Characterization of a cmc1 null mutant. (A) The respiration-competent wild-type strain BY4741 (WT) and a strain carrying a null allele of cmc1 (Δcmc1) were grown overnight in liquid YPD medium. Tenfold 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. (B) Respiratory properties of cmc1 mutant cells. KCN-sensitive endogenous cell respiration was measured polarographically in the presence of galactose. Oxygen consumption was also assayed in mitochondria prepared from the BY4741 wild-type strain and the Δcmc1 null mutant in the presence of NADH as the substrate as described in Materials and Methods. The bars indicate the means ± SD from at least three independent sets of measurements. (C) Total mitochondrial cytochrome spectra. Mitochondria from the wild-type strain BY4741 and the Δcmc1 null mutant were extracted at a protein concentration of 5 mg/ml with potassium deoxycholate under conditions that quantitatively solubilize all the cytochromes (39). 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 maxima at 603 nm; the maxima for cytochrome b and for cytochromes c and c₁ are 560 and 550 nm, respectively. (D) Mitochondrial respiratory chain enzyme spectrophotometric measurements in isolated mitochondria. COX and NADH cytochrome c reductase (NCCR) were measured as described in Materials and Methods. (E) Steady-state concentrations of COX subunits estimated by Western blot analyses of proteins separated by 12% Tris-glycine SDS-PAGE are shown in the upper panel. In the lower panel, extracts of 100 μg of mitochondrial proteins, prepared as described in Materials and Methods, were loaded on a BN-polyacrylamide gel. The gel was stained for COX activity. With the conditions used, only the monomeric COX is detected.

Homology of Cmc1p with other proteins involved in copper delivery to COX. (A) Hydropathy plot of Cmc1p obtained by using the algorithm of Kyte and Doolittle (23). The positions of cysteine residues in the Cmc1p sequence are shown by arrows. (B) Protein alignments of Cmc1p and other proteins with conserved CX₉C twin motifs, Cox17p and Cox19p, required for COX assembly and previously shown to bind Cu(I). Conserved cysteines are indicated by asterisks. The known copper binding cysteines in Cox17p (3) are underlined.

Purification of recombinant Cmc1p and reconstitution of CuCmc1p. (A) Recombinant His₆-Cmc1p was affinity purified using a combination of metal affinity and size exclusion chromatography as described in Materials and Methods. Subsequent fractions were analyzed by SDS-PAGE and Coomassie blue staining. CL, cell lysate; FT, flow through the metal column; E, pool of elution fractions; TH, thrombin-cleaved recombinant Cmc1p. (B) Cu(I) fluorescence of CuCmc1p. An excitation of 310 nm exhibits an emission peak with a maximum at ∼560 nm, indicative of Cu(I)-thiolate complexes that are not solvent accessible. The addition of 5 mM KCN, a Cu(I) chelator, to CuCmc1p significantly eliminates the observed emission. (C) Titration of reduced apoCmc1p with Cu(I). Maximal emission is obtained at one equivalent of copper.

Properties and expression of the human gene homologue of yeast CMC. (A) CMC1 is conserved across kingdoms, from yeast to human. A sequence alignment of human (Homo sapiens), bovine (Bos taurus), mouse (Mus musculus), fruit fly (Drosophila melanogaster), and plant (Arabidopsis thaliana) homologues of S. cerevisiae CMC1 is shown. Red arrows indicate conserved cysteine residues. (B) Localization and genomic structure of the hCMC1 gene based on the result of a BLAST search of the human genome for homology to the hCMC1 cDNA sequence. The blue and gray bars depict the exon and intron regions, respectively. (C) Subcellular localization of hCmc1p in human HeLa cells. Cells were transiently transfected with a cDNA coding for an HA-tagged hCmc1p. The protein was visualized by indirect immunofluorescence using fluorescently conjugated antibodies to human Cox I (red) and to the HA epitope of hCMC1p-HA (green). A merged image is shown on the right.

