Allotopic expression of COX6 elucidates Atco-driven co-assembly of cytochrome oxidase and ATP synthase.

The Cox6 subunit of Saccharomyces cerevisiae cytochrome oxidase (COX) and the Atp9 subunit of the ATP synthase are encoded in nuclear and mitochondrial DNA, respectively. The two proteins interact to form Atco complexes that serve as the source of Atp9 for ATP synthase assembly. To determine if Atco is also a precursor of COX, we pulse-labeled Cox6 in isolated mitochondria of a cox6 nuclear mutant with COX6 in mitochondrial DNA. Only a small fraction of the newly translated Cox6 was found to be present in Atco, which can explain the low concentration of COX and poor complementation of the cox6 mutation by the allotopic gene. This and other pieces of evidence presented in this study indicate that Atco is an obligatory source of Cox6 for COX biogenesis. Together with our finding that atp9 mutants fail to assemble COX, we propose a regulatory model in which Atco unidirectionally couples the biogenesis of COX to that of the ATP synthase to maintain a proper ratio of these two complexes of oxidative phosphorylation.

The Contribution of Tissue Inhibitor of Metalloproteinase-2 Genotypes to Breast Cancer Risk in Taiwan.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinase-2 and is highly expressed in breast cancer (BC) cases at diagnosis. However, the genetic investigations for the association of TIMP-2 genotypes with BC risk are rather limited. In this study, contribution of TIMP-2 rs8179090, rs4789936, rs2009196 and rs7342880 genotypes to BC risk was examined among Taiwan's BC population. TIMP-2 genotypic profiles were revealed among 1232 BC cases and 1232 controls about their contribution to BC using a PCR-based RFLP methodology. The TIMP-2 rs8179090 homozygous variant CC genotype was significantly higher in BC cases than controls (odds ratio (OR) = 2.76, 95% confidence interval (95%CI) = 1.78-4.28, p = 0.0001). Allelic analysis showed that C allele carriers have increased risk for BC (OR = 1.39, 95%CI = 1.20-1.62, p = 0.0001). Genotypic together with allelic analysis showed that TIMP-2 rs4789936, rs2009196 or rs7342880 were not associated with BC risk. Stratification analysis showed that TIMP-2 rs8179090 genotypes were significantly associated with BC risk among younger (≤55) aged women, not among those of an elder (>55) age. Last, rs8179090 genotypes were also associated with triple negative BC. This study sheds light into the etiology of BC in Taiwanese women. Rs8179090 may be incorporated into polygenic risk scores and risk prediction models, which could aid in stratifying individuals for targeted breast cancer screening.

The Contribution of Interleukin-8 Rs4073 Genotypes to Triple Negative Breast Cancer Risk in Taiwan.

BACKGROUND/AIM: Triple negative breast cancer (TNBC) is one of the most challenging breast cancer types. Interleukin-8 (IL-8) is a pro-tumorigenic cytokine, promoting tumor proliferation and migration. This study aimed to examine the contribution of IL-8 rs4073 genotypes to breast cancer risk and provide a summary of related literature. MATERIALS AND METHODS: IL-8 genotypic profiles were determined among 1,232 breast cancer cases and 1,232 controls via polymerase chain reaction-restriction fragment length polymorphism methodology. RESULTS: The IL-8 rs4073 AT and AA genotypes had significantly lower prevalence in the case group compared to control group. Allelic frequency analysis showed that individuals carrying the A allele have relatively decreased risk for breast cancer. The stratification analysis showed that IL-8 rs4073 genotypes were protective markers for those with younger (≤55) age. CONCLUSION: IL-8 rs4073 A allele is a novel predictor for breast cancer, especially TNBC.

Contribution of Interleukin-10 Genotype to Triple Negative Breast Cancer Risk.

