Insights Into the COX Assembly Process

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Biochimica et Biophysica Acta 1793 (2009) 97–107

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Biochimica et Biophysica Acta

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Review Suppression mechanisms of COX assembly defects in yeast and human: Insights into the COX assembly process

Antoni Barrientos a,b,⁎, Karine Gouget a, Darryl Horn b, Ileana C. Soto b, Flavia Fontanesi a

a Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA b Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA

article info abstract

Article history: Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is Received 11 March 2008 a multimeric enzyme formed by subunits of dual genetic origin whose assembly is intricate and highly Received in revised form 29 April 2008 regulated. In addition to the structural subunits, a large number of accessory factors are required to build the Accepted 5 May 2008 holoenzyme. The function of these factors is required in all stages of the assembly process. They are relevant Available online 15 May 2008 to human health because devastating human disorders have been associated with mutations in nuclear genes encoding conserved COX assembly factors. The study of yeast strains and human cell lines from patients Keywords: Mitochondria carrying mutations in structural subunits and COX assembly factors has been invaluable to attain the current Cytochrome c oxidase state of knowledge, even if still fragmentary, of the COX assembly process. After the identification of the Suppression genes involved, the isolation and characterization of genetic and metabolic suppressors of COX assembly Mitochondrial disorder defects, reviewed here, have become a profitable strategy to gain insight into their functions and the COX assembly pathways in which they operate. Additionally, they have the potential to provide useful information for devising therapeutic approaches to combat human disorders associated with COX deficiency. © 2008 Elsevier B.V. All rights reserved.

1. Eukaryotic cytochrome c oxidase assembly in health and disease enzyme from other terminal oxidases that use quinol rather than cytochrome c as the electron donor [2,5,6]. The electron transfer The fundamental mechanism by which respiratory energy trans- reaction is coupled to the vectorial transfer of protons from the matrix duction occurs in mitochondria and aerobic bacteria is the coupling of to the intermembrane space thus contributing to the generation of the electron transfer reactions to the formation of transmembrane electro- proton gradient which is subsequently used by the F1F0 mitochondrial chemical gradients [1]. In eukaryotes, cytochrome c oxidase (COX) or ATPase to drive the synthesis of ATP. The mechanism of proton complex IV, the terminal enzyme of the mitochondrial respiratory pumping has been the focus of intense study and has been recently chain, is one of the primary coupling sites. COX is a multimeric copper- reviewed elsewhere ([7] and [8]). heme A metalloenzyme that functions as an electron-driven proton Eukaryotic COX is formed by 11–13 subunits (11 in the yeast Sac- pump. It catalyzes the transfer of electrons from ferrocytochrome c to charomyces cerevisiae and 13 in mammals) of dual genetic origin. molecular oxygen via its four redox active metal co-factors. Fig. 1 Subunits 1, 2 and 3 form the catalytic core of the enzyme and in the summarizes the catalytic metal centers and the electron transfer majority of eukaryotes are encoded in the mitochondrial DNA. The pathway in COX. Electrons enter COX through a mixed valence core is surrounded by a set of nuclear-encoded small subunits that are dinuclear copper center, the CuA site, located in subunit 2. Electrons important for both the assembly and function of the enzyme as well as are transferred from CuA to a low-spin heme a located in subunit 1 and its dimerization (reviewed in [9,10]). These subunits are also involved are subsequently transferred intra-molecularly to the active site where in the modulation of the catalytic activity and in the protection of the a high-spin heme a3 and CuB form a binuclear center for oxygen core from reactive oxygen species. A list of COX homologue subunits in binding. The mechanism of electron transfer through COX has been yeast and mammals is shown in Table 1. extensively studied (reviewed in [2–4]). From an evolutionary point of As a consequence of its central role in oxidative metabolism, COX ﬁ view, the presence of the CuA center de nes COX and distinguishes this has been intensively studied by biochemical, genetic, spectroscopic, and crystallographic means [11–13]. From these studies it is known that other metals such as zinc and magnesium are also bound to the enzyme, although the basis for their speciﬁc requirements are largely ⁎ Corresponding author. Departments of Neurology and Biochemistry and Molecular unknown. The zinc atom could play a role in structural stability of the Biology, The John T. Macdonald Center for Medical Genetics, Universtiy of Miami, Miller complex [14]. The magnesium/manganese site is in close proximity to School of Medicine, 1600 NW 10th Avenue, RMSB # 2067, Miami, FL 33136, USA. Tel.: +1 305 243 86 83; fax: +1 305 243 39 14. the H2O exit channel and is thought to aid in the stability and release E-mail address: [email protected] (A. Barrientos). of H2O produced during the reduction of O2 [15].

model for COX assembly by studying the accumulation of subassembly intermediates in the absence of speciﬁc COX assembly factors [35–37]. These studies, described below, have provided information concerning the assembly step either catalyzed or affected by the mutated factor. In contrast, the study of assembly intermediates in yeast has been restricted since they do not seem to accumulate in detectable amounts [37]. The accumulation of assembly intermediates in most yeast COX mutants is probably limited by the small amount of Cox1p

Table 1 Homologue COX subunits and COX assembly factors in yeast and mammals

Yeast Mammals Function

Gene Protein Gene Protein Catalytic core (mtDNA-encoded structural subunits) COX1 Cox1p MTCOXI COX1 Catalytic subunits COX2 Cox2p MTCOXII COX2 COX3 Cox3p MTCOXIII COX3 Catalytic core stability?

