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.     

Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast

Surf1p is a protein involved in the assembly of mitochondrial respiratory chain complexes. However its exact role in this process remains to be elucidated. We studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better understanding of the molecular pathogenesis of cytochrome c oxidase (COX) deficiency in SURF1 mutant cells from Leigh syndrome patients. Assembly of COX was analysed in a shy1 null mutant strain by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Steady-state levels of the enzyme were found to be strongly reduced, the total amount of assembled complex being approximately 30% of control. The presence of a significant amount of holo-COX in the SHY1-disruptant strain suggests that Shy1p may either facilitate assembly of the enzyme, or increase its stability. However, our observations, based on 2D-PAGE analysis of mitochondria labelled in vitro, now provide the first direct evidence that COX assembly is impaired in a Deltashy1 strain. COX enzyme assembled in the absence of Shy1p appears to be structurally and enzymically normal. The in vitro labelling studies additionally indicate that mitochondrial translation is significantly increased in the shy1 null mutant strain, possibly reflecting a compensatory mechanism for reduced respiratory capacity. Protein interactions of both Shy1p and Surf1p are implied by their appearance in a high molecular weight complex of about 250 kDa, as shown by 2D-PAGE.     

Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is shown to be dependent on a new assembly factor designated Coa1 that associates with the mitochondrial inner membrane. Translation of the mitochondrial-encoded subunits of CcO occurs normally in coa1Delta cells, but these subunits fail to accumulate. The respiratory defect in coa1Delta cells is suppressed by high-copy MSS51, MDJ1 and COX10. Mss51 functions in Cox1 translation and elongation, whereas Cox10 participates in the biosynthesis of heme a, a key cofactor of CcO. Respiration in coa1Delta and shy1Delta cells is enhanced when Mss51 and Cox10 are coexpressed. Shy1 has been implicated in formation of the heme a3-Cu(B) site in Cox1. The interaction between Coa1 and Cox1, and the physical and genetic interactions between Coa1 and Mss51, Shy1 and Cox14 suggest that Coa1 coordinates the transition of newly synthesized Cox1 from the Mss51:Cox14 complex to the heme a cofactor insertion involving Shy1. coa1Delta cells also display a mitochondrial copper defect suggesting that Coa1 may have a direct link to copper metallation of CcO.     

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.     

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.     

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.     

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.     

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.     

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.     

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.     

Identification and characterization of cytochrome bc(1) subcomplexes in mitochondria from yeast with single and double deletions of genes encoding cytochrome bc(1) subunits

We have examined the status of the cytochrome bc(1) complex in mitochondrial membranes from yeast mutants in which genes for one or more of the cytochrome bc(1) complex subunits were deleted. When membranes from wild-type yeast were resolved by native gel electrophoresis and analyzed by immunodecoration, the cytochrome bc(1) complex was detected as a mixed population of enzymes, consisting of cytochrome bc(1) dimers, and ternary complexes of cytochrome bc(1) dimers associated with one and two copies of the cytochrome c oxidase complex. When membranes from the deletion mutants were resolved and analyzed, the cytochrome bc(1) dimer was not associated with the cytochrome c oxidase complex in many of the mutant membranes, and membranes from some of the mutants contained a common set of cytochrome bc(1) subcomplexes. When these subcomplexes were fractionated by SDS/PAGE and analyzed with subunit-specific antibodies, it was possible to recognize a subcomplex consisting of cytochrome b, subunit 7 and subunit 8 that is apparently associated with cytochrome c oxidase early in the assembly process, prior to acquisition of the remaining cytochrome bc(1) subunits. It was also possible to identify a subcomplex consisting of subunit 9 and the Rieske protein, and two subcomplexes containing cytochrome c(1) associated with core protein 1 and core protein 2, respectively. The analysis of all the cytochrome bc(1) subcomplexes with monospecific antibodies directed against Bcs1p revealed that this chaperone protein is involved in a late stage of cytochrome bc(1) complex assembly.     

Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly

Respiratory chain (RC) complexes are organized into supercomplexes forming 'respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly.     

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.     

Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System

The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex.     

A Highlights from MBoC Selection: A complex of Cox4 and mitochondrial Hsp70 plays an important role in the assembly of the cytochrome c oxidase

The formation of the mature cytochrome c oxidase (complex IV) involves the association of nuclear- and mitochondria-encoded subunits. The assembly of nuclear-encoded subunits like cytochrome c oxidase subunit 4 (Cox4) into the mature complex is poorly understood. Cox4 is crucial for the stability of complex IV. To find specific biogenesis factors, we analyze interaction partners of Cox4 by affinity purification and mass spectroscopy. Surprisingly, we identify a complex of Cox4, the mitochondrial Hsp70 (mtHsp70), and its nucleotide-exchange factor mitochondrial GrpE (Mge1). We generate a yeast mutant of mtHsp70 specifically impaired in the formation of this novel mtHsp70-Mge1-Cox4 complex. Strikingly, the assembly of Cox4 is strongly decreased in these mutant mitochondria. Because Cox4 is a key factor for the biogenesis of complex IV, we conclude that the mtHsp70-Mge1-Cox4 complex plays an important role in the formation of cytochrome c oxidase. Cox4 arrests at this chaperone complex in the absence of mature complex IV. Thus the mtHsp70-Cox4 complex likely serves as a novel delivery system to channel Cox4 into the assembly line when needed.     

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.