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.

FIGURE 1.

The Cox1 carboxyl-terminal end is required to down-regulate Cox1 synthesis in a cox2Δ mutant. A, model of the S. cerevisiae Cox1 protein, based on the crystal structure of the bovine CcO. The model was constructed using SWISS MODEL, and visualized with MacPymol. Alignment of the yeast and bovine Cox1 sequences revealed that S. cerevisiae has ∼23 additional residues in the C-terminal region, from Lys⁴⁸³ to Asn⁵⁰⁵, which are located at −52 to −29 with respect to the C-terminal end of Cox1 (Loop). Arrows indicate residues on the Cox1 C-terminal end where deletions start. The number in parentheses indicates the position of these residues with respect to the last amino acid of Cox1. B, mitochondrial translation products were labeled with [³⁵S]methionine in the presence of cycloheximide, and proteins were analyzed as described under “Experimental Procedures.” Cells carried either the wild-type Cox1 protein (Cox1), or the Cox1 protein lacking 15 (Cox1ΔC15), 11 (Cox1ΔC11), or 5 (Cox1ΔC5) amino acids of the carboxyl-terminal end. The cox2Δ mutation (Δ) was introduced as indicated. Abbreviations are as follows: cytochrome c oxidase subunit 1, Cox1; subunit 2, Cox2; subunit 3, Cox3; cytochrome b, Cytb; subunit 6 of ATPase, Atp6; and the ribosomal protein, Var1.

FIGURE 2.

The C-terminal end of Cox1 is necessary for stable interaction of Mss51 and Cox14. A, translation in isolated mitochondria from wild-type Cox1 or Cox1ΔC15 strains was performed in the presence of [³⁵S]methionine. Mitochondria were washed and solubilized with 1% digitonin. Mitochondrial extracts were immunoprecipitated with a Myc-specific antibody to precipitate Cox14-Myc or (B) HA-specific antibody to precipitate Mss51-HA. As control, strains lacking the Myc or HA epitopes in Cox14 and Mss51, respectively, were included as indicated. Translation products were separated by SDS-PAGE, and transferred to PVDF membrane before autoradiography. The membranes were incubated with HA-specific antibody to detect Mss51-HA or Myc-specific antibody to detect Cox14-Myc as indicated. The immunoprecipitated fractions in the Western blot with anti-Myc antibody showed an additional, unspecific band, which is probably due to the IgG light chain of the antibody used for co-immunoprecipitation (IP) (*). Total samples represent 5% of the aliquots used for immunoprecipitation.

FIGURE 3.

Interaction of Mss51 with Cox14 is not stable in CcO assembly mutants lacking the C-terminal end of Cox1. Mitochondria from Cox1 or Cox1ΔC15 in the presence of either wild-type COX4 (WT) or cox4Δ (Δ) mutation were solubilized with 1% dodecyl maltoside and immunoprecipitated with a Myc-specific antibody. The immunoprecipitated (IP) fraction was analyzed by Western blot with an antibody to HA, the membrane was stripped and then reprobed with an antibody to Myc. The total fractions represent 5% of mitochondria before solubilization. Western blot with anti-Myc antibody showed a doublet, which is probably due to partial cleavage of the triple Myc epitope.

FIGURE 4.

The Cox1 C-terminal end regulates Cox1 synthesis in many CcO mutants. A, Cox1 (−) or Cox1ΔC15 (+) cells with a deletion in the indicated genes were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide, and proteins were analyzed as described under “Experimental Procedures.” B, quantification of the Cox1 signals from A. The level of Cox1 labeling was normalized to the Cox3/Atp6 signal, and was expressed as a percentage of the wild-type, Cox1 signal (except for signals from the pet54Δ and pet122Δ mutants, which were normalized with respect to Cytb). Error bars indicate standard deviations from 3 independent experiments. We also compared the signal of the cytochrome b to the Cox3/Atp6 signal (or the signal from Cox2 to the cytochrome b in the pet54Δ and pet122Δ mutants), and in those cases no significant difference was observed (data not shown). C, translation of the mitochondrial reporter gene COX1(1–512)::ARG8^m was analyzed by growth of the indicated mutants on media lacking (−Arg) or containing arginine (+Arg). In this construct the precursor of Arg8 was fused to the C-terminal end of the complete Cox1. Cells were spotted as serial dilutions and grown for 3 days at 30 °C.

FIGURE 5.

Synthesis of Cox1 is reduced in a cox6Δ mutant. A, translation of COX1(1–512)::ARG8^m in the indicated mutants was analyzed as described in the legend to Fig. 4C. B, mitochondrial translation products of cells from A were obtained in the presence of cycloheximide and [³⁵S]methionine. In addition, a strain with wild type COX1 was included as control. The Cox1-Arg8 precursor protein is indicated with an arrow. For unknown reasons this fusion is detected as a doublet. C, Cox1 or Cox1ΔC15 cells with a deletion in the indicated genes were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide, and proteins were analyzed as described under “Experimental Procedures.”

FIGURE 6.

Model for assembly-feedback translational regulation of the COX1 mRNA. See text for details. The C-terminal 15 residues of Cox1 are indicated with a thick black line. A thick gray line represents the rest of the Cox1 protein.