Core protective shield (nDNA encoded structural subunits) COX4 Cox4p COXVb COX5b Subunits required for COX assembly COX5a Cox5ap COXIV-1 COX4-1 and function COX5b Cox5bp COXIV-2 COX4-2 COX6 Cox6p COXVa COX5a Fig. 1. Cytochrome c oxidase. Representation of the complex, showing the two catalytic COX7 Cox7p COXVIIa COX7a subunits (subunits 1 and 2) involved in electron transfer from cytochrome c to COX9 Cox7ap COXVIc COX6c molecular oxygen. The mechanism of electron transport, coupled to proton pumping, is – – COXVIIb COX7b depicted. Subunit 2 contains the binuclear Cu center that receives electrons from A –– COXVIII COX8 cytochrome c. In subunit I, a low-spin heme (heme a) accepts electrons from Cu and A COX8 Cox8p COXVIIc COX7c transfers them to a binuclear center consisting of a high-spin heme (heme a ) and a 3 COX12 Cox9p COXVIb COX6b Non-essential subunits copper atom (Cu ). Within the binuclear center, molecular oxygen is bound to heme a B 3 COX13 Cox10p COXVIa COX6a and sequentially reduced to water. IMS, intermembrane space; MIM, mitochondrial inner membrane. Membrane insertion and processing of catalytic core subunits OXA1 Oxa1p OXA1 OXA1 Membrane insertion of COX subunits, COX biogenesis probably occurs with the different subunits and co- cytochrome b and ATPase proteolipid factors being added in an ordered manner. Data obtained from COX20 Cox20p COX20 COX20 Cox2p chaperone. Presentation of analyses of the human enzyme performed by Blue-Native electro- Cox2p-precursor to the IMP complex COX18 Cox18p COX18 COX18 Export of the Cox2p C-terminus tail phoresis suggests an assembly pathway characterized by the MSS2 Mss2p ––Export of the Cox2p C-terminus tail sequential incorporation of COX subunits. Assembly is initiated PNT1 Pnt1p ––Export of the Cox2p C-terminus tail around a seed formed by subunit 1 and proceeds with the formation IMP1 Imp1p ––Responsible for the maturation of Cox2p of several discrete assembly intermediates probably representing IMP2 Imp2p IMMP2L IMMP2L Necessary for the stability and activity of Imp1 SOM1 Som1p ––Third component of the yeast IMP complex. rate-limiting steps in the process [16]. It could play a role in substrate recognition Analysis of assembly intermediates has given insights into the overview of the assembly process, but it has provided very limited Copper metabolism and insertion information about additional players involved and their specific roles. COX17 Cox17p COX17 COX17 Delivery of copper to Sco1p and Cox11p To disclose the non-structural ancillary factors involved in COX SCO1 Sco1p SCO1 SCO1 Transfer of copper to COX and/or reduction fi SCO2 SCO2 of cysteine residues in subunit 2 assembly, a pro table strategy has been to systematically analyze COX11 Cox11p COX11 COX11 Stable formation of the Cu(B) and yeast mutants defective in COX assembly. This approach was followed magnesium centers with the goal of identifying the functions of the gene products COX19 Cox19p COX19 COX19 CX9C proteins. They could play roles in responsible for the COX defective phenotype to subsequently recon- COX23 Cox23p COX23 COX23 redox control and copper trafficking in PET191 Pet191p PET191 PET191 the intermembrane space struct the different steps of the assembly pathway. Screens of nuclear CMC1 Cmc1p CMC1 CMC1 respiratory deficient mutants have revealed the existence of a large number of nuclear genes coding for accessory factors that selectively Heme A biosynthesis affect expression of this respiratory complex in yeast [17,18]. Their COX10 Cox10p COX10 COX10 Farnesylation of protoheme functions, required for all steps of the process and significantly con- COX15 Cox15p COX15 COX15 Hydroxylation of heme O served from yeast to humans, are summarized in Table 1 and have been Assembly/unknown previously reviewed [9,19,20]. COX16 Cox16p COX16 COX16 Unknown function Over the last 15 years, COX biogenesis has received significant PET117 Pet117p ––Unknown function attention because of its medical relevance. Defective COX biogenesis PET100 Pet100p ––Formation of assembly intermediates containing Cox7p, Cox8p, and Cox9p results in mitochondrial diseases frequently involving brain, skeletal SHY1 Surf1p SURF1 SURF1 Catalyzes an assembly step in which – muscle and heart tissues (reviewed in [21 23]). To date, all Mendelian Cox1p is one of the partners disorders presenting COX deficiency have been assigned to mutations MSS51 Mss51p ––Required for translation of COX1 mRNA. in ancillary factors. Specifically, mutations have been found in SURF1, Additionally, binds Cox1p and is required required for the formation of early assembly intermediates [24,25], for its stability/ maturation/assembly –– – COX14 Cox14p Binds Cox1p and is required for its stability/ SCO1 and SCO2, required for COX copper metallation [26 30], COX10 maturation/assembly and COX15, essential for heme A biosynthesis [31–33], and LRPPRC, COA1 Coa1p ––Binds Cox1p and is required for its stability/ required for the expression of COX subunits [34]. maturation/assembly fi Mutant broblast cell lines from patients suffering from some Genes involved in expression of the mitochondrial DNA-encoded subunits and in of these disorders have been used to refine the proposed sequential mitochondrial import of nuclear-encoded subunits are not included. A. Barrientos et al. / Biochimica et Biophysica Acta 1793 (2009) 97–107 99 that is newly synthesized in the absence of fully assembled COX [38]. pharmacological suppressors have the potential to provide useful However, subassemblies have been recently detected in yeast cox2 information for devising therapeutic approaches to combat human [39] and pet100 mutants [40] and seem to be similar to those observed disorders associated with COX deficiency. in mammalian cells. The aim of this review is to summarize some of the understanding After the identification of the genes involved, the further concerning COX assembly that has been obtained by studying dif- exploitation of the yeast paradigm by isolating and characterizing ferent classes of suppressors. The information obtained by the study of genetic and metabolic suppressors of COX assembly defects, reviewed suppressors of mitochondrial gene expression [41] and of defects in here, has become a rewarding effort to gain insight into the functions the membrane insertion and processing of mtDNA-encoded COX of these genes and the pathways in which they operate. The genetic subunits ([42] and listed on Table 2) has been reviewed elsewhere. We suppressors can be classified in two general groups, extragenic and have focused on describing some of the knowledge obtained by intragenic. Intragenic suppressors restore function to a protein at a site studying suppressors of structural, heme A biosynthesis, copper different from the primary mutation. They are very useful for example metabolism and insertion defects and alterations in the formation of to understand the roles of several domains in a protein or the role of a assembly intermediates. particular residue and their tertiary structure neighbors