BACKGROUND/AIM: Triple negative breast cancer (TNBC) is characterized by increased recurrence and poor survival. Mounting evidence suggests that interleukin-10 (IL-10) plays a role in carcinogenesis, however, little is known about the contribution of IL-10 to TNBC. The study evaluated the contribution of IL-10 promoter A-1082G (rs1800896), T-819C (rs3021097), A-592C (rs1800872) genotypes to the risk of TNBC. MATERIALS AND METHODS: IL-10 genotypes were examined among 1,232 breast cancer patients and 1,232 controls and evaluated. RESULTS: The percentages of AG and GG for IL-10 A-1082G genotypes were higher in the breast cancer patient group than in the control group. The GG genotype carriers were of higher risk for breast cancer [odds ratio (OR)=2.02, 95% confidence interval (CI)=1.28-3.21, p=0.0021]. Interestingly, G allele carriers were of higher risk of TNBC (OR=1.25, 95%CI=1.07-1.46, p=0.0050). CONCLUSION: The G allele of IL-10 A-1082G genotype may serve as a predictor for TNBC risk. The finding should be validated in other populations.

Interleukin-13 Promoter Genotypes and Taiwanese Breast Cancer Susceptibility.

BACKGROUND/AIM: The current study aimed at evaluating the contribution of IL-13 promoter rs1881457 and rs1800925 genotypes to the risk of breast cancer in Taiwan. MATERIALS AND METHODS: A total of 1,232 breast cancer cases and 1,232 age-matched controls were genotyped by typical polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. RESULTS: As for IL-13 rs1881457, the rates of AA, AC and CC genotypes were 54.8, 37.9 and 7.3% among the cases, and 53.8, 38.7 and 7.5% among the healthy controls, respectively; there were no statistically significant differences between the two groups (p for trend=0.8889). Also, regarding IL-13 rs1800925, there were no statistically significant differences between the two groups either (p for trend=0.6803). Furthermore, the allelic frequencies for IL-13 rs1881457 and rs1800925 were not differentially distributed between the case and control groups (p=0.6515 and 0.8753, respectively). CONCLUSION: The rs1881457 and rs1800925 IL-13 promoter polymorphisms may not serve as breast cancer susceptibility determinants for Taiwanese.

Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase.

Mitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX). Pulse-chase experiments indicated a correlation between the loss of newly translated Atp9 complexed to Cox6 and an increase of newly formed Atp9 ring, but did not exclude the possibility of an alternate source of Atp9 for ring formation. Here we have extended studies on the functions and structure of this complex, referred to as Atco. We show that Atco is the exclusive source of Atp9 for the ATP synthase assembly. Pulse-chase experiments show that newly translated Atp9, present in Atco, is converted to a ring, which is incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship. Even though Atco does not contain the ring form of Atp9, cross-linking experiments indicate that it is oligomeric and that the inter-subunit interactions are similar to those of the bona fide ring. We propose that, by providing Atp9 for biogenesis of ATP synthase, Atco complexes free Cox6 for assembly of COX. This suggests that Atco complexes may play a role in coordinating assembly and maintaining proper stoichiometry of the two oxphos enzymes.

Modular assembly of yeast mitochondrial ATP synthase and cytochrome oxidase.

The respiratory pathway of mitochondria is composed of four electron transfer complexes and the ATP synthase. In this article, we review evidence from studies of Saccharomyces cerevisiae that both ATP synthase and cytochrome oxidase (COX) are assembled from independent modules that correspond to structurally and functionally identifiable components of each complex. Biogenesis of the respiratory chain requires a coordinate and balanced expression of gene products that become partner subunits of the same complex, but are encoded in the two physically separated genomes. Current evidence indicates that synthesis of two key mitochondrial encoded subunits of ATP synthase is regulated by the F1 module. Expression of COX1 that codes for a subunit of the COX catalytic core is also regulated by a mechanism that restricts synthesis of this subunit to the availability of a nuclear-encoded translational activator. The respiratory chain must maintain a fixed stoichiometry of the component enzyme complexes during cell growth. We propose that high-molecular-weight complexes composed of Cox6, a subunit of COX, and of the Atp9 subunit of ATP synthase play a key role in establishing the ratio of the two complexes during their assembly.

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.

Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast Saccharomyces cerevisiae, COX is composed of 11-13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450-550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.

Contribution of excision repair cross-complementing group 1 genotypes to triple negative breast cancer risk.

