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 aI5β 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.

Keywords: Cox1; Mss51; cytochrome c oxidase (Complex IV); mitochondria; mitochondrial DNA (mtDNA); translation initiation; translation regulation; yeast.

FIGURE 1.

Down-regulation of Cox1 synthesis in Δpet54 mutants is independent of the Cox1 C-terminal end. A, mitochondrial translation products from strains carrying the wild-type Cox1 (WT) or the Cox1 lacking the last 15 residues of the C-terminal end (ΔC15) were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide and analyzed by SDS-PAGE and autoradiography. The Δpet54, Δpet122, and Δpet494 mutants were introduced as indicated. Cytb, cytochrome b; Atp6, subunit 6 of ATPase; Var1, ribosomal protein. B, the intensity of the Cox1 labeling in A was quantified using the ImageJ software and normalized to the cytochrome b signal. It was expressed as a percentage of the wild-type Cox1 signal (♦). Error bars, S.D. values from three independent experiments. We also compared the signals of Cox2 with the cytochrome b signal. In these cases, no significant difference was observed. The relevant significant differences between strains (*) were determined by Student's t test. A p value of <0.01 was considered statistically significant.

FIGURE 2.

Down-regulation of Cox1 synthesis in Δpet54 mutants is independent of Cox14 and Coa3. Cox1 or Cox1ΔC15 cells with a deletion in PET54, COX14 (A), or COA3 (B), as indicated, were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide. The mitochondrial products were analyzed by SDS-PAGE and autoradiography. C, growth phenotype of strains bearing the COX1(1–512)::ARG8^m mitochondrial construct. The ARG8^m gene was fused in-frame to the complete COX1 codons, and the black triangle indicates the processing site for the pre-Arg8 matrix-targeting signal. Mutation Δpet54, Δcox14, or Δcox6 was introduced as indicated. Cells were spotted as serial dilutions on medium with arginine (+ARG) or lacking arginine (−ARG) and grown for 2 days at 30 °C.

FIGURE 3.

Pet54 plays a role in COX1 mRNA untranslated regions that depends on the presence of the Cox1 protein. A, the cox1Δ::ARG8^m construct was inserted at the COX1 locus, where the ARG8^m reporter replaced the coding sequence of COX1. In addition, the COX1 coding sequence flanked by the COX2 UTRs was inserted at an ectopic site, 295 bp upstream of COX2 on the mtDNA (20). Mitochondria from cells carrying the wild-type mtDNA or the ectopic chimeric mtDNA were analyzed by SDS-PAGE and Western blotting. The membrane was probed with an antibody for Cox1 and afterward for citrate synthase (CS) as a loading control. The Δpet111 mutation was introduced as indicated. B, serial dilutions of the indicated mutants bearing the ectopic chimeric mtDNA were spotted on medium lacking (−ARG) or containing (+ARG) arginine and were grown for 2 days at 30 °C. C, cells carrying the ectopic chimeric mtDNA and either an empty plasmid or a plasmid expressing a high copy number of PET111 were grown on medium lacking (−ARG) or containing (+ARG) arginine. Serial dilutions were grown for 2 days at 30 °C. D, the COX1 coding region was replaced by the reporter gene ARG8^m (cox1Δ::ARG8^m); however, in this construct, no chimeric COX1 construct was inserted. The indicated mutants bearing this mtDNA were grown on serial dilutions as in B.

FIGURE 4.

Pet54 acts on the COX1 5′-UTR. Cells containing wild-type Pet54 or the Δpet54 mutation were spotted on serial dilutions on medium lacking (−ARG) or containing (+ARG) arginine and incubated for 2–4 days at 30 °C. The cells contained similar mitochondrial genomes as the one shown in Fig. 3A, except that the cox1Δ::ARG8^m gene was flanked by native COX1 5′- and 3′-UTRs (top construct), flanked by the native COX1 5′-UTR and the COX2 3′-UTR (middle), or flanked by the COX2 5′-UTR and the native COX1 3′-UTR (bottom). Black bars, COX1 untranslated regions; gray bars, COX2 untranslated regions. For COX1 3′-UTR replacement, 525 bp of the COX1 downstream sequence were replaced by 118 bp of the COX2 downstream sequence. For COX1 5′-UTR replacement, 505 bp of the COX1 upstream sequence were replaced by 73 bp of the COX2 upstream sequence (40).

FIGURE 5.

Pet54 does not affect the levels of Pet309 and Mss51 proteins and shows a mild genetic interaction with Mss51. A sample of 10 and 50 μg of mitochondrial protein from strains expressing the Mss51–3xHA (A) or Pet309–3xHA (B) as well as the untagged versions of these genes were separated by SDS-PAGE and analyzed by Western blotting. The membranes were probed with antibodies anti-HA and anti-citrate synthase (CS) as a loading control. C, cells carrying the COX1(1512)::ARG8^m construct were transformed with empty plasmid (vector) or with the indicated genes cloned on YEp352 plasmid. The transformants were spotted on synthetic complete medium lacking (−ARG) or containing (+ARG) arginine. 10-Fold serial dilutions were grown for 3 days at 30 °C. The black arrow indicates the processing site for the pre-Arg8 matrix-targeting signal.