within a protein. Extragenic suppressors, instead, reconstitute function in a 2. Suppression of cytochrome c oxidase biogenesis defects in yeast system either by mutating a protein to work with another mutated and human protein or by bypassing the requirement of the first protein. Here, we will review exclusively some cases of extragenic suppression of COX 2.1. Suppressors of structural defects assembly defects. Extragenic suppressors of strains carrying a null allele of a particular gene can arise naturally by a spontaneous Some nuclear-encoded subunits have evolved isoforms that allow mutation in a gene that compensates for the absence of the first gene. the enzyme to regulate its activity depending on environmental These natural suppressors are rare. In the cases of the respiratory factors. For example, subunit Cox5p in the yeast S. cerevisiae exists in deficient COX assembly mutants, only a few yeast strains sponta- two isoforms, Cox5ap and Cox5bp, which are expressed according to neously revert to a respiratory-competent phenotype (Table 2). The oxygen availability. In humans, tissue specific isoforms have been hunt for genetic suppressors is designed to identify additional reported for four nuclear-encoded subunits, including COX4, the mutations that restore a particular aspect of COX assembly and/or homologue of yeast Cox5p [43,44]. Studies on suppressors of yeast its respiratory capacity to the initial mutant. The characterization of cox5a mutants have given insight into oxygen regulation of COX these suppressor mutations has lead to the identification of new genes subunits and their switch to modulate COX enzymatic activity. with functions related to the initial mutated gene. These studies have Yeast Cox5ap and Cox5bp share 66% sequence homology [45]. The also suggested interactions between proteins that participate in a presence of either one or the other isoform confers different kinetic common pathway. Another often-used means for uncovering inter- properties to the holoenzyme. Moreover, the transmembrane α-helix acting proteins in a common pathway is suppression by overexpres- of Cox5p interacts with Cox1p and it is able to alter, depending on the sion of wild-type alleles. Suppression caused by overexpression can isoform, the protein environment around the binuclear center. The occur by many different mechanisms but in general the necessity of Cox5bp-containing enzyme has a higher maximal turnover, since the mutated protein is bypassed. Additionally, the characterization of electron transfer from heme a to heme a3 occurs three to four times these genetic suppressors in combination with the identification of faster than in the Cox5ap-containing isoenzyme [46].Theexpressionof COX5a and COX5b genes is regulated by oxygen availability [47].In normoxia, the expression of COX5a is induced by the heme-dependent Table 2 transcriptional activator Hap2p/3p/4p/5p complex, while COX5b expres- Genetic and metabolic suppressors of COX deficiencies in cellular and animal models as sion is repressed, also in a heme-dependent fashion [48].Whenthe well as in human patients environmental oxygen concentration decreases below 0.5 μM, a switch Mutated Genetic suppressors Metabolic/ in Cox5p isoform expression takes place [49]. The change results from gene pharmacologic the consequence of low heme biosynthesis, a process that requires Spontaneous extragenic High copy suppressors suppressors suppressors oxygen [50]. Due to the lack of induction of COX5a and the lack of repression of COX5b, Cox5bp is expressed and used to assemble a more Yeast cultures efficient COX holoenzyme. By this means, ATP production is maximized COX5a ROX1 [51] ORD1 [52] under hypoxia while minimizing ROS generation. In this way, by OXA1 CYT1 [144] HAP4 [132] regulating COX isoforms composition, yeast cells can rapidly respond to OMS1 [145] RMD9 [146] changes in environmental oxygen concentration. IMP1 SOM1 [147] The oxygen regulation of these genes has been exhaustively studied COX17 SCO1 [71] SCO2 + Copper [61] in the yeast S. cerevisiae taking advantage of the ability of a respiratory Copper [71] deficient Δcox5a strain to spontaneously revert to a respiratory- SCO1 SCO2 [71] COX23 COX17+Copper [68] competent phenotype. Two different revertant complementation SHY1 MSS51 [112] MSS51 [112] HAP4 [128] Copper [128] groups were isolated and analyzed [51,52]. In both cases, the reversion COX5a + COX6 [128] was accounted for by recessive mutations in a single nuclear gene. These COA1 MSS51 [123] COX10 [123] mutations do not bypass the subunit 5 requirement. The mechanism of MDJ1 [123] reversion rather consists of an increase in COX5b expression in fi Human cell cultures normoxia. The cloning of the suppressor genes allowed the identi ca- SURF1 NF-YA [128] tion of two new players in the oxygen regulatory pathway: Reo1p/Rox1p SCO2 Copper [91,148] and Ordlp/Ixr1p, two transcriptional repressors of COX5b gene [51,52]. COX10 PPAR agonists Rox1p is a general hypoxic regulator that represses a large group of [104] COX1 PGC1-α [143] PGC1-α [143] hypoxic genes, including COX5b, during aerobic growth. In normoxia, ROX1 is induced by the heme-binding transcriptional activator Hap1p Human patients [53]. In the absence of oxygen, ROX1 expression is down-regulated and SCO2 Copper [93] the repression of COX5b is released [51]. Rox1p levels are tightly Intragenic suppressors are not listed. regulated in the cell and additional regulatory factors of ROX1 have been 100 A. Barrientos et al. / Biochimica et Biophysica Acta 1793 (2009) 97–107 described [54]. Furthermore, the synergistic activity of Rox1p and enous copper suppression studies were very useful to place Cox17p Mot3p, another general repressor of hypoxic genes, has been recently in the COX copper delivery pathway. The copper source for COX reported, enlarging our understanding of the complex pathway of metallation seems to be a matrix pool of copper bound by a low oxygen-dependent gene regulation [55]. In contrast to Rox1p, Ord1p is molecular weight non-proteinaceous ligand [65,66]. The ligand from an oxygen dependent but Hap1p independent specific COX5b repressor the copper complex has been found in the cytoplasm and it has been [52]. Both Rox1p and Ord1p bind to the same 44 bp Upstream suggested that it may recruit, in place of a copper chaperone, the Repression Sequence in the promoter of COX5b gene [52]. Currently, it copper that is translocated and stored within the mitochondrial matrix remains unknown how Ord1p expression or function is regulated [66]. by oxygen and which kind of functional interactions (competition/ At least two homologues of Cox17p are present in the IMS, the cooperation) exist between Rox1p and Ord1p. The physiological sig- small soluble proteins Cox19p [67] and Cox23p [68], which also nificance for the existence of multiple COX5b repressors probably lies in contain twin Cx9C metal binding motifs and are required for COX the necessity to achieve a large range of gene expression variations in assembly. Cox23p