Coa2 is an assembly factor for yeast cytochrome c oxidase biogenesis that facilitates the maturation of Cox1

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is dependent on a new assembly factor designated Coa2. Coa2 was identified from its ability to suppress the respiratory deficiency of coa1Delta and shy1Delta cells. Coa1 and Shy1 function at an early step in maturation of the Cox1 subunit of CcO. Coa2 functions downstream of the Mss51-Coa1 step in Cox1 maturation and likely concurrent with the Shy1-related heme a(3) insertion into Cox1. Coa2 interacts with Shy1. Cells lacking Coa2 show a rapid degradation of newly synthesized Cox1. Rapid Cox1 proteolysis also occurs in shy1Delta cells, suggesting that in the absence of Coa2 or Shy1, Cox1 forms an unstable conformer. Overexpression of Cox10 or Cox5a and Cox6 or attenuation of the proteolytic activity of the m-AAA protease partially restores respiration in coa2Delta cells. The matrix-localized Coa2 protein may aid in stabilizing an early Cox1 intermediate containing the nuclear subunits Cox5a and Cox6.     

Analysis of Leigh Syndrome Mutations in the Yeast SURF1 Homolog Reveals a New Member of the Cytochrome Oxidase Assembly Factor Family

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F²⁴⁹T and Y³⁴⁴D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G¹³⁷E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G¹³⁷E Shy1 mutant phenocopied shy1Δ cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G¹³⁷E Shy1 mutant revealed Coa2, Cox10, and a novel factor designated Coa4. Coa2 and Cox10 are previously characterized CcO assembly factors. Coa4 is a twin CX₉C motif mitochondrial protein localized in the intermembrane space and associated with the inner membrane. Cells lacking Coa4 are depressed in CcO activity but show no impairment in Cox1 maturation or formation of the Shy1-stabilized Cox1 assembly intermediate. To glean insights into the functional role of Coa4 in CcO biogenesis, an unbiased suppressor screen of coa4Δ cells was conducted. Respiratory function of coa4Δ cells was restored by the overexpression of CYC1 encoding cytochrome c. Cyc1 is known to be important at an ill-defined step in the assembly and/or stability of CcO. This new link to Coa4 may begin to further elucidate the role of Cyc1 in CcO biogenesis.Leigh syndrome (LS) is a highly progressive neurological disorder of infancy characterized by necrotizing lesions in the midbrain and brain stem (32). Humans afflicted with LS have compromised oxidative phosphorylation (OXPHOS) function due to mutations in nuclear or mitochondrial genes encoding respiratory chain components or their assembly factors. Although LS infants are born with a normal appearance, neurological lesions develop within months and dysfunction extends to other organs, resulting in a high mortality rate. LS patients typically have mutations affecting complex I or complex IV (cytochrome c oxidase [CcO]) of the OXPHOS pathway (14). Patients with a specific CcO deficiency most often have mutations in the SURF1 gene that encodes a CcO assembly factor (9, 15, 41).SURF1 is not absolutely required for CcO biogenesis in humans, since SURF1-deficient patient fibroblasts retain 10 to 15% of residual CcO activity (32). The yeast homolog of SURF1 is Shy1 (SURF1 homolog in yeast) and has a conserved function in CcO biogenesis (24). Yeast lacking Shy1 retain residual CcO activity, but growth of the mutant strain is compromised on respiratory, nonfermentable carbon sources (4).Insights into the function of SURF1 in human cells have been gleaned through the characterization of stalled CcO assembly intermediates in cells isolated from SURF1 LS patients using blue native (BN) gel electrophoresis. One intermediate, designated S2, which accumulates in SURF1-deficient patient fibroblasts, consists of Cox1 in association with two nuclear CcO subunits, CoxIV and Va (38, 45, 47). A similar stalled assembly intermediate accumulates in CcO-deficient patients with mutations in two other assembly factors, SCO1 and SCO2. These assembly proteins function in the maturation of the mitochondrially encoded Cox2 subunit and the binuclear copper (Cu_A) site within this subunit. In contrast, studies with patient fibroblasts harboring mutations in the genes encoding Cox10 and Cox15 proteins, which are involved in the biosynthesis of the heme a cofactor used exclusively by CcO (at the heme a and heme a₃:Cu_B sites), show only free Cox1 by BN analysis (1, 2). These data suggest that CcO biogenesis commences with the