Compared with other subgroups of breast cancer, triple negative breast cancer (TNBC) is considered to be the one with the greatest invasiveness and metastatic mobility, and the highest recurrence rate. Considering the lack of predictive markers for TNBC, we aimed to examine the contribution of excision repair cross complementing-group 1 (ERCC1) genotypes to TNBC. The rs11615 and rs3212986 of ERCC1 were investigated and evaluated for their associations with susceptibility to breast cancer, especially TNBC, in Taiwan. In this study, 1,232 breast cancer patients (104 were TNBC) and 1,232 healthy controls were recruited and their genotypes at ERCC1 rs11615 and rs3212986 were revealed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Our results indicated that genotypes of ERCC1 rs11615 (Ptrend = 2.2*10E-9), but not rs3212986 (Ptrend = 0.6181), were associated with breast cancer risk. In the allelic frequency distribution analysis, breast cancer patients carried the T allele of ERCC1 rs11615 a higher rate than the control subjects, further supporting the idea that ERCC1 rs11615 TT genotype is positively associated with breast cancer susceptibility. More importantly, the frequency of the ERCC1 rs11615 TT genotype was even higher among TNBC patients than among other subtypes of breast cancer patients (P = 0.0001, odds ratio = 1.73, 95% confidence interval = 1.15-2.63). The genotypes of ERCC1 rs11615 were not associated with Ki67 status. Our findings firstly show that the T allele of ERCC1 rs11615 can serve as a predictive biomarker for breast cancer and TNBC. We believe that ERCC1 could serve as a target for personalized treatment of breast cancer, especially for TNBC.

The Association of Matrix Metalloproteinase-1 Promoter Polymorphisms with Breast Cancer.

BACKGROUND/AIM: The family of matrix metalloproteinases (MMPs) are responsible for the homeostasis of extracellular matrix components and their genetic polymorphisms may be associated with cancer susceptibility. The serum levels of MMP-1 have been reported to be lower in breast cancer patients than healthy subjects. In the current study, we aimed at investigating the contribution of a polymorphism in the promoter region of MMP-1 to breast cancer in Taiwan. MATERIALS AND METHODS: The MMP-1 rs1799705 polymorphic genotypes were genotyped among 1,232 breast cancer patients and 1,232 healthy controls by the typical polymerase chain reaction-restriction fragment length polymorphism methodology. RESULTS: The percentages of 2G/2G, 1G/2G, and 1G/1G for MMP1 -1607 genotypes were 35.4, 40.6 and 24.0% in the breast cancer group and 34.1, 43.6, and 22.3% in the healthy control group (p trend=0.3025), respectively. The odds ratios (ORs) after adjusting for age, smoking and alcohol drinking status for those carrying 1G/2G and 1G/1G genotypes at MMP1 -1607 were 0.93 (95%CI=0.76-1.11, p=0.2390) and 1.01 (95%CI=0.77-1.23, p=0.7377), respectively, compared to those carrying the wild-type 2G/2G genotype. Supporting this finding, the adjusted OR for those carrying the 1G allele at MMP-1 -1607 was 1.03 (95%CI=0.91-1.18, p=0.8860), compared to those carrying the wild-type 2G allele. Our findings suggest that the polymorphic genotypes at MMP1 promoter -1607 investigated in the current study, may not play a major role in determining cancer susceptibility to breast cancer in Taiwan. Other early diagnostic and predictive markers are urgently needed for personalized and precise breast cancer detection and therapy.

Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase.

Mitochondrial cytochrome oxidase (COX) catalyzes the last step in the respiratory pathway. In the yeast Saccharomyces cerevisiae, this inner membrane complex is composed of 11 protein subunits. Expression of COX is assisted by some two dozen ancillary proteins that intercede at different stages of the assembly pathway. One such protein, Cox16p, encoded by COX16, was shown to be essential for the activity and assembly of COX. The function of Cox16p, however, has not been determined. We present evidence that Cox16p is present in Cox1p assembly intermediates and in COX. This is based on the finding that Cox16p, tagged with a dual polyhistidine and protein C tag, co-immunopurified with Cox1p assembly intermediates. The pulldown assays also indicated the presence of Cox16p in mature COX and in supercomplexes consisting of COX and the bc1 complex. From the Western signal strengths, Cox16p appears to be substoichiometric with Cox1p and Cox4p, which could indicate that Cox16p is only present in a fraction of COX. In conclusion, our results indicate that Cox16p is a constituent of several Cox1p assembly intermediates and of COX.