does not physically interact with Cox17p in a stable response to oxygen availability. complex but the COX defect in cox23 mutants is suppressed by Noteworthy, COX isoform oxygen-dependent expression is highly exogenous copper in combination with COX17 overexpression. These conserved from lower to higher eukaryotes. In mammals, COX subunit 4, data have suggested that both proteins function in a common pathway the homologue of yeast subunit 5, also exists in two isoforms, COX4-1 with Cox17p acting downstream of Cox23p [68]. Cox19p is a copper- and COX4-2. As their yeast counterparts, they are regulated by oxygen binding protein [69] and could be part of the same copper distribution and confer different kinetic properties to the holoenzyme [56].The pathway. regulation of the mammalian isoform switch seems to be regulated Cox17p transfers copper ions to two additional chaperones [70] that differently than in yeast. Homologues of the main yeast regulatory facilitate copper insertion into the COX CuA and CuB active sites, Sco1p elements have not been found in mammals. The regulation of COX4 [71] and Cox11p [72,73], respectively. These proteins are anchored to isoform expression in response to oxygen availability is instead mediated the mitochondrial inner membrane (MIM) through a transmembrane by the hypoxia-inducible factor 1α (HIF-1 α)(reviewedin[57]). In α-helix and expose their copper-binding sides in the IMS where copper aerobic conditions, HIF-1α is hydroxylated in an oxygen-dependent transfer occurs [62,74]. In vitro experiments have shown that soluble reaction and degraded by the proteosome [58]. During hypoxia, truncated forms of both Cox11p and Sco1p are able to bind copper hydroxylation is inhibited; HIF-1α is not degraded and accumulates in donated from Cox17p [75,76]. It is still not clear, however, how the the cell. Consequently, HIF-1α induces transcription of both, the COXIV-2 transfer of copper occurs between Cox17p and these proteins because gene and the gene encoding for the mitochondrial protease LON, physical interactions among them have not been detected [70]. responsible for the degradation of COX4-1 [59]. Like in yeast, the COX4 High copy suppressor screens were essential to place Sco1p into isoform switch in mammals is a fundamental cellular adaptive response the COX copper delivery pathway. SCO1 was originally identified as to maintain the efficiency of respiration under conditions of reduced essential for COX assembly in yeast [77] and subsequently as a oxygen availability. Additionally, while COXIV-1 is ubiquitously expressed multicopy suppressor of a cox17 null mutant [61]. Sco1p transfers in all tissues, COXIV-2 is expressed at particularly high levels in the lungs copper from Cox17p to Cox2p and has been shown to directly interact and trachea [56].Huttemannetal.haveproposedthattheexpressionof with Cox2p [78]. Sco1p has a metal binding thioredoxin-like CX3C COX4-2 isoform in these highly oxygenated tissues can have a motif analogous to the copper-binding motif of Cox2p and this motif is physiological protective function by increasing electron transfer effi- essential for its function as demonstrated by site-direct mutagenesis ciency and consequently decreasing ROS formation [60]. [79]. The mechanism of copper transfer from Cox17p and to Cox2p has been recently reviewed [80]. Although the experimental data could 2.2. Suppressors of copper insertion defects support a role for Sco1p in copper insertion, considering its structural similarity to the protein family of disulfide reductases, it has been The analyses of suppressors of copper insertion defects have proposed that it could rather be involved in the reduction of cysteines allowed for the identification of factors involved in mitochondrial in the Cox2p copper-binding site [81,82]. This reduction is necessary copper metabolism and the COX copper insertion pathway as well as for the co-factor incorporation [83,84]. Sco1p has the ability to form for the development of therapeutic approaches. homodimeric complexes [78] which could facilitate the performance COX copper metallation involves the function of several conserved of both functions by the collaborative action of each monomer. proteins located in the mitochondrial intermembrane space (IMS) that Yeast SCO1 has a highly conserved homologue, SCO2 [85]. However, were initially identified in yeast. The first protein involved in this deletion of sco2 does not affect COX assembly [71]. SCO2 overexpres- function was the IMS chaperone Cox17p [61],asmallsoluble sion does not suppress the COX assembly defect of a sco1 null mutant hydrophilic protein which binds copper(I) ions [62] through a CCxC strain but it is able to partially rescue a sco1 point mutant [71]. SCO2 metal binding motif. Cox17p also contains a twin Cx9C structural overexpression also suppresses cox17 mutations, although less effi- motif. Mutational analyses in yeast have shown that while the first ciently than SCO1, and only when the growth media is supplemented cysteine in the Cx9C motif is part of the copper-binding motif, the with copper [71]. These data were interpreted to indicate that yeast remaining three conserved cysteines are not important for Cox17p Sco1p and Sco2p have overlapping but not identical functions [71]. copper-binding function [63]. However, structural studies on human Remarkably, humans also have two homologues of yeast Sco1p, COX17 have recently shown that the two adjacent cysteines in the SCO1 and SCO2 [86], both of which are essential for COX assembly. CCxC domain are actually the copper-binding cysteines [64].Cox17p Mutations in SCO1 [26] and SCO2 [27–29] result in severe mitochon- has a dual location in the IMS and in the cytoplasm. The respiratory drial disorders described below. Functional complementation studies deficient phenotype of cox17 null mutant strains can be rescued by have shown that expression of either human SCO1 or SCO2 does supplementation of high copper concentrations to the media or by not complement a yeast strain carrying a null allele of sco1 [87]. significantly lower concentrations in combination with overexpres- Interestingly, the expression of a chimeric protein with the N terminus sion of CTR1 (the structural gene for the plasma membrane copper of yeast and the C-terminus (that contains the CX3C copper-binding pump) [61]. This suppression was the basis to propose that Cox17p domain) of human SCO1 (but not of SCO2) is able to complement the could be shuttling copper from the cytoplasm into the mitochondrial yeast mutant [87]. The functional differences among the two yeast and intermembrane space [61,62]. This hypothesis has been challenged by human isoforms is explained by the fact that the two genes probably the fact that the tethering of Cox17p to the inner mitochondrial originated from a duplication that occurred separately in the two membrane does not affect COX assembly [65]. Nevertheless, the exog- organisms [27]. A. Barrientos et al. / Biochimica et Biophysica Acta 1793 (2009) 97–107 101