mitochondrial synthesis and maturation of Cox1, while the other two mitochondrially encoded subunits, Cox2 and Cox3, are added at later stages. The absence of the S2 intermediate in cells with mutations in COX10 or COX15 is consistent with the prediction that the S2 assembly intermediate contains Cox1 with at least the heme a center formed.The first major clue to the function of SURF1 came from studies with the bacterium Rhodobacter sphaeroides, in which surf1 mutant cells showed impairment in the formation of the heme a₃:Cu_B bimetallic center within Cox1 (33). Specifically, heme a and Cu_B were observed spectroscopically with surf1 mutant cells, but heme a₃ was not present. The Cu_B site had an altered spectroscopic signature to compensate for the loss of heme a₃, as the two cofactors typically coordinate with each other. This study suggests Surf1 is involved in the maturation of the heme a₃ site in CcO. In lower eukaryotes, impairment of CcO assembly results in proteolytic degradation of the stalled intermediates (16). Thus, it is not possible to isolate the CcO complex in shy1Δ yeast cells to identify any missing cofactors. However, Shy1 was shown to have a key role in formation of the heterobimetallic Cu_B:heme a₃ center in yeast Cox1 (18). Furthermore, it was recently shown that Surf1 in bacteria is a heme-binding protein (10), although these findings have yet to be confirmed in eukaryotes.Additional insights into the function of SURF1/Shy1 came from the isolation of genetic suppressors of shy1Δ respiratory deficiency in yeast (3). Respiratory function can be partially restored in shy1Δ cells by enhancing Cox1 translation through the overexpression of MSS51 (6), a dual-function protein that acts as a COX1 translational activator in addition to binding to the newly synthesized Cox1 polypeptide. Suppression of the shy1Δ respiratory defect is also observed with enhanced expression levels of the two CcO subunits Cox5a and Cox6 corresponding to the human S2-containing subunits CoxIV and Va (15). Overexpression of COA2, a recently identified CcO assembly factor shown to interact with Shy1, can also suppress the shy1Δ respiratory defect (30). Finally, overexpression of the COX10 gene that encodes the hydroxyfarnesyl transferase, which generates heme o as the first step in heme a biosynthesis, can partially restore respiratory function in shy1Δ cells. Although overexpression of COX10 has only very weak suppressor activity, a marked synergistic effect was apparent in the overexpression of both MSS51 and COX10 (29).Shy1 has a secondary function in yeast in the maintenance of the conserved mitochondrial copper storage pool that is used in the copper metallation of Cox1 and Cox2 during CcO biogenesis. Yeast cells lacking Shy1 contain mitochondria with a partially depleted matrix copper storage pool, and the respiratory defect of shy1Δ cells can be partially reversed by growth in the presence of exogenous copper (29). Similarly, liver and muscle samples from patients with SURF1 mutations exhibit a cellular copper deficiency (37). Maintenance of the matrix copper pool is postulated to be linked to active CcO biogenesis in general, as patient tissue with mutations to two other CcO assembly factors, SCO1 and SCO2, result in a cellular copper deficiency as well (22).Human SURF1 and yeast Shy1 are both mitochondrial proteins tethered to the inner membrane (IM) by two transmembrane (TM) helices with a large central domain projecting into the intermembrane space (IMS). Most LS patients with SURF1 mutations have gene deletions or rearrangements. Missense mutations in SURF1 are quite rare, with only a limited number being reported. These mutations tend to be associated with a mild clinical phenotype, and patient survival is prolonged (28). We selected a subset of known missense mutations, two of which lie within the IMS globular domain and a third that maps to the second TM domain. In an attempt to gain further insights into which functional step of SURF1 was compromised by the missense mutations, we engineered and characterized the corresponding mutations in conserved residues of yeast SHY1. In doing so, we have additionally identified a new member of the CcO assembly factor family, Coa4, that may be linked to the role of cytochrome c in CcO assembly. We show that the respiratory defect of cells lacking Coa4 is specifically suppressed by the overexpression of the IMS electron carrier cytochrome c (CYC1). This is the first time CYC1 has been found as a suppressor of a CcO assembly mutant.     