Matrix Metalloproteinase-1 Genetic Polymorphism in Breast Cancer in Taiwanese.

AIM: Metalloproteinases (MMPs) are a family of enzymes involved in many physiological processes, such as skeletal development, wound healing, and scar formation, as well as carcinogenesis. However, the contribution of MMP1 genotype to breast cancer has not been elucidated. This study aimed to evaluate the contribution of commonly studied MMP1 promoter 1607 genotype to breast cancer risk. MATERIALS AND METHODS: In this hospital-based case-control study, contribution of MMP1 genotype to breast cancer risk was evaluated among 1,232 patients with breast cancer and 1,232 gender-matched healthy controls. RESULTS: The distribution of 2G/2G, 1G/2G and 1G/1G for MMP1 promoter 1607 genotype was 36.0%, 41.3% and 22.7% in the breast cancer group and 34.2%, 44.5% and 21.3% in the non-cancer group, respectively (p for trend=0.2820). We also analyzed the allelic frequency distributions and found that the variant 1G allele of MMP1 promoter 1607 conferred similar breast cancer susceptibility as the wild-type 2G allele (odds ratio=0.99, 95% confidence interval=0.89-1.11, p=0.8858). There was no interaction between MMP1 promoter 1607 genotype and cigarette smoking or alcohol drinking habits. CONCLUSION: The genotype of MMP1 promoter 1607 may not be a major determining factor for breast cancer risk. The contribution of MMP1 promoter 1607 genotype to prognosis and subtypes of breast cancer needs further investigation.

The contributions of the tissue inhibitor of metalloproteinase-1 genotypes to triple negative breast cancer risk.

The tissue inhibitors of metalloproteinases (TIMPs) are a family of multifunctional proteins which have been shown to be upregulated in various types of cancers. However, the contribution of TIMPs in breast cancer is not fully understood, not to mention triple negative breast cancer (TNBC). This study's aim was to evaluate the contribution of TIMP-1 rs4898, rs6609533, and rs2070584 genotypes to the risk of breast cancer, especially the subtype of TNBC. The contributions of these TIMP-1 genotypes to cancer risk were examined among 1232 breast cancer patients and 1232 healthy controls, and several clinicopathologic factors were also analyzed. The results showed that the percentages of CC, CT, and TT of TIMP-1 rs4898 were differentially distributed at 28.5%, 33.1% and 38.4% in the breast cancer patient group and 34.5%, 41.0% and 24.5% in the control group, respectively (P for trend = 7.99*10(-13)). It was also found that the CC genotype carriers were of increased risk for breast cancer (odds ratio = 1.90, 95% confidence interval = 1.55-2.33, P = 0.0001) than the TT genotype carriers. In addition, we analyzed the allelic frequency distributions of all three TIMP-1s, and the results showed that the C allele of TIMP-1 rs4898 contributes to an increase in breast cancer susceptibility (P = 2.41*10(-12)). On the other hand, there was no difference found in the distribution of genotypic or allelic frequencies among the patients and the controls for TIMP-1 rs6609533 and rs2070584. Thus, it is our conclusion that the CC genotype of TIMP-1 rs4898 compared to the TT wild-type genotype may increase the risk for breast cancer, especially TNBC in Taiwan, and may serve as an early detective and predictive marker.

Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p.

Expression of the mitochondrially encoded ATP6 and ATP8 genes is translationally regulated by F1 ATPase. We report a translational repressor (Smt1p) of the ATP6/8 mRNA that, when mutated, restores translation of the encoded Atp6p and Atp8p subunits of the ATP synthase. Heterozygous smt1 mutants fail to rescue the translation defect, indicating that the mutations are recessive. Smt1p is an intrinsic inner membrane protein, which, based on its sedimentation, has a native size twice that of the monomer. Affinity purification of tagged Smt1p followed by reverse transcription of the associated RNA and PCR amplification of the resultant cDNA with gene-specific primers demonstrated the presence in mitochondria of Smt1p-ATP8/ATP6 and Smt1p-COB mRNA complexes. These results indicate that Smt1p is likely to be involved in translational regulation of both mRNAs. Applying Occam's principle, we favor a mechanistic model in which translation of the ATP8/ATP6 bicistronic mRNA is coupled to the availability of F1 for subsequent assembly of the Atp6p and Atp8p products into the ATP synthase. The mechanism of this regulatory pathway is proposed to entail a displacement of the repressor from the translationally mute Smt1-ATP8/ATP6 complex by F1, thereby permitting the Atp22p activator to interact with and promote translation of the mRNA.