In contrast with their yeast homologues, human SCO1 and SCO2 two latter enzymes are probably necessary for the supply of electrons have been shown to perform independent, cooperative functions in to the oxygenase Cox15p [98–100]. Interestingly, in the yeast Schizo- the process of COX assembly [88]. This is suggested by the fact that saccharomyces pombe COX15 and YAH1 are fused in a single gene [98]. overexpression of each SCO protein in ﬁbroblasts from patients with At present, the identity of the putative gene product involved in the mutation in the other SCO protein results in a dominant negative oxidation of the alcohol resulting from the Cox15p action to the phenotype [88]. In a proposed model, human COX17 delivers copper corresponding aldehyde to yield heme A remains unknown. to SCO2 which in turn transfers it directly to the CuA site in COX The biosynthesis of heme A is regulated by downstream events in subunit II in a reaction facilitated by SCO1 [88]. SCO2 could form a the COX assembly process. By measuring the amount of heme A and heterodimer with SCO1, which could be required to create the Cu(II)– heme O in different yeast COX mutants it was observed that all the

Cu(I) dinuclear CuA site [89]. Recently, SCO1 and SCO2 were shown to mutants analyzed showed a drastic reduction of steady-state levels of have additional regulatory roles in the maintenance of cellular copper heme A, with the exception of shy1, cox20 and cox5a null mutants, in homeostasis cooperating to regulate copper efflux under conditions of which heme A was still detectable at 10–25%. This amount is probably excessive cellular copper [90]. associated with residual assembled COX [101]. The overexpression of A better understanding of the pathophysiology of human disorders COX15 acted as a suppressor of the heme A accumulation defect in associated with mutations in genes required for copper insertion in COX mutants including mutants in which Cox1p was not synthesized. COX has suggested new avenues for treatment of these disorders. This observation suggested that the absence of heme A in the mutants Specifically, mutations in SCO2 produce childhood onset cardioence- is not due to a rapid turnover of the co-factor in the absence of COX phalomyopathy [27,28,30]. The addition of CuCl2 [91] or copper- subunit 1, but rather to a feedback regulation of the heme A synthesis histidine (Cu-His) [91] to cultured cells from patients with SCO2 when the COX assembly process is blocked. The COX mutants analyzed, mutations improved the biochemical defect. The mechanism of with the obvious exception of cox10, also showed an accumulation of suppression remains unclear. As pointed out by Jaksch et al. [91], heme O, indicating that this compound is stable. In addition, the cox15 suppression could result from the very low residual levels of SCO2 in null mutant presented a very low amount of heme O, a phenotype that the patient cells, direct addition of copper to the CuA site, or by was not rescued by the overexpression of COX10. This observation another copper-binding protein. Whatever the mechanism, copper suggested that the first step of the heme A biosynthesis is also posi- supplementation has suggested a possible therapy for this mitochon- tively regulated in a Cox15p dependent manner [101]. This regulatory drial disease. Cu-His has been used in the treatment of Menke's system could be different in mammalian cells. Analyses of mitochon- disease (a neurological disorder produced by the loss of the copper- drial heme content in COX15 deficient fibroblasts from a human transporting ATPase ATP7A) with some success [92] and the same patient suffering from hypertrophic cardiomyopathy showed levels of treatment may be effective for children with SCO2 mutations. Actually, heme O significantly higher than in control fibroblasts [33]. subcutaneous injection of Cu-His, a physiological compound found in Pharmacological suppressor studies of the respiratory defect of blood, in a patient affected by severe hypertrophic cardiomyopathy human fibroblasts from patients with defects in components of the associated with SCO2 mutations did induce hypertrophic regression mitochondrial respiratory chain, including mutants of COX10 [31],have and ventricular function improvement with a significantly longer allowed the identification of agents for possible use in therapeutic survival in comparison with all previously reported patients [93]. approaches to combat these mitochondrial diseases. Specifically, it was

