S. cerevisiae CMC2 codes for a protein required for respiratory growth. A, sequence alignment of S. cerevisiae CMC1 and CMC2. Gray arrows indicate conserved cysteine residues. B, the respiratory competent wild-type strain W303-1A (WT), a strain carrying a null allele of cmc2 (Δcmc2 or Δ), and a Δ strain overexpressing either CMC1 or CMC2 were grown overnight in liquid YPD media. 10-Fold serial dilutions of the two strains were plated on solid YPD or YPEG medium and incubated at 30 °C. Pictures were taken after 3 days of incubation. C, KCN-sensitive endogenous cell respiration measured polarographically in the presence of galactose. The bars indicate the mean ± S.D. from at least three independent sets of measurements.

Mitochondrial localization and abundance of Cmc2. A, Cmc2 is a mitochondrial protein. Mitochondria (Mt) and the postmitochondrial supernatant (PMS) fraction were isolated from the W303-1A strain. Samples of the two fractions corresponding to 40 μg of protein were analyzed by Western blotting using antibodies against Cmc2 and the cytosolic marker 3-phosphoglycerate kinase subunit 1 (Pgk1). B, Cmc2 is a membrane protein. Soluble (S) and membrane-bound (P) mitochondrial proteins were separated from 40 μg of total mitochondria by brief/gentle sonication followed by centrifugation at 35,000 × g for 30 min at 4 °C. Samples were analyzed by Western blotting using antibodies against Cmc2, the soluble intermembrane space protein cytochrome b₂, and the inner membrane intrinsic protein Shy1. C, Cmc2 is an extrinsic protein. The pellet from B was resuspended in 0.6 m sorbitol, 20 mm HEPES buffer containing 0.1 m Na₂CO₃, pH 11, and 50 mm EDTA and incubated on ice for 30 min. Centrifugation at 35,000 × g for 30 min at 4 °C allows the separation of the extrinsic proteins present in the supernatant (CS) from the intrinsic proteins in the pellet (CP). The different samples were analyzed by Western blotting using antibodies against Cmc2 and Shy1. D, Cmc2 is a membrane protein facing the intermembrane space. Four aliquots of 40 μg of mitochondrial protein were pelleted and resuspended in buffer containing either 20 mm HEPES or 0.6 m sorbitol, 20 mm HEPES. One aliquot in each buffer was supplemented with a final 100 μg/ml of proteinase K (PK) and incubated on ice for 60 min. The reaction was stopped with 2 mm phenylmethylsulfonyl fluoride. Mitochondria and mitoplasts were recovered by centrifugation at 40,000 × g for 15 min at 4 °C. The pelleted fraction was resuspended in gel buffer and loaded on a 10% Tris Tricine gel. Western blots were probed with antibodies against Cmc2, Sco1, Mss51, and cytochrome b₂. E, Cmc2 is an inner mitochondrial membrane protein. Isolated mitochondria were fractionated into inner and outer membranes by sonication plus sucrose gradient sedimentation as described (48). Whole mitochondria (M), inner membranes (IM), and outer membranes (OM) were analyzed by Western blotting using antibodies against porin (outer membrane marker), Cox3 (inner membrane marker), and Cmc2. F, purification of recombinant Cmc2. Recombinant Cmc2 was affinity purified using a combination of GST pulldown and size exclusion chromatography as described under ”Experimental Procedures.“ Subsequent fractions were analyzed by SDS-PAGE and Coomassie staining. L, cell lysate; G, material eluted from the GST column; C, digestion with precision protease to cleave GST off Cmc2; P, size exclusion chromatography yielding purified recombinant Cmc2, respectively. G, concentration of Cmc2 and Cmc1 in wild-type mitochondria. Western blots of the indicated amounts of W303-1A mitochondria and purified Cmc2 (panel F) and Cmc1 (19). The density of the signals was used to estimate the concentration of Cmc1 and Cmc2 in the mitochondrial samples.

Mitochondrial superoxide dismutase 1 activity depends upon Cmc2 levels. A, SOD activity in mitochondria isolated from wild-type, Δcmc2 mutant, and CMC2 overexpressing cells. B, the same samples were solubilized in SDS-sample buffer and separated on a 12% PAGE gel. Mitochondrial Sod1 and porin, a loading control, were visualized by immunoblotting. C, quantification of experiment in panels A and B. D and E, similar to A–C but including mitochondria isolated from wild-type, Δcmc1, and Δcmc2 single mutants and a Δcmc1Δcmc2 double mutant strain. The images were digitalized and densitometry was performed using the histogram function of the Adobe Photoshop program. Data from three independent experiments were included. *, indicates p < 0.01.