Contribution of DNA Double-strand Break Repair Gene XRCC3 Genotypes to Triple-negative Breast Cancer Risk.

AIM: The DNA-repair gene X-ray repair cross-complementing group 3 (XRCC3) is important in DNA double-strand break repair and plays a critical part in initiation of carcinogenesis. Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype with no existing gene-targeting drugs and little knowledge on its genetic etiology. This study aimed to investigate the contribution of the XRCC3 genotype to individual TNBC susceptibility. MATERIALS AND METHODS: A total of 2,464 Taiwan citizens consisting of 1,232 breast cancer cases and 1,232 controls were enrolled in this case-control study, and genotyping of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539 and rs28903081 were performed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). We also conducted risk-stratified sub-group analyses to determine the association between the genotype and age- and hormone-related characteristics of breast cancer sub-groups. RESULTS: There was no significant difference between breast cancer and control groups in the distributions of the genotypic or allelic frequencies as for the XRCC3 rs1799794 (p=0.5195 and 0.9545), rs45603942 (p=0.3478 and 0.1449), rs861530 (p=0.4567 and 0.5081), rs3212057 (p=1.0000 and 1.0000), rs1799796 (p=0.8487 and 0.7315) and rs28903081 (p=1.0000 and 1.0000), respectively. However, the XRCC3 rs861539 TT genotype was more prevalent in patients with breast cancer [odds ratio (OR)=2.99, 95% confidence interval (CI)=1.62-5.55; p=0.0002], and especially among those who were younger than 55 years (OR=2.61, 95% CI=1.82-3.73; p=0.0001), with first menarche earlier than 12.2 years (OR=2.47, 95% CI=1.74-3.52; p=0.0001), with menopause at 49.0 years old or later (OR=2.53, 95% CI=1.76-3.62; p=0.0001), or with TNBC (OR=2.05, 95% CI=1.46-4.28; p=4.63*10(-4)). CONCLUSION: XRCC3 rs861539 TT is a potential predictive marker for TNBC in Taiwanese women and investigations in other populations are warranted for further universal application in cancer detection and prediction.

Contribution of personalized Cyclin D1 genotype to triple negative breast cancer risk.

Aim: Cell cycle regulator cyclin D1 (CCND1) is a pivotal regulator for G1/S phase transition, playing a critical part in initiation of carcinogenesis. Triple negative breast cancer comprises a very heterogeneous group of cancer cells, but little is known about what is wrong in the genome of these patients. This study investigated contribution of CCND1 genotype to individual triple negative breast cancer susceptibility. Materials: In all, 2464 native Taiwan subjects consist of 1232 breast cancer cases and 1232 controls were enrolled in a hospital-based, case-control study. CCND1 A870G (rs9344) genotyping was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Risk-stratified analyses correlated genotype and age-related characteristics of breast cancer subgroups. Results: No significant difference was found between patient and control groups in distribution of genotypic and allelic frequencies in CCND1 genotype, yet CCND1 A870G (rs9344) GG genotype was far less prevalent in breast cancer patients younger than 55 years (OR=0.62, 95%CI=0.43-0.89, P=0.0362), with first menarche earlier than 12.2 years (OR=0.61, 95% CI=0.42-0.87, P=0.0241), with menopause earlier than 49.0 years (OR=0.57, 95%CI=0.39-0.82, P=0.0093), or showing triple-negative breast cancer (OR=0.28, 95%CI=0.13-0.62, P=0.0006). Such valuable findings suggest CCND1 A870G (rs9344) as a predictive marker for triple negative breast cancer in Taiwanese women; the authors sincerely hope these help us fight the toughest subtype in clinical management.