Selective Oma1 protease-mediated proteolysis of Cox1 subunit of cytochrome oxidase in assembly mutants

Stalled biogenesis of the mitochondrial cytochrome c oxidase (CcO) complex results in degradation of subunits containing redox cofactors. The conserved Oma1 metalloproteinase mediates facile Cox1 degradation in cells lacking the Coa2 assembly factor, but not in a series of other mutants stalled in CcO maturation. Oma1 is activated in coa2Δ cells, but the selective Cox1 degradation does not arise merely from its activation. Oma1 is also active in cells with dysfunctional mitochondria and cox11Δ cells impaired in CcO maturation, but this activation does not result in Oma1-mediated Cox1 degradation. The facile and selective degradation of Cox1 in coa2Δ cells, relative to other CcO assembly mutants, is likely due to impaired hemylation and subsequent misfolding of the subunit. Specific Cox1 proteolysis in coa2Δ cells arises from a combination of Oma1 activation and a susceptible conformation of Cox1.     

Oligomerization of Heme o Synthase in Cytochrome Oxidase Biogenesis Is Mediated by Cytochrome Oxidase Assembly Factor Coa2

The synthesis of the heme a cofactor used in cytochrome c oxidase (CcO) is dependent on the sequential action of heme o synthase (Cox10) and heme a synthase (Cox15). The active state of Cox10 appears to be a homo-oligomeric complex, and formation of this complex is dependent on the newly synthesized CcO subunit Cox1 and the presence of an early Cox1 assembly intermediate. Cox10 multimerization is triggered by progression of Cox1 from the early assembly intermediate to downstream intermediates. The CcO assembly factor Coa2 appears important in coupling the presence of newly synthesized Cox1 to Cox10 oligomerization. Cells lacking Coa2 are impaired in Cox10 complex formation as well as the formation of a high mass Cox15 complex. Increasing Cox1 synthesis in coa2Δ cells restores respiratory function if Cox10 protein levels are elevated. The C-terminal segment of Cox1 is important in triggering Cox10 oligomerization. Expression of the C-terminal 54 residues of Cox1 appended to a heterologous matrix protein leads to efficient Cox10 complex formation in coa2Δ cells, but it fails to induce Cox15 complex formation. The state of Cox10 was evaluated in mutants, which predispose human patients to CcO deficiency and the neurological disorder Leigh syndrome. The presence of the D336V mutation in the yeast Cox10 backbone results in a catalytically inactive enzyme that is fully competent to oligomerize. Thus, Cox10 oligomerization and catalytic activation are separate processes and can be uncoupled.     

Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly

Cytochrome c oxidase (complex IV) of the respiratory chain is assembled from nuclear and mitochondrially-encoded subunits. Defects in the assembly process lead to severe human disorders such as Leigh syndrome. Shy1 is an assembly factor for complex IV in Saccharomyces cerevisiae and mutations of its human homolog, SURF1, are the most frequent cause for Leigh syndrome. We report that Shy1 promotes complex IV biogenesis through association with different protein modules; Shy1 interacts with Mss51 and Cox14, translational regulators of Cox1. Additionally, Shy1 associates with the subcomplexes of complex IV that are potential assembly intermediates. Formation of these subcomplexes depends on Coa1 (YIL157c), a novel assembly factor that cooperates with Shy1. Moreover, partially assembled forms of complex IV bound to Shy1 and Cox14 can associate with the bc1 complex to form transitional supercomplexes. We suggest that Shy1 links Cox1 translational regulation to complex IV assembly and supercomplex formation.     

Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae

Mutations in SURF1, the human homologue of yeast SHY1, are responsible for Leigh's syndrome, a neuropathy associated with cytochrome oxidase (COX) deficiency. Previous studies of the yeast model of this disease showed that mutant forms of Mss51p, a translational activator of COX1 mRNA, partially rescue the COX deficiency of shy1 mutants by restoring normal synthesis of the mitochondrially encoded Cox1p subunit of COX. Here we present evidence showing that Cox1p synthesis is reduced in most COX mutants but is restored to that of wild type by the same mss51 mutation that suppresses shy1 mutants. An important exception is a null mutation in COX14, which by itself or in combination with other COX mutations does not affect Cox1p synthesis. Cox14p and Mss51p are shown to interact with newly synthesized Cox1p and with each other. We propose that the interaction of Mss51p and Cox14p with Cox1p to form a transient Cox14p-Cox1p-Mss51p complex functions to downregulate Cox1p synthesis. The release of Mss51p from the complex occurs at a downstream step in the assembly pathway, probably catalyzed by Shy1p.     

Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria

Regulation of eukaryotic cytochrome oxidase assembly occurs at the level of Cox1 translation, its central mitochondria-encoded subunit. Translation of COX1 messenger RNA is coupled to complex assembly in a negative feedback loop: the translational activator Mss51 is thought to be sequestered to assembly intermediates, rendering it incompetent to promote translation. In this study, we identify Coa3 (cytochrome oxidase assembly factor 3; Yjl062w-A), a novel regulator of mitochondrial COX1 translation and cytochrome oxidase assembly. We show that Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for Mss51 association with these complexes. Mss51 exists in equilibrium between a latent, translational resting, and a committed, translation-effective, state that are represented as distinct complexes. Coa3 and Cox14 promote formation of the latent state and thus down-regulate COX1 expression. Consequently, lack of Coa3 or Cox14 function traps Mss51 in the committed state and promotes Cox1 synthesis. Our data indicate that Coa1 binding to sequestered Mss51 in complex with Cox14, Coa3, and Cox1 is essential for full inactivation.     

The Carboxyl-terminal End of Cox1 Is Required for Feedback Assembly Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

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.     

Evidence for a pro-oxidant intermediate in the assembly of cytochrome oxidase

The hydrogen peroxide sensitivity of cells lacking two proteins, Sco1 and Cox11, important in the assembly of cytochrome c oxidase (CcO), is shown to arise from the transient accumulation of a pro-oxidant heme A-Cox1 stalled intermediate. The peroxide sensitivity of these cells is abrogated by a reduction in either Cox1 expression or heme A formation but exacerbated by either enhanced Cox1 expression or heme A production arising from overexpression of COX15. Sco1 and Cox11 are implicated in the formation of the Cu(A) and Cu(B) sites of CcO, respectively. The respective wild-type genes suppress the peroxide sensitivities of sco1Delta and cox11Delta cells, but no cross-complementation is seen with noncognate genes. Copper-binding mutant alleles of Sco1 and Cox11 that are nonfunctional in promoting the assembly of CcO are functional in suppressing the peroxide sensitivity of their respective null mutants. Likewise, human Sco1 that is nonfunctional in yeast CcO assembly is able to suppress the peroxide sensitivity of yeast sco1Delta cells. Thus, a disconnect exists between the respiratory capacity of cells and hydrogen peroxide sensitivity. Hydrogen peroxide sensitivity of sco1Delta and cox11Delta cells is abrogated by overexpression of a novel mitochondrial ATPase Afg1 that promotes the degradation of CcO mitochondrially encoded subunits. Studies on the hydrogen peroxide sensitivity in CcO assembly mutants reveal new aspects of the CcO assembly process.     

Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) biogenesis is translationally regulated. Mss51, a specific COX1 mRNA translational activator and Cox1 chaperone, drives the regulatory mechanism. During translation and post-translationally, newly synthesized Cox1 physically interacts with a complex of proteins involving Ssc1, Mss51, and Cox14, which eventually hand over Cox1 to the assembly pathway. This step is probably catalyzed by assembly chaperones such as Shy1 in a process coupled to the release of Ssc1-Mss51 from the complex. Impaired COX assembly results in the trapping of Mss51 in the complex, thus limiting its availability for COX1 mRNA translation. An exception is a null mutation in COX14 that does not affect Cox1 synthesis because the Mss51 trapping complexes become unstable, and Mss51 is readily available for translation. Here we present evidence showing that Cox25 is a new essential COX assembly factor that plays some roles similar to Cox14. A null mutation in COX25 by itself or in combination with other COX mutations does not affect Cox1 synthesis. Cox25 is an inner mitochondrial membrane intrinsic protein with a hydrophilic C terminus protruding into the matrix. Cox25 is an essential component of the complexes containing newly synthesized Cox1, Ssc1, Mss51, and Cox14. In addition, Cox25 is also found to interact with Shy1 and Cox5 in a complex that does not contain Mss51. These results suggest that once Ssc1-Mss51 are released from the Cox1 stabilization complex, Cox25 continues to interact with Cox14 and Cox1 to facilitate the formation of multisubunit COX assembly intermediates.     

Mss51 and Ssc1 Facilitate Translational Regulation of Cytochrome c Oxidase Biogenesis

The intricate biogenesis of multimeric organellar enzymes of dual genetic origin entails several levels of regulation. In Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) assembly is regulated translationally. Synthesis of subunit 1 (Cox1) is contingent on the availability of its assembly partners, thereby acting as a negative feedback loop that coordinates COX1 mRNA translation with Cox1 utilization during COX assembly. The COX1 mRNA-specific translational activator Mss51 plays a fundamental role in this process. Here, we report that Mss51 successively interacts with the COX1 mRNA translational apparatus, newly synthesized Cox1, and other COX assembly factors during Cox1 maturation/assembly. Notably, the mitochondrial Hsp70 chaperone Ssc1 is shown to be an Mss51 partner throughout its metabolic cycle. We conclude that Ssc1, by interacting with Mss51 and Mss51-containing complexes, plays a critical role in Cox1 biogenesis, COX assembly, and the translational regulation of these processes.Translational regulation is a fundamental mechanism used to control the accumulation of key proteins in a large variety of biogenetic and physiological processes in both prokaryotic and eukaryotic cells (20, 23). Translational autoregulation is a particular form of regulation exerted by the protein being translated. It is a well-established control mechanism for bacteriophage and prokaryotic systems (15), and it has also been reported in eukaryotes (4). Usually, the newly synthesized protein binds to its own mRNA to repress translation (20). However, repression can also be exerted by nascent chains interacting with the ribosome (49).Translational autoregulation also occurs in semiautonomous eukaryotic organelles of ancestral bacterial origin, namely, mitochondria and chloroplasts. During evolution, these organelles have retained a few genes in their own genomes, which are transcribed within the organelle, and the mRNAs are translated on organellar ribosomes. Most proteins synthesized within the organelles are part of large multimeric enzyme complexes devoted to energy production. These complexes are formed by subunits of dual genetic origin, nuclear and organellar, and assemble in the organellar membranes. Interestingly, intraorganellar translation of certain subunits has been proposed to be regulated by the availability of their assembly partners (1, 39, 54, 55). A distinctive characteristic of these systems is the involvement of ternary factors, mRNA-specific translational activators whose availability would be regulated by the specific gene products. The players and mechanisms involved remain largely unknown.We have focused on the characterization, in the yeast Saccharomyces cerevisiae, of an assembly-controlled translational regulatory system that operates during the biogenesis of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain. The three subunits forming the COX catalytic core (1, 2, and 3) are encoded in the mitochondrial DNA (mtDNA), and the remaining eight subunits are encoded in the nuclear DNA. Subunits 1 and 2 coordinate the heme A and copper prosthetic groups of the enzyme. COX biogenesis requires the assistance of a large number of ancillary factors acting at all the levels of the process (11). COX assembly is thought to be linear, consisting of the sequential addition of subunits to an initial seed formed by the mtDNA-encoded subunit 1 (Cox1) in both mammalian and yeast cells (11).The concerted accumulation of COX subunits is regulated by posttranslational degradation of most unassembled Cox1 and the other highly hydrophobic core subunits (27). Recently, we along with others have proposed an additional level of regulation, namely, an assembly-controlled synthesis of Cox1 (1, 2, 39, 56). In S. cerevisiae, COX1 mRNA translation is under the control of Mss51 and Pet309 (8, 30). Mss51 is a key element of the regulatory system. Mss51 acts on the 5′ untranslated region (UTR) of COX1 mRNA to promote translation initiation (39, 56) and additionally acts on a target in the protein coding sequence of COX1 mRNA, perhaps to promote elongation (39). Mss51 and newly synthesized Cox1 form a transient complex (2, 39) that is stabilized by Cox14 (2). We have postulated that these interactions downregulate Cox1 synthesis when COX assembly is impaired by trapping Mss51 and limiting its availability for COX1 mRNA translation (2). According to this model, the release of Mss51 from the ternary complex and its availability for Cox1 synthesis probably occur when Cox1 acquires its prosthetic groups or interacts with other COX subunits, a step possibly catalyzed by Shy1, a protein involved in maturation and/or assembly of Cox1 (2, 10, 34). Coa1 could also participate in Cox1 maturation and stabilize the ternary Cox1/Mss51/Cox14 complex until it interacts with Shy1 (34, 40). Further studies are required to understand how Mss51 is recycled from its posttranslational function to become available for COX1 mRNA translation and to fully clarify how this regulatory mechanism operates.In this study, we have analyzed protein-interacting partners of Mss51 in the wild type and a collection of COX assembly mutants. We found that the native molecular weight (MW) of Mss51 is dependent on both the status of COX assembly and the synthesis of Cox1. The mitochondrial Hsp70 (mtHsp70) chaperone Ssc1 interacts with Mss51 and with several high-molecular weight Mss51-containing complexes involving the COX1 mRNA translational apparatus, Cox1, and several Cox1 assembly factors. Mutants defective in Cox1 maturation or in other aspects of COX biogenesis accumulate distinct ratios of these complexes. In this way, Cox1 regulates its own translation through the action of Mss51 and Ssc1.     

Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae

Expression of yeast mitochondrial genes depends on specific translational activators acting on the 5'-untranslated region of their target mRNAs. Mss51p is a translational factor for cytochrome c oxidase subunit 1 (COX1) mRNA and a key player in down-regulating Cox1p expression when subunits with which it normally interacts are not available. Mss51p probably acts on the 5'-untranslated region of COX1 mRNA to initiate translation and on the coding sequence itself to facilitate elongation. Mss51p binds newly synthesized Cox1p, an interaction that could be necessary for translation. To gain insight into the different roles of Mss51p on Cox1p biogenesis, we have analyzed the properties of a new mitochondrial protein, mp15, which is synthesized in mss51 mutants and in cytochrome oxidase mutants in which Cox1p translation is suppressed. The mp15 polypeptide is not detected in cox14 mutants that express Cox1p normally. We show that mp15 is a truncated translation product of COX1 mRNA whose synthesis requires the COX1 mRNA-specific translational activator Pet309p. These results support a key role for Mss51p in translationally regulating Cox1p synthesis by the status of cytochrome oxidase assembly.     

Dual Functions of Mss51 Couple Synthesis of Cox1 to Assembly of Cytochrome c Oxidase in Saccharomyces cerevisiae Mitochondria

Functional interactions of the translational activator Mss51 with both the mitochondrially encoded COX1 mRNA 5′-untranslated region and with newly synthesized unassembled Cox1 protein suggest that it has a key role in coupling Cox1 synthesis with assembly of cytochrome c oxidase. Mss51 is present at levels that are near rate limiting for expression of a reporter gene inserted at COX1 in mitochondrial DNA, and a substantial fraction of Mss51 is associated with Cox1 protein in assembly intermediates. Thus, sequestration of Mss51 in assembly intermediates could limit Cox1 synthesis in wild type, and account for the reduced Cox1 synthesis caused by most yeast mutations that block assembly. Mss51 does not stably interact with newly synthesized Cox1 in a mutant lacking Cox14, suggesting that the failure of nuclear cox14 mutants to decrease Cox1 synthesis, despite their inability to assemble cytochrome c oxidase, is due to a failure to sequester Mss51. The physical interaction between Mss51 and Cox14 is dependent upon Cox1 synthesis, indicating dynamic assembly of early cytochrome c oxidase intermediates nucleated by Cox1. Regulation of COX1 mRNA translation by Mss51 seems to be an example of a homeostatic mechanism in which a positive effector of gene expression interacts with the product it regulates in a posttranslational assembly process.     