Mitochondrial localization of Cmc1p-HA. (A) Expression of an HA-tagged version of Cmc1p complements the respiratory growth defect of Δcmc1 mutant. The respiration-competent wild-type strain BY4741 (WT), a strain carrying a null allele of cmc1 (Δcmc1), and the mutant strain with a plasmid expressing HA-tagged Cmc1p (CMC1-HA) were grown overnight in liquid YPD medium. Tenfold 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. (B) Cmc1p-HA is a mitochondrial protein. Mitochondria (Mt) and the postmitochondrial supernatant (PMS) fraction were isolated from the CMC1-HA strain. Samples of the two fractions corresponding to 40 μg of protein were separated by SDS-PAGE on a 12% polyacrylamide gel, transferred to nitrocellulose, and probed with antibodies against HA (Cmc1p-HA) or the cytosolic marker 3-phosphoglycerate kinase subunit 1 (Pgk1p). (C) Cmc1p-HA is an extrinsic 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 rpm for 30 min. at 4°C. The pellet (P) 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 rpm 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 loaded on a 10% Tris-Tricine gel, and Western blotting was performed against cytochrome b₂, HA (Cmc1p-HA), Shy1p, and Mss51p. (D) Cmc1p-HA is a membrane protein facing the IMS. 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 final concentration of 100 μg/ml proteinase K (PK) and incubated on ice for 60 min. The reaction was stopped with 2 mM phenylmethylsulfonyl fluoride. Mitochondria (Mt) and mitoplasts (Mp) were recovered by centrifugation at 40,000 rpm 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 Cmc1p-HA, Sco1, porin, cytochrome b₂, and α-ketoglutarate dehydrogenase. (E) Cmc1p is an inner mitochondrial membrane protein. Isolated mitochondria were fractionated into inner and outer membranes by sonication plus sucrose gradient sedimentation as described previously (24). Whole mitochondria (M), inner membranes (IM), and outer membranes (OM) were loaded on a 10% Tris-Tricine gel, and the levels of porin (outer membrane marker), Cox3p (inner membrane marker), and Cmc1p-HA were detected by immunoblotting. (F) Cartoon depicting the submitochondrial localization and topology of Cmc1p and other mitochondrial proteins used as references. (G) Sedimentation properties of Cmc1p-HA in a linear 7 to 20% sucrose gradient were analyzed as explained in Materials and Methods. The gradient was calibrated with hemoglobin (Hb) (67 kDa) and lactate dehydrogenase (LDH) (130 kDa). Following centrifugation, the gradient was collected in 13 equal fractions. Each fraction was assayed for Hb by absorption at 410 nm and for LDH activity by measuring NADH-dependent conversion of pyruvate to lactate. The distribution of Cmc1p was assayed by Western blot analysis. The mass of Cmc1p was determined from the positions of the respective peaks relative to those of the markers.

Copper supplementation alleviates respiratory deficiency in a cmc1 mutant. (A) Wild-type (WT) and Δcmc1 cells were serially diluted 10-fold and then spotted on YPD and YPEG plates and on YPEG plates supplemented with 2.5 and 5 mM CuSO₄. Pictures were taken after the indicated time of incubation. (B) Cells were grown overnight on respiratory medium supplemented with 2.5 or 5 mM CuSO₄. Cells were then harvested, and KCN-sensitive endogenous cell respiration was measured polarographically as described in Materials and Methods. (C) Cells grown as for panel B were harvested, converted to spheroplasts by treatment with Zymolyase, washed, and disrupted by two freezing-thawing cycles. COX activity was measured spectrophotometrically as oxidation of cytochrome c as described in Materials and Methods. The bars indicate the means ± SD from at least three independent sets of measurements.

Mitochondrial Sod1p activity depends upon Cmc1p levels. (A) SOD activity in whole cells and in isolated mitochondria from the indicated strains was measured in an in-gel assay as described in Materials and Methods. WT, wild type. (B) Quantification of the SOD activity staining gel in panel A. The images were digitalized and densitometry performed using the histogram function of the Adobe Photoshop program. (C) SOD activity in mitochondria isolated from wild-type, Δcmc1 mutant, and CMC1-overexpressing cells (upper panel). The same samples were solubilized in SDS sample buffer and separated on a 12% polyacrylamide gel. Mitochondrial Sod1p and porin, a loading control, were visualized by immunoblotting (lower panel). (D) Quantification of the experiment in panel C was performed as for panel B by including data from three independent experiments. *, P < 0.01.