FIGURE 6.

Pet54 migrates into a high molecular weight complex that is different from that of the Mss51 complexes. A, a sample of 100 μg of mitochondrial proteins from cells carrying the Mss51–3xHA and Pet54–3xMyc or the untagged proteins was solubilized with 1% digitonin and separated on a 5–13% acrylamide blue native gel. Western blotting was performed with the indicated antibodies after consecutive antibody-stripping treatments. An antibody against Tom40 was used as a loading control. B, mitochondria from the strains in A were analyzed by SDS-PAGE and Western blotting using antibodies against the c-Myc epitope (to detect Pet54–3xMyc) and citrate synthase as a loading control (CS). C, yeast two-hybrid plasmids containing the Pet54, Mss51, and Pet122 coding regions fused in frame with the activation domain (AD) or binding domain (BD) of Gal4 were co-transformed into the yeast two-hybrid strain Pj69-4a (34) as indicated. The double transformants were selected on medium lacking leucine and uracil or leucine and tryptophan. Growth was tested on medium lacking histidine (in the presence of 3 mm 3-aminotriazole) and medium lacking adenine. 10-Fold serial dilutions were grown at 30 °C for 7 days. Growth of cells containing Pet122-AD and Pet54-BD was used as a positive control of two proteins previously demonstrated to interact (29, 33).

FIGURE 7.

Absence of Pet54 has a wild-type migration pattern of Mss51 complexes on blue native PAGE. Mitochondria bearing the untagged Mss51 or the Mss51–3xHA proteins with the indicated mutations Δpet54, Δpet122, or Δcox11 were separated by BN-PAGE. The membrane was probed with antibodies against the HA epitope, Coa3, Cox1, and Tom40 antibodies.

FIGURE 8.

Mss51 is present in the translational active complex but is not competent for efficient synthesis of Cox1 in the absence of Pet54. A, wild type, Δpet54, Δpet122, or the combined mutants with Δcox14 were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide. The mitochondrial products were analyzed by SDS-PAGE and autoradiography. B, mitochondria from the strains in A were separated by BN-PAGE and analyzed by Western blotting using antibodies against the HA epitope (to detect Mss51), Cox1, and Tom40 (as a loading control). C, a sample of 25 μg of mitochondrial proteins was analyzed by SDS-PAGE and Western blotting using antibodies against Cox1, Cox2, Cox3, cytochrome b (Cytb), and citrate synthase as loading control (CS). D, mitochondria from the indicated strains were analyzed by BN-PAGE as in B.

FIGURE 9.

Pet54 might modulate the conformation and/or hemylation of Mss51. Wild-type or Δpet54 cells carrying either the wild-type Mss51 or the mss51^F199I variant were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide. The mitochondrial products were resolved by SDS-PAGE and subjected to autoradiography as in Fig. 1.

FIGURE 10.

Pet54 interacts with COX1 mRNA, and this interaction is independent of Mss51. A, mitochondria were solubilized with dodecyl maltoside, and Pet54–3xMyc or untagged Pet54 (−) was subjected to immunoprecipitation with antibody anti-Myc. One-fourth of the immunoprecipitate (IP) was resolved by SDS-PAGE and transferred to a PVDF membrane for Western blotting. The membrane was probed with anti-Myc antibody and with anti-citrate synthase antibody (CS) as a negative control for interaction. The total fraction represents 5% of the mitochondrial extract used for immunoprecipitation. *, nonspecific bands from the immunoglobulin heavy chain used for immunoprecipitation. ♦, nonspecific bands when the anti-Myc antibody was used. B, RNA was extracted from the total (T) and immunoprecipitate fractions. Each fraction was divided in two, and cDNA was prepared in the presence (+) or absence (−) of reverse transcriptase (RT) using primers for the COX1 and VAR1 5′-UTRs. The (−) RT lanes represent a negative control for DNA contamination. The PCR products were run on an agarose gel. ●, bands due to primer dimers. C and D, mitochondria from cells carrying Pet54–3xMyc or the untagged Pet54 and either the wild type MSS51 or the Δmss51 deletion were treated and analyzed as in A and B.

FIGURE 11.

Model of the role of Pet54 on regulation of Cox1 synthesis. Interaction between translational-effective Mss51^TE and Cox1 renders Mss51 inactive (Mss51^TI, depicted in gray). This might happen in a heme- and/or conformation-dependent manner (step 1). Upon CcO assembly, Mss51^TI is released from COA complex (step 2). Pet54 interacts with the COX1 mRNA 5′-UTR (step 3). Via its interaction with Mss51^TI, Pet54 may drive Mss51^TI reactivation, thus bridging an interaction of Mss51^TE with COX1 mRNA (step 4). In the absence of Pet54, Mss51^TI is released from the COA complex but is unable to be recycled toward a Mss51^TE conformation.