The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast.

Yeast cytochrome c oxidase contains three large subunits made in mitochondria and at least six smaller subunits made in the cytoplasm. There is evidence that the catalytic centers (heme a and copper) are associated with the mitochondrially-made subunits, but the role of the cytoplasmically-made subunits has remained open. Using a gene interruption technique, we have now constructed a Saccharomyces cerevisiae mutant which lacks the largest of the cytoplasmically-made subunits (subunit IV). This mutant is devoid of cyanide-sensitive respiration, the absorption spectrum of cytochrome aa3 and cytochrome c oxidase activity. It still contains the other cytochrome c oxidase subunits but these are not assembled into a stable complex. Active cytochrome c oxidase was restored to the mutant by introducing a plasmid-borne wild-type subunit IV gene; no restoration was seen with a gene carrying an internal deletion corresponding to amino acid residues 28-66 of the mature subunit. Subunit IV is thus necessary for proper assembly of cytochrome c oxidase.     

Regulation of the nuclear-coded peptides of yeast cytochrome c oxidase

We have analyzed the catabolite regulation of cytochrome oxidase by assaying changes in the synthesis of precursors of the nuclear-coded peptides (IV--VII) of cytochrome c oxidase in an in vitro reticulocyte cell-free system programmed with RNA isolated from cells grown in either glucose or raffinose. As a first step, we have characterized antibodies which bind to the precursors of subunits V and VI. Initial translation products for subunits IV and VII have also been tentatively identified by utilizing these antibodies. The messenger RNAs coding for the precursors of the nuclear-coded subunits fall in the expected size range of 8--15 S. Catabolite repression of the nuclear-coded oxidase peptides appears to be regulated by the abundance of their messenger RNAs. Translation of messenger RNA isolated from yeast cells grown on glucose indicates a coordinate and uniform increase in precursor synthesis during glucose derepression. In contrast, when RNA isolated from raffinose (derepressed) grown cells is used to direct cell-free translation, precursor abundance is high throughout growth, although the synthesis of some of the species changes in a complex pattern of ratio and abundance. These data indicate that the abundance of the messengers for the nuclear-coded precursors is regulated in a fashion dependent on the physiologic state of the cell.     

Thermal stability of membrane-reconstituted yeast cytochrome c oxidase

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit IV of human cytochrome c oxidase, polymorphism and a putative isoform.

As part of our study of isoenzyme forms of human cytochrome c oxidase, we purified subunit IV from human heart and skeletal muscle with reversed-phase HPLC and determined the N-terminal amino acid sequences and the electrophoretic mobility. The N-terminus of human heart subunit IV proved to be ragged with 30% of the protein lacking the first three residues. Also a Tyr/Phe polymorphism was observed at residue 16. No differences in N-terminal sequence and electrophoretic mobility were observed between subunit IV of cytochrome c oxidase from human heart and skeletal muscle. Therefore, our results suggest that identical subunits IV are present in cytochrome c oxidase from human heart and skeletal muscle. A putative isoform of subunit IV with a blocked N-terminus was purified from human heart cytochrome c oxidase, which proved to have a different retention time on a reversed-phase column and also a slightly higher electrophoretic mobility on an SDS-polyacrylamide gel compared to the native subunit IV. We could not demonstrate the existence of isoforms of subunit IV in human skeletal muscle.     

Construction of a cDNA clone for a nuclear-coded subunit of cytochrome c oxidase from rat liver

A cDNA library for 6S-9S poly(A)-containing RNA from rat liver was constructed in E. coli. Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3'-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.     

New isoforms of cytochrome c oxidase subunit IV in tuna fish

In the present study, the cDNA sequences of cytochrome c oxidase subunit IV isoforms from tuna fish are reported. The cDNAs share 57% identity among each other and the deduced amino acid sequences of the mature proteins 56% identity. Until now, only in yeast are two isoforms of the corresponding subunit V known, which are expressed in response to the oxygen supply. The hypothetical function of the new isoforms in fish for adaptation to different oxygen partial pressures in tissues of higher organisms is discussed.     

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.     

Synthesis and assembly of cytochrome c oxidase in synchronous cultures of yeast

Yeast cells growing synchronously in glucose medium accumulate in the cytosol, the cytosolically made subunits of cytochrome oxidase, during the G1 and early-S phases. The mitochondrially made subunits, on the other hand, are detected only after the mid-S phase. The cytosolically synthesized subunits are integrated into the membrane after the mid-S phase.     

Intramitochondrial sorting of the precursor to yeast cytochrome c oxidase subunit Va

We have continued our studies on the import pathway of the precursor to yeast cytochrome c oxidase subunit Va (pVa), a mitochondrial inner membrane protein. Previous work on this precursor demonstrated that import of pVa is unusually efficient, and that inner membrane localization is directed by a membrane-spanning domain in the COOH- terminal third of the protein. Here we report the results of studies aimed at analyzing the intramitochondrial sorting of pVa, as well as the role played by ancillary factors in import and localization of the precursor. We found that pVa was efficiently imported and correctly sorted in mitochondria prepared from yeast strains defective in the function of either mitochondrial heat shock protein (hsp)60 or hsp70. Under identical conditions the import and sorting of another mitochondrial protein, the precursor to the beta subunit of the F1 ATPase, was completely defective. Consistent with previous results demonstrating that the subunit Va precursor is loosely folded, we found that pVa could be efficiently imported into mitochondria after translation in wheat germ extracts. This results suggests that normal levels of extramitochondrial hsp70 are also not required for import of the protein. The results of this study enhance our understanding of the mechanism by which pVa is routed to the mitochondrial inner membrane. They suggest that while the NH2 terminus of pVa is exposed to the matrix and processed by the matrix metalloprotease, the protein remains anchored to the inner membrane before being assembled into a functional holoenzyme complex.     

The nuclear-coded subunits of yeast cytochrome c oxidase. II. The amino acid sequence of subunit VIII and a model for its disposition in the inner mitochondrial membrane

The amino acid sequence of subunit VIII from yeast cytochrome c oxidase is reported. This 47-residue (Mr = 5364) amphiphilic polypeptide has a polar NH2 terminus, a hydrophobic central section, and a dilysine COOH terminus. An analysis of local hydrophobicity and predicted secondary structure along the peptide chain predicts that the hydrophobic central region is likely to be transmembranous. Subunit VIII from yeast cytochrome c oxidase exhibits 40.4% homology to bovine heart cytochrome c oxidase subunit VIIc , at the level of primary structure. Secondary structures and hydrophobic domains predicted from the sequences of both polypeptides are also highly conserved. From the location of hydrophobic domains and the positions of charged amino acid residues we have formulated a topological model for subunit VIII in the inner mitochondrial membrane.     

Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme.

Yeast cytochrome c oxidase has been isolated by ion exchange chromatography using lauryl maltoside (n-dodecyl beta-D-maltoside) as the solubilizing detergent. The enzyme prepared in this way has a heme aa3 concentration of 8-9 nmol/mg of protein and a turnover number in the range of 180-210 s-1 at pH 6.2 in 0.01% lauryl maltoside at 20 degrees C. Yeast cytochrome c oxidase prepared by any of several previously published methods which use Triton X-100 contains nine subunits. The enzyme isolated in lauryl maltoside contains these same nine different polypeptides and three others, including homologues of subunits VIa and VIb of the mammalian enzyme.