The CuB site located in Cox1p is formed by one copper ion recently reported that treatment with bezafibrate significantly rescues coordinated by three histidine ligands and present in close proximity the respiratory defects in the mutant cells. Bezafibrate is a widely used to the heme a moiety. The metallochaperone for the formation of the hypolipidemic drug that acts as a highly specific Peroxisome Prolif-

CuB site of Cox1p is the product of COX11 [72,73]. Cox11p was initially erator-Activated Receptor (PPAR) agonist [102].PPARsareligand- described in yeast as necessary for COX assembly [94] but its role in activated nuclear receptors involved in the regulation of several

CuB formation was first shown in the prokaryote Rhodobacter energy metabolism genes, such as the ones encoding mitochondrial sphaeroides [72]. The COX assembly defect in cox11 yeast mutants is fatty acid β-oxidation enzymes (reviewed in [103]). Supplementation not suppressed by exogenous copper and genetic suppressors of the with 400 μMbezafibrate for 72 h significantly increases COX activity in null mutants have not been obtained. However, Cox11p was shown to COX10 mutant fibroblasts. This partial restoration of COX activity is bind Cu(I) [95]. The soluble C-terminal domain of Cox11p forms a accompanied by complete restoration of cell respiration to normal levels dimer that coordinates one Cu(I) per monomer. The two Cu(I) ions in [104]. In the bezafibrate-treated COX10 deficient cells, the levels of both the dimer exist in a binuclear cluster and appear to be ligated by three mRNA and protein of the COX mitochondrial encoded subunit COX2 and conserved cysteine residues [95]. The mechanism of copper transfer to the COX nuclear-encoded subunit COX4 are increased to normal levels.

