Purification and characterization of yeast Sco1p, a mitochondrial copper protein

The present studies were undertaken to further characterize the properties of Sco1p, a constituent of the mitochondrial inner membrane implicated in copper transfer to cytochrome oxidase. We report a procedure capable of yielding Sco1p of >95% purity. Sco1p has been purified from strains of Saccharomyces cerevisiae that overexpress the protein. The amino-terminal sequence of purified Sco1p indicates that the first 40 amino acids of the primary translation product constitute a mitochondrial targeting sequence that is proteolytically cleaved during import. We estimate that Sco1p constitutes 0.08% total mitochondrial proteins in wild type yeast and 5% in the transformant used for the purification. Sco1p contains approximately 1 mol of copper/mol protein. The copper is not removed by the treatment of Sco1p with EDTA, indicating that it is bound with high affinity. Purified Sco1p sediments identical to Sco1p in crude extracts of mitochondria from wild type yeast or from a strain transformed with SCO1 on a high copy plasmid. Native Sco1p has an estimated mass of 88 kDa, suggesting that it is a homotrimer. Sco1p expressed as a soluble protein lacking the internal 17 amino acids of the membrane-anchoring domain has been localized in the matrix. The protein has also been targeted to the intermembrane space. Neither soluble matrix nor intermembrane-localized Sco1p is able to complement a sco1 mutant, suggesting that only the membrane form with the carboxyl-terminal domain facing the intermembrane space is able to exert its normal function.     

Mapping the functional interaction of Sco1 and Cox2 in cytochrome oxidase biogenesis

Sco1 is implicated in the copper metallation of the Cu(A) site in Cox2 of cytochrome oxidase. The structure of Sco1 in the metallated and apo-conformers revealed structural dynamics primarily in an exposed region designated loop 8. The structural dynamics of loop 8 in Sco1 suggests it may be an interface for interactions with Cox17, the Cu(I) donor and/or Cox2. A series of conserved residues in the sequence motif (217)KKYRVYF(223) on the leading edge of this loop are shown presently to be important for yeast Sco1 function. Cells harboring Y219D, R220D, V221D, and Y222D mutant Sco1 proteins failed to restore respiratory growth or cytochrome oxidase activity in sco1Delta cells. The mutant proteins are stably expressed and are competent to bind Cu(I) and Cu(II) normally. Specific Cu(I) transfer from Cox17 to the mutant apo-Sco1 proteins proceeds normally. In contrast, using two in vivo assays that permit monitoring of the transient Sco1-Cox2 interaction, the mutant Sco1 molecules appear compromised in a function with Cox2. The mutants failed to suppress the respiratory defect of cox17-1 cells unlike wild-type SCO1. In addition, the mutants failed to suppress the hydrogen peroxide sensitivity of sco1Delta cells. These studies implicate different surfaces on Sco1 for interaction or function with Cox17 and Cox2.     

Characterization of the peroxide sensitivity of COX-deficient yeast strains reveals unexpected relationships between COX assembly proteins

A number of distinct cuproproteins of the mitochondrial inner membrane are required for the assembly of cytochrome oxidase (COX), thought to function in a “bucket brigade” fashion to provide copper to the Cu_A and Cu_B sites. In yeast, the loss of two these proteins, Sco1p and Cox11p, leads to respiratory deficiency and a specific inability to survive exposure to hydrogen peroxide (H₂O₂). Using a quantitative assay, we have identified subtle differences in the peroxide-sensitive phenotypes between sco1 and cox11 mutant strains. Interestingly, the peroxide sensitivity of the sco1 null strain can be suppressed by overexpressing either SCO2 or COX11, although overexpression of neither SCO1 nor SCO2 can rescue the cox11 null strain. We also find that overexpression of either CTT1, encoding the cytosolic catalase T, or CTA1, encoding the mitochondrial matrix catalase, suppresses the peroxide sensitivity in both the sco1 and the cox11 null mutants. Direct measurement of peroxide metabolism shows that sco1 and cox11 null strains fail to degrade a significant amount of exogenously provided H₂O₂. Taken together, our data demonstrate that although Cox11p and Sco1p play distinct roles in COX assembly, they seem to play overlapping or related roles in peroxide metabolism that require further investigation.     

Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His-239, Cys-148 or Cys-152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.     

