assembly intermediates.…”

“…CcO assembly mutants overaccumulate Mss51 in the COA complexes and underaccumulate Mss51 in translational activator form, decreasing the rate of Cox1 synthesis (24,27,29,33,34). We examined the distribution of Mss51 in the Cox1 point mutants when CcO assembly was blocked by deletion of Pet111 (causing the absence of Cox2).…”

“…Progression of CcO assembly promotes release of Mss51 and Ssc1 from the COA complexes (24,26), making Mss51 available for further translational activation. At this point, Pet54 is required to render Mss51 competent for efficient translational activation of the COX1 mRNA (27). If CcO assembly is blocked, then Cox1 synthesis decreases because Mss51 is sequestered in the COA complexes and thus is unavailable for translational activation (21,26).…”