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis

Eukaryotic cytochrome c oxidase (CcO), the terminal enzyme of the energy-transducing mitochondrial electron transport chain is a hetero-oligomeric, heme–copper oxidase complex composed of both mitochondrially and nuclear-encoded subunits. It is embedded in the inner mitochondrial membrane where it couples the transfer of electrons from reduced cytochrome c to molecular oxygen with vectorial proton translocation across the membrane. The biogenesis of CcO is a complicated sequential process that requires numerous specific accessory proteins, so-called assembly factors, which include translational activators, translocases, molecular chaperones, copper metallochaperones and heme a biosynthetic enzymes. Besides these CcO-specific protein factors, the correct biogenesis of CcO requires an even greater number of proteins with much broader substrate specificities. Indeed, growing evidence indicates that mitochondrial ATP-dependent proteases might play an important role in CcO biogenesis. Out of the four identified energy-dependent mitochondrial proteases, three were shown to be directly involved in proteolysis of CcO subunits. In addition to their well-established protein-quality control function these oligomeric proteolytic complexes with chaperone-like activities may function as molecular chaperones promoting productive folding and assembly of subunit proteins. In this review, we summarize the current knowledge of the functional involvement of eukaryotic CcO-specific assembly factors and highlight the possible significance for CcO biogenesis of mitochondrial ATP-dependent proteases.     

Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix

The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2Delta cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.     

Copper trafficking to the mitochondrion and assembly of copper metalloenzymes

Copper is required within the mitochondrion for the function of two metalloenzymes, cytochrome c oxidase (CcO) and superoxide dismutase (Sod1). Copper metallation of these two enzymes occurs within the mitochondrial intermembrane space and is mediated by metallochaperone proteins. Cox17 is a key copper donor to two accessory proteins, Sco1 and Cox11, to form the two copper centers in the mature CcO complex. Ccs1 is the necessary metallochaperone for the copper metallation of Sod1 in the IMS as well as within the cytoplasm where the bulk of Sod1 resides. Copper ions used in the metallation of CcO and Sod1 appear to be provided by a novel copper pool within the mitochondrial matrix. This review documents copper ion shuttling within the mitochondrion and the proteins that mediate assembly of active CcO and Sod1.     

Cytochrome c oxidase dysfunction in oxidative stress

Cytochrome c oxidase (CcO) is the terminal oxidase of the mitochondrial electron transport chain. This bigenomic enzyme in mammals contains 13 subunits of which the 3 catalytic subunits are encoded by the mitochondrial genes. The remaining 10 subunits with suspected roles in the regulation, and/or assembly, are coded by the nuclear genome. The enzyme contains two heme groups (heme a and a3) and two Cu(2+) centers (Cu(2+) A and Cu(2+) B) as catalytic centers and handles more than 90% of molecular O(2) respired by the mammalian cells and tissues. CcO is a highly regulated enzyme which is believed to be the pacesetter for mitochondrial oxidative metabolism and ATP synthesis. The structure and function of the enzyme are affected in a wide variety of diseases including cancer, neurodegenerative diseases, myocardial ischemia/reperfusion, bone and skeletal diseases, and diabetes. Despite handling a high O(2) load the role of CcO in the production of reactive oxygen species still remains a subject of debate. However, a volume of evidence suggests that CcO dysfunction is invariably associated with increased mitochondrial reactive oxygen species production and cellular toxicity. In this paper we review the literature on mechanisms of multimodal regulation of CcO activity by a wide spectrum of physiological and pathological factors. We also review an array of literature on the direct or indirect roles of CcO in reactive oxygen species production.