Human Sco1 and Sco2 function as copper-binding proteins

The function of human Sco1 and Sco2 is shown to be dependent on copper ion binding. Expression of soluble domains of human Sco1 and Sco2 either in bacteria or the yeast cytoplasm resulted in the recovery of copper-containing proteins. The metallation of human Sco1, but not Sco2, when expressed in the yeast cytoplasm is dependent on the co-expression of human Cox17. Two conserved cysteines and a histidyl residue, known to be important for both copper binding and in vivo function in yeast Sco1, are also critical for in vivo function of human Sco1 and Sco2. Human and yeast Sco proteins can bind either a single Cu(I) or Cu(II) ion. The Cu(II) site yields S-Cu(II) charge transfer transitions that are not bleached by weak reductants or chelators. The Cu(I) site exhibits trigonal geometry, whereas the Cu(II) site resembles a type II Cu(II) site with a higher coordination number. To identify additional potential ligands for the Cu(II) site, a series of mutant proteins with substitutions in conserved residues in the vicinity of the Cu(I) site were examined. Mutation of several conserved carboxylates did not alter either in vivo function or the presence of the Cu(II) chromophore. In contrast, replacement of Asp238 in human or yeast Sco1 abrogated the Cu(II) visible transitions and in yeast Sco1 attenuated Cu(II), but not Cu(I), binding. Both the mutant yeast and human proteins were nonfunctional, suggesting the importance of this aspartate for normal function. Taken together, these data suggest that both Cu(I) and Cu(II) binding are critical for normal Sco function.     

Crystal structure of yeast Sco1

The Sco family of proteins are involved in the assembly of the dinuclear Cu_A site in cytochrome c oxidase (COX), the terminal enzyme in aerobic respiration. These proteins, which are found in both eukaryotes and prokaryotes, are characterized by a conserved CXXXC sequence motif that binds copper ions and that has also been proposed to perform a thiol:disulfide oxidoreductase function. The crystal structures of Saccharomyces cerevisiae apo Sco1 (apo-ySco1) and Sco1 in the presence of copper ions (Cu–ySco1) were determined to 1.8- and 2.3-Å resolutions, respectively. Yeast Sco1 exhibits a thioredoxin-like fold, similar to that observed for human Sco1 and a homolog from Bacillus subtilis. The Cu–ySco1 structure, obtained by soaking apo-ySco1 crystals in copper ions, reveals an unexpected copper-binding site involving Cys181 and Cys216, cysteine residues present in ySco1 but not in other homologs. The conserved CXXXC cysteines, Cys148 and Cys152, can undergo redox chemistry in the crystal. An essential histidine residue, His239, is located on a highly flexible loop, denoted the Sco loop, and can adopt positions proximal to both pairs of cysteines. Interactions between ySco1 and its partner proteins yeast Cox17 and yeast COX2 are likely to occur via complementary electrostatic surfaces. This high-resolution model of a eukaryotic Sco protein provides new insight into Sco copper binding and function.     

A human SCO2 mutation helps define the role of Sco1p in the cytochrome oxidase assembly pathway

Deficiencies in cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, are most often caused by an inability to complete assembly of the enzyme. Pathogenic mutations in SCO2, which encodes a cytochrome oxidase assembly factor, were recently described in several cases of fatal infantile cardioencephalomyopathy. To determine the molecular etiology of these disorders, we describe the generation and characterization of the parallel mutations in the homologous yeast SCO1 gene. We show that the E155K yeast sco1 mutant is respiration-competent, whereas the S240F mutant is not. Interestingly, the S240F mutation allows partial but incorrect assembly of cytochrome oxidase, as judged by an altered cytochrome aa(3) peak. Immunoblot analysis reveals a specific absence of subunit 2 from the cytochrome oxidase in this mutant. Taken together, our data suggest that Sco1p provides copper to the Cu(A) site on subunit 2 at a step occurring late in the assembly pathway. This is the first instance of a yeast cytochrome oxidase assembly mutant that is partially assembled. The S240F mutant also represents a powerful new tool with which to elucidate further steps in the cytochrome oxidase assembly pathway.     

Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria

The genome of Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family (MCF) and 58 MCF members are coded by the genome of Arabidopsis thaliana, most of which have been functionally characterized. Here two members of this family, Ymc2p from S. cerevisiae and BOU from Arabidopsis, have been thoroughly characterized. These proteins were overproduced in bacteria and reconstituted into liposomes. Their transport properties and kinetic parameters demonstrate that Ymc2p and BOU transport glutamate, and to a much lesser extent L-homocysteinesulfinate, but not other amino acids and many other tested metabolites. Transport catalyzed by both carriers was saturable, inhibited by mercuric chloride and dependent on the proton gradient across the proteoliposomal membrane. The growth phenotype of S. cerevisiae cells lacking the genes ymc2 and agc1, which encodes the only other S. cerevisiae carrier capable to transport glutamate besides aspartate, was fully complemented by expressing Ymc2p, Agc1p or BOU. Mitochondrial extracts derived from ymc2Δagc1Δ cells, reconstituted into liposomes, exhibited no glutamate transport at variance with wild-type, ymc2Δ and agc1Δ cells, showing that S. cerevisiae cells grown in the presence of acetate do not contain additional mitochondrial transporters for glutamate besides Ymc2p and Agc1p. Furthermore, mitochondria isolated from wild-type, ymc2Δ and agc1Δ strains, but not from the double mutant ymc2Δagc1Δ strain, swell in isosmotic ammonium glutamate showing that glutamate is transported by Ymc2p and Agc1p together with a H⁺. It is proposed that the function of Ymc2p and BOU is to transport glutamate across the mitochondrial inner membrane and thereby play a role in intermediary metabolism, C1 metabolism and mitochondrial protein synthesis.     

HCC1, the Arabidopsis homologue of the yeast mitochondrial copper chaperone SCO1, is essential for embryonic development

The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomyces cerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phenotype of the hcc1-1 line. Localization of the SNAP-tagged HCC1 in transgenic lines identified HCC1 as a mitochondrial protein. To determine if HCC1 is a functional homologue to Sco1p, the respiratory-deficient yeast sco1 mutant was transformed with chimeric constructs containing different combinations of HCC1 and SCO1 sequences. One of the resulting chimeric proteins restored respiration in the yeast mutant. This protein had the N-terminal mitochondrial targeting signal and the single transmembrane domain derived from Sco1p and the C-terminal half (including the copper-binding motif) derived from HCC1. Growth of the complemented yeast mutant was enhanced by the addition of copper to the medium. The data demonstrate that HCC1 is essential for embryo development in Arabidopsis, possibly due to its role in cytochrome c oxidase assembly.     

Multiple proteins and phosphorylations regulate Saccharomycescerevisiae Cdc24p localization

Targeting of Saccharomyces cerevisiae Cdc24p to polarized growth sites is essential for its function. Localization of GFP-tagged Cdc24 proteins or fragments was assayed in deletion mutants of Cdc24p-interacting proteins. The boi2Δ, ent2Δ, and hua1Δ mutants showed localization defects. The tos2Δ skg6Δ double mutant displayed aberrant pre-anaphase localization to the mother-bud neck region. The same aberrant pattern was seen when potential phosphorylation sites Ser697, Thr704, and Tyr200 were mutated. The S697A mutation also resulted in phosphorylation defects in vivo. These data support roles for Boi2p, Ent2p, Hua1p, Tos2p, and for Cdc24p phosphorylation in targeting Cdc24p to growth sites.     

Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase

The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the Cu(B) and Cu(A) sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins.     

Mdm35p imports Ups proteins into the mitochondrial intermembrane space by functional complex formation

Ups1p, Ups2p, and Ups3p are three homologous proteins that control phospholipid metabolism in the mitochondrial intermembrane space (IMS). The Ups proteins are atypical IMS proteins in that they lack the two major IMS‐targeting signals, bipartite presequences and cysteine motifs. Here, we show that Ups protein import is mediated by another IMS protein, Mdm35p. In vitro import assays show that import of Ups proteins requires Mdm35p. Loss of Mdm35p led to a decrease in steady state levels of Ups proteins in mitochondria. In addition, mdm35Δ cells displayed a similar phenotype to ups1Δups2Δups3Δ cells. Interestingly, unlike typical import machineries, Mdm35p associated stably with Ups proteins at a steady state after import. Demonstrating that Mdm35p is a functional component of Ups–Mdm35p complexes, restoration of Ups protein levels in mdm35Δ mitochondria failed to restore phospholipid metabolism. These findings provide a novel mechanism in which the formation of functional protein complexes drives mitochondrial protein import.     

A Sco homologue plays a role in defence against oxidative stress in pathogenic Neisseria

Sco proteins are found in mitochondria and in a variety of oxidase positive bacteria. Although Sco is required for the formation of the Cu(A) centre in a cytochrome oxidase of the aa(3) type, it was observed that oxidases with a Cu(A) centre are not present in many bacteria that contain a Sco homologue. Two bacteria of this type are the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. The sco genes of N. gonorrhoeae strain 1291 and N. meningitidis strain MC58 were cloned, inactivated by inserting a kanamycin resistance cassette and used to make knockout mutants by allelic exchange. Both N. gonorrhoeae and N. meningitidis sco mutants were highly sensitive to oxidative killing by paraquat, indicating that Sco is involved in protection against oxidative stress in these bacteria.     