Evaluation of the contribution of cyclooxygenase 2 genotypes to breast cancer in Taiwan.

Overexpression of cyclooxygenase 2 (COX-2) has been suggested to be associated with breast carcinogenesis. The aim of the present study was to evaluate the contribution of genotypic polymorphisms in COX-2 to breast cancer risk of Taiwanese females. In total, 1,232 breast cancer patients and 1,232 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Six polymorphic variants of COX-2, including G-1195A (rs689466), G-765C (rs20417), T8473C (rs5275), intron 1 (rs2745557), intron 5 (rs16825748) and intron 6 (rs2066826) were examined. The results showed that the GC genotype of COX-2, G-765C was associated with a lower risk compared to the wild-type GG genotype (odds ratio(OR)=0.66, 95% confidence interval(CI)=0.53-0.83, p=0.0005). The C allele of COX-2 G-765C was significantly more frequently found in controls than in cancer patients (p=0.0006). In addition, the OR of the GG/AG+AA, GC/GG and GC/AG+AA at G-765C/Intron 1 combined genotypes compared to wild-type GG/GG genotype were 0.79 (95%CI=0.66-0.96; p=0.0166), 0.61 (95%CI=0.48-0.78; p=0.0001), and 0.71 (95%CI=0.36-1.37; p=0.3040), respectively. As for the combination of G-765C and intron 6, the OR of the GG/AG+AA, GC/GG and GC/AG+AA combined genotypes compared with wild-type GG/GG reference genotype were 0.79 (95%CI=0.62-1.01; p=0.0561), 0.63 (95%CI=0.50-0.81; p=0.0003), and 0.68 (95%CI=0.38-1.21; p=0.1897), respectively. Our results indicate that the C allele of COX-2, G-765C was associated with a decreased risk of breast cancer in Taiwan, and could serve as an early detection and predictive marker for breast cancer risk.

Assembly of the rotor component of yeast mitochondrial ATP synthase is enhanced when Atp9p is supplied by Atp9p-Cox6p complexes.

The Atp9p ring is one of several assembly modules of yeast mitochondrial ATP synthase. The ring, composed of 10 copies of Atp9p, is part of the rotor that couples proton translocation to synthesis or hydrolysis of ATP. We present evidence that before its assembly with other ATP synthase modules, most of Atp9p is present in at least three complexes with masses of 200-400 kDa that co-immunopurify with Cox6p. Pulse-labeling analysis disclosed a time-dependent reduction of radiolabeled Atp9p in the complexes and an increase of Atp9p in the ring form of wild type yeast and of mss51, pet111, and pet494 mutants lacking Cox1p, Cox2p, and Cox3p, respectively. Ring formation was not significantly different from wild type in an mss51 or atp10 mutant. The atp10 mutation blocks the interaction of the Atp9p ring with other modules of the ATP synthase. In contrast, ring formation was reduced in a cox6 mutant, consistent with a role of Cox6p in oligomerization of Atp9p. Cox6p involvement in ATP synthase assembly is also supported by studies showing that ring formation in cells adapting from fermentative to aerobic growth was less efficient in mitochondria of the cox6 mutant than the parental respiratory-competent strain or a cox4 mutant. We speculate that the constitutive and Cox6p-independent rate of Atp9p oligomerization may be sufficient to produce the level of ATP synthase needed for maintaining a membrane potential but limiting for optimal oxidative phosphorylation.

The Cox3p assembly module of yeast cytochrome oxidase.

Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis. This suggests that Cox4p, which contacts Cox1p and Cox3p in the holoenzyme, can be incorporated into COX by two alternative pathways. In addition to subunits of COX, some Cox3p intermediates contain Rcf1p, a protein associated with the supercomplex that stabilizes the interaction of COX with the bc1 (ubiquinol-cytochrome c reductase) complex. Finally, our results indicate that although assembly of the Cox1p module is not contingent on the presence of Cox3p, the converse is not true, as none of the Cox3p subassemblies were detected in a mutant blocked in translation of Cox1p. These studies support our proposal that Cox3p and Cox1p are separate assembly modules with unique compositions of ancillary factors and subunits derived from the nuclear genome.