Biochemical properties of Δcmc2 cells. A, total mitochondrial cytochrome spectra. Mitochondria from wild-type strain W303, the null mutant Δcmc2, and Δcmc2 cells overexpressing CMC2 were extracted at a protein concentration of 5 mg/ml with potassium deoxycholate under conditions that quantitatively solubilize all the cytochromes (upper panel (54)), or only with salt to exclusively extract cytochrome c (lower panel (40)). Difference spectra of the reduced (sodium dithionite) versus oxidized (potassium ferricyanide) extracts were recorded at room temperature. The absorption bands corresponding to cytochromes a and a₃ have maximum at 603 nm (a and a₃), the maximums for cytochrome b (b) and cytochrome c and c₁ (c, and c₁) are 560 and 550 nm, respectively. B, mitochondrial respiratory chain enzyme spectrophotometric measurements in isolated mitochondria. COX, NADH cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), ubiquinol cytochrome c reductase (QCCR), NADH dehydrogenase, succinate quinone dichlorophenol indophenol reductase (SQDR), and cytochrome c oxidase (COX) were measured as described under ”Experimental Procedures.“ C, steady-state concentrations of COX and bc₁ complex subunits estimated by Western blot analyses of proteins separated in a 12% Tris glycine SDS-PAGE. D, N-acetyl-l-cysteine (NAC)-treated and untreated cells were used to spectrophotometrically measure COX, NCCR, and citrate synthase activities as described (55). The bars indicate the mean ± S.D. from three independent sets of measurements. E, in vivo mitochondrial protein synthesis in the presence of cycloheximide in the indicated strains. The mitochondrial products are identified in the margin.

Physical and genetic interaction of Cmc2 and Cmc1. A, sedimentation properties of Cmc1-HA in a linear 7–20% sucrose gradient were analyzed as described under ”Experimental Procedures.“ The gradient was calibrated with hemoglobin (Hb, 67 kDa) and lactate dehydrogenase (LDH, 130 kDa). Following centrifugation the gradient was collected in 14 equal fractions. Each fraction was assayed for Hb by absorption at 410 nm, and LDH activity by measuring NADH-dependent conversion of pyruvate to lactate. The distribution of Cmc1 was assayed by Western blot analysis. The mass of Cmc2 and Cmc1 were determined from the positions of the respective peaks relative to those of the markers. B, Cmc2 physically interacts with Cmc1. Mitochondria extracted from Δcmc1 and Δcmc2 cells expressed from chromosomally integrated plasmids CMC1-HA and CMC2-3HA, respectively, were extracted with 0.4% lauryl maltoside and 200 mm KCl. The extract (E) was used for immunoprecipitation assays using anti-HA-associated Sepharose-4B. The unbound material in the supernatant (S) and the material bound to the beads (B) were separated by centrifugation and analyzed by Western blotting using antibodies against Cmc1 and Cmc2. The bands shown in the Western blots were quantified by densitometry. In the lower panel, the open bars represent the percentage of the corresponding protein bound to the beads, and the filled bars represent the percentage of unbound protein recovered in the supernatant fraction. C, Cmc1 and Cmc2 interact genetically. Steady-state levels of Cmc1 and Cmc2 in the presence and absence of each protein estimated by Western blotting. The graph on the right panel shows the quantification of the Western blot presented on the left panel. The results of two independent experiments did not differ by more than 5%. The bars indicate the mean ±S.D. from three independent sets of measurements.

Properties and expression of metazoans homologues of yeast CMC2. A, CMC2 is conserved across kingdoms, from yeast to human. Sequence alignment of S. cerevisiae CMC2 homologues from human (Homo sapiens), fish (Danio rerio), frog (Xenopus laevis), fruit fly (Drosophila melanogaster), worm (C. elegans), and plant (Arabidopsis thaliana). Red arrows indicate conserved cysteine residues. B, histochemical staining for cytochrome c oxidase activity in cmc2-silenced C. elegans. Worms fed HT115DE(3) bacteria transformed with empty vector pL4440 were used as negative controls. Silenced (cmc2) and control (pL4440) rrf-3(pk1426) nematodes were fixed, permeabilized, and stained for COX. Control reactions contained the COX-specific inhibitor KCN. Photographs of representative examples of the whole controls and silenced nematodes are shown. C, sequence alignment of human CMC1 and CMC2 proteins. D, localization and genomic structure of the human hCMC2 gene based on the result of a BLAST search of the human genome for homology to the hCMC2 cDNA sequence. The dark blue and gray bars depict the exon and intron regions, respectively. E, subcellular localization of CMC2 in human HeLa cells. Cells were transiently transfected with a cDNA coding for a hemagglutinin-tagged CMC2. The protein was visualized by indirect immunofluorescence using Mitotracker Red and a fluorescently conjugated antibody to human to the hemagglutinin epitope of CMC2-HA (green). A merged image is shown on the right.