The yeast O-acyltransferase Gup1p interferes in lipid metabolism with direct consequences on the sphingolipid-sterol-ordered domains integrity/assembly

Saccharomyces cerevisiae Gup1p is a membrane-bound O-acyltransferase. Previous works involved GUP1 in a wide range of crucial processes for cell preservation and functioning. These include cytoskeleton polarization and secretory/endocytic pathway, GPI-anchor remodelling, wall composition and integrity, and membrane lipids, with a reduction in phospholipids and an increase in acylglycerols. DRM fractions were found in considerably lower amounts in gup1Δ than in wt strain. Additionally, the proteins presumably associated with lipid micro domains, Gas1p and Pma1p, were present in much smaller amounts in the mutant DRMs. Pma1p is also found in minor quantities in the whole cells extracts of the gup1Δ mutant. Accordingly, H⁺-ATPase activity was reduced in about 40%. Deletion of GUP1 resulted in higher sensibility to specific sphingolipid biosynthesis inhibitors and a notorious resistance to ergosterol biosynthesis inhibitors. Furthermore, the majority of mutant cells displayed an even (less punctuated) sterol distribution. The present work presents improvements to DRMs extraction methodology and filipin-sterol staining, provides evidence supporting that Gup1p is involved in lipid metabolism and shows the direct consequences of its absence on the plasma membrane sphingolipid-sterol-ordered domains integrity/assembly.     

The antioxidant function of Sco proteins depends on a critical surface-exposed residue

BackgroundBesides their role in copper metabolism, Sco proteins from different organisms have been shown to play a defensive role against oxidative stress. In the present study, we set out to identify crucial amino acid residues for the antioxidant activity.MethodsNative and mutated Sco proteins from human, Arabidopsis thaliana and the yeast Kluyveromyces lactis were expressed in the model organism Saccharomyces cerevisiae. The oxidative stress resistance of the respective transformants was determined by growth and lipid peroxidation assays.ResultsA functionally important site, located 15 amino acids downstream of the well-conserved copper binding CxxxC motif, was identified. Mutational analysis revealed that a positive charge at this position has a detrimental effect on the antioxidant capacity. Bioinformatic analysis predicts that this site is surface-exposed, and according to Co-IP data it is required for binding of proteins that are connected to known antioxidant pathways.ConclusionThis study shows that the antioxidant capacity of eukaryotic Sco proteins is conserved and depends on the presence of functional site(s) rather than the extent of overall sequence homology.General significanceThese findings provide an insight into the conserved functional sites of eukaryotic Sco proteins that are crucial for combating oxidative stress. This capacity is probably not due to an enzymatic activity but rather is indirectly mediated by interaction with other proteins.     

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.     

Pleiotropic role of the Sco1/SenC family copper chaperone in the physiology of Streptomyces

Antibiotic production and cell differentiation in Streptomyces is stimulated by micromolar levels of Cu²⁺. Here, we knocked out the Sco1/SenC family copper chaperone (ScoC) encoded in the conserved gene cluster ‘sco’ (the S treptomycesco pper utilization) in Streptomyces coelicolor A3(2) and S. griseus. It is known that the Sco1/SenC family incorporates Cu²⁺ into the active centre of cytochrome oxidase (cox). The knockout caused a marked delay in antibiotic production and aerial mycelium formation on solid medium, temporal pH decline in glucose‐containing liquid medium, and significant reduction of cox activity in S. coelicolor. The scoC mutant produced two‐ to threefold higher cellular mass of the wild type exhibiting a marked cox activity in liquid medium supplied with 10 µM CuSO₄, suggesting that ScoC is involved in not only the construction but also the deactivation of cox. The scoC mutant was defective in the monoamine oxidase activity responsible for cell aggregation and sedimentation. These features were similarly observed with regard to the scoC mutant of S. griseus. The scoC mutant of S. griseus was also defective in the extracellular activity oxidizing N,N′‐dimethyl‐p‐phenylenediamine sulfate. Addition of 10 µM CuSO₄ repressed the activity of the conserved promoter preceding scoA and caused phenylalanine auxotrophy in