CuB remains to be elucidated. More importantly, the expression of the COX10 mutated gene, encoding for a partially functional protein, is also increased suggesting that the 2.3. Suppression of heme A biogenesis and insertion defects increase in COX activity is due to improved COX assembly. Bastin et al. dissected the bezafibrate-mediated suppression mechanism. It was The study of suppressors of heme A biosynthesis defects in yeast previously reported that fibrates are able to induce PPARδ-mediated and human cell lines has provided information of biological and expression of PGC-1α (PPAR coactivator-1α)inmicemuscle[105].This medical relevance. is in agreement with the existence of a PPAR response element in the Heme A is a unique heme compound present exclusively in COX. It PGC-1α promoter [106].PGC-1α up-regulates a large group of nuclear differs from protoheme (heme B) because it has a farnesyl instead of a genes encoding respiratory chain components or proteins involved in vinyl group at carbon C2 and a formyl instead of a methyl group at mitochondria biogenesis, such as the mitochondrial transcription factor carbon C8 [96]. The first step of the heme A biosynthetic pathway is A (Tfam). PGC-1α does not bind to the DNA, but rather activates the the conversion of heme B to heme O, a reaction that is catalyzed by the nuclear respiratory factors 1 and 2 (NRF1 and NRF2) already bound to farnesyl-transferase Cox10p [97]. The subsequent oxidation of heme O the promoter of the target genes, namely genes involved in oxidative to heme A occurs in two discrete monooxygenase steps. The first phosphorylation (review in [107]). It is also known that PGC-1α induces consists of a monooxygenase-catalyzed hydroxylation of the methyl the expression of NRF1 and NRF2 [108]. In the bezafibrate-treated COX10 group at carbon position 8 resulting in an alcohol that would be deficient cells, PGC-1α expression increases about 2.5 fold compared to further oxidized to the aldehyde by a dehydrogenase. The first step is untreated cells while an additional slight increase in the mRNA levels of catalyzed by Cox15p. In S. cerevisiae, Cox15p acts in concert with NRF1 and Tfam is also observed [104].Bezafibrate treatment activates ferredoxin (Yah1p) and the putative ferredoxin reductase Arh1p. The PPARs which in turn directly induces PCG-1α expression and thereby 102 A. Barrientos et al. / Biochimica et Biophysica Acta 1793 (2009) 97–107 starts an activation cascade of nuclear and mitochondrial transcriptional the inner mitochondrial membrane where it is part of the high molec- activators leading to increased COX structural and assembly gene ular weight complex containing Cox14p, Mss51p and newly synthe- expression. It seems clear that bezafibrate suppression of COX10 defects sized Cox1p [121,123]. The interaction Cox14p-Mss51p is unaffected in does not bypass COX10 function, but it is mediated by the increased a Δcoa1 mutant. In the absence of Cox14p, however, the binding of amounts of the partially functional COX10 concomitant with a general Coa1p-Mss51p is disrupted suggesting that the interaction of Coa1p increase in the expression of COX structural subunits. These results with the Cox1p-Cox14p-Mss51p complex is mediated through Cox14p support the possibility that therapeutic approaches aiming to increase (27). Shy1p is not part of the Cox1p-Cox14p-Mss51p-Coa1p complex mitochondrial biogenesis can rescue to some extent partial oxidative (27), but it has been shown to co-precipitate with Coa1p [121,123], phosphorylation defects, including COX assembly deficiencies. suggesting that these two factors probably interact once Mss51p has disengaged from the complex (27). As for cox14 mutants, mutations in 2.4. Suppression of defects affecting the formation of assembly coa1 do not affect the synthesis but the accumulation of Cox1p in the intermediates holoenzyme suggesting that Coa1p also plays a role in the feedback regulation of Cox1p expression [121,123]. The COX assembly defect Suppression studies of defects affecting the formation of assembly in a coa1 null mutant strain can be suppressed by overexpression of intermediates have provided novel information concerning COX Mss51p and Cox10p [97]. Both suppressors can act synergistically assembly regulation and interactions among several COX assembly when the two proteins are co-expressed and thus link Coa1p to both factors. translational regulation and maturation of Cox1p [123]. Pierrel et al. COX assembly is characterized by a concerted accumulation of its have speculated that Coa1p could stabilize the Cox1p-Cox14p-Mss51p constitutive subunits. Data mainly obtained from studies performed complex until Shy1p interacts with Coa1p in a step involving heme A with yeast mutants indicate that most unassembled subunits, insertion into Cox1p and further progression in the assembly process particularly the ones forming the catalytic core, are post-translation- [123]. While Cox1p maturation certainly occurs within these initial ally degraded [110,111]. Recently, another contribution to the Cox1p complexes, the precise role of each factor remains to be stoichiometric accumulation of subunits during COX biogenesis in elucidated. the yeast S. cerevisiae has been reported. It consists of a regulatory At present, it is not clear if Cox1p translation in other organisms is mechanism by which the synthesis of subunit 1 is down-regulated in also subject to regulation by downstream events. Mammalian homo- the absence of its assembly partners [112–115]. Decreased Cox1p logues of Mss51p, Cox14p and Coa1p have not been identified to date. synthesis relative to other mitochondrial translation products was The precise function of SURF1/Shy1p in COX assembly is currently previously reported as a secondary observation in several yeast COX unknown. However, data gathered by studying spontaneous suppres- assembly mutants [116,117]. This phenotype could not be accounted sor mutations and high copy suppressors of yeast shy1 mutants have for by a defect in the translation system because the mutations were indicated that SURF1/Shy1p plays a role in the formation of an early not in proteins related to this mitochondrial activity [116,117]. A major COX assembly intermediate containing subunit 1 as mentioned above. step in the characterization of this regulatory mechanism came from Analysis of COX by Blue Native gel electrophoresis has indicated that studies aiming to understand the function of the COX assembly factor assembly of COX in SURF1 deficient fibroblasts is blocked at an early Shy1p [118]. Shy1p function is of significant interest because the step, most likely before the incorporation of subunit II into the nascent human homologue of SURF1 is responsible for cases of Leigh's intermediates composed of subunit 1 alone or subunit 1 plus subunit 4 syndrome (LS) associated with COX deficiency, a severe neurological and 5a [124– 126]. In the COX assembly model originally proposed by disorder of childhood [24,25]. Nitjmans et al. [16], the first subassembly (S1) is formed exclusively by The study of respiratory deficient strains carrying a null allele of subunit 1, which acts as a seed for sequential incorporation of COX shy1 that spontaneously reverted to a respiratory-competent pheno- subunits. In this model, the addition of subunits COX4 and COX5a type allowed the identification of MSS51 as a extragenic suppressor of (yeast Cox5ap and Cox6p, respectively) to S1 results in the progression shy1 mutations. Both mutant forms of mss51 and overexpression of to the second assembly intermediate (S2). Insertion of heme A into the wild-type gene act as suppressors of shy1 null mutants by COX1 probably occurs before the addition of subunits 4 and 5a, as increasing the levels of newly synthesized Cox1p [112]. Mss51p is suggested by the accumulation of the COX1–COX4–COX5a intermedi- required for COX1 mRNA translation [114,119] and also plays a post- ate in SCO1 and SCO2 mutant fibroblasts [36,126] but not in COX10 and translational role [113,114] as discussed below. Screens of a collection COX15 mutant cells [32,33,126]. These findings suggest that the pres- of strains carrying null mutations of COX biogenesis factors showed ence of heme A in COX1 might stabilize its binding to COX4 and COX5a. that the amount of newly synthesized Cox1p is not only reduced in After the formation of the COX1–COX4–COX5a subassembly, the COX shy1 mutants, but also in most COX assembly mutants. In all these assembly process continues with the formation of the third proposed mutants, the Cox1p synthesis defect is restored by the mss51 sup- intermediate (S3) by the addition of the remaining structural subunits pressors of shy1 or by mutations in cox14, which codes for another with the exception of COX6a and COX7a/b (yeast subunits 10 and 7). COX assembly factor [120]. Mss51p and Cox1p were shown to form a These subunits are added later to complete the holoenzyme [16,126]. transient complex [113,114] that is stabilized by Cox14p [38]. These This model has been refined by recent observations. New analysis of interactions have been postulated to down-regulate Cox1p synthesis subassemblies in fibroblasts from patients with SCO2 and SURF1 when COX assembly is impaired [38]. According to this model, the suggested that mammalian COX4 and COX5a subunits form a dimer release of Mss51p from the ternary complex and its availability for before their incorporation into S1 [36]. Noticeably, the equivalent yeast additional Cox1p synthesis occur at a downstream step in the Cox5p–Cox6p dimer was also detected in a yeast COX mutant in which assembly pathway, and is likely catalyzed by Shy1p [113,121]. The assembly is compromised in the latter stages of the process [40]. This unique properties of this regulatory mechanism offer a means to result is consistent with the observation that in yeast the presence catalyze multiple-subunit assembly [113,114,121]. In addition, reduced of subunit 6 is required for subunit 5 stability [127]. An additional Cox1p synthesis in the absence of COX assembly and resulting intermediate seems to exist between S2 and S3 and consists of at least degradation of the other core subunits will also limit the accumulation mammalian subunits COX1–COX2–COX4–COX5a [36].COX2may of partially matured core subunits that, as recently proposed for yeast associate with the COX1–COX4–COX5a intermediate upon its copper subunit 1, could contribute to the production of unstable pro-oxidant metallation by the specific SCO1–SCO2 copper chaperones as sug- intermediates [122]. gested by the observation that SCO1 and SCO2 mutant fibroblasts from Coa1p was recently described as a new player in the subunit 1 patients with COX deficiency accumulate the COX1–COX4–COX5a synthesis regulatory loop [121,123]. Coa1p is a protein associated with intermediate [36,126]. A. Barrientos et al. / Biochimica et Biophysica Acta 1793 (2009) 97–107 103

Recent studies of novel high copy suppressors of shy1 mutants cox10, cox11, cox14, cox15, cox16, cox17, oxa1, mss51 and pet191 failed to have provided insight into SURF1/Shy1p role within the COX assembly induce any suppression of their respiratory growth defect. This obser- process. However, it remains unclear whether the primary Shy1p vation indicated that the suppression by HAP4 was specifictotheshy1 function is required for either Cox1p maturation, assembly or both. null mutant and suggested a specificmechanismofHAP4 suppression in Mss51p mediated suppression of yeast shy1 mutants [112] has been this strain. shown to be enhanced by both co-overexpression of COX10 [123] and It was previously reported that overexpression of the HAP4 gene co-overexpression of the COX subunits Cox5p and Cox6p [128]. The was able to suppress the respiratory defect of a strain carrying a presence of SURF1 orthologues in terminal oxidase operons of several deletion of the matrix domain of Oxa1p although it did not compensate prokaryotes [129] in which COX contains the evolutionary conserved for the total absence of Oxa1p [132], in agreement with our results. core subunits (Cox1p, Cox2p, Cox3p) but not the other structural Oxa1p is a key component of the machinery for the insertion of subunits such as Cox5p and Cox6p suggests a role for SURF1/Shy1p in membrane proteins in mitochondria [133]. The protein interacts with forming the catalytic core of the enzyme. Studies in R. sphaeroides nascent mitochondrial polypeptides [133] and it has been proposed to have suggested that bacterial SURF1 is required for either insertion of mediate the co-translational insertion of mitochondrially encoded heme A at the a3 center or stabilization of the a3-CuB binuclear center subunits through an interaction of its C-terminal tail located in the in COX1 [130]. The respiratory defect of yeast shy1 mutants is not mitochondrial matrix with the mitochondrial ribosome [134,135]. rescued by supplementation of the growth media with hemin [128]. Additionally, Oxa1p seems essential for the translocation of the Analyses of human COX assembly intermediates have shown that the hydrophilic domain of Cox2p [136–138]. The mechanism of the oxa1 SURF1 deficient fibroblasts accumulate the S2 assembly intermediate. suppression by HAP4 remains to be elucidated. Hlavacek et al. reported They are similar in this respect to SCO1 and SCO2 deficient fibroblast an increase in the expression of Cox2p and proposed that this fact but differ from COX10 and COX15 deficient fibroblasts in which S2 is alone or in combination with an increase in the expression of other not formed and only S1 accumulates as mentioned above [36,125,126]. respiratory subunits by HAP4 overexpression could compensate for the These observations apparently argue against a primary role of Surf1p defect in co-translational membrane insertion of these subunits due to in heme A insertion. Exogenous copper, however, partially suppresses the oxa1 mutation [132]. the respiratory defect of a yeast shy1 null mutant strain by a In the case of the shy1 null mutants, the specificity of HAP4 mechanism that remains to be characterized [128]. Although Shy1p suggested that it could act by either increasing the expression of a could play a direct or indirect role in Cox1p maturation, the possibility protein with an overlapping function with Shy1p or the amount of of a SURF1/Shy1p role in facilitating the addition of subunit 2 to the specific COX subunits and/or assembly factors favoring a more efficient Cox1p–5p–6p complex cannot be excluded. Further support for the assembly of COX. In this case, the shy1 suppression by HAP4 was role of Shy1p as an assembly factor was recently reported by showing demonstrated to be mediated by a specific increase in the expression of that Shy1p promotes COX biogenesis through association with subunits Cox5p and Cox6p, the partners of Cox1p in early COX different protein modules, which are potential COX assembly inter- subassemblies [128]. These results give support to the notion that mediates containing Cox1p and Cox5p, among other proteins [121].In Shy1p could act by promoting/stabilizing the formation of assembly addition, Shy1p and Cox14p were also found to interact with partially intermediates. Some of the described suppressor mechanisms of shy1 and fully assembled forms of COX associated with complex III of the mutants are summarized in Fig. 2. mitochondrial respiratory chain [121], which suggests that these two The HAP complex is conserved from yeast to mammals where the COX assembly factors play chaperone roles beyond the early stages of human complex, termed NF-Y, contains three subunits, NF-YA, B and C. COX assembly. The proteins share homology to Hap2p, 3p and 5p, respectively. Some Finally, our group has recently reported that overexpression of HAP4 of the subunits are interchangeable across species. For example, suppresses the respiratory deficient phenotype of yeast shy1 null and human NF-YA, which contains both a DNA binding domain and a point mutant strains [128]. Hap4p is the catalytic subunit of the CCAAT Q-rich activation domain, complements the respiratory defect of a binding site transcriptional activator Hap2p,3p,4p,5p (HAP) complex hap2 yeast mutant [139]. Interestingly, overexpression of NF-YA, the which globally activates transcription of nuclear genes involved in catalytic subunit of the human NF-Y complex, is able to increase mitochondrial respiration during the transition from fermentation to mitochondrial COX activity in SURF1 deficient fibroblasts through a respiration [131]. HAP4 overexpression in strains carrying null alleles of mechanism that remains to be characterized [128].

Fig. 2. Mechanisms of suppression of the COX assembly defect of yeast shy1 mutants. After Cox1p synthesis, this subunit is matured by insertion of heme A and copper prosthetic groups, before or during the formation of an assembly intermediate containing Cox5ap and Cox6p. This ternary complex will subsequently assemble with metallated Cox2p. Shy1p could play a role in either Cox1p maturation or assembly. Mutant and additional copies of mss51 act as shy1 suppressors by increasing the amount of newly synthesized Cox1p, while overexpression of Hap4p suppresses largely by increasing the amount of Cox5ap–Cox6p. This ﬁgure has been reproduced with permission from [128]. 104 A. Barrientos et al. / Biochimica et Biophysica Acta 1793 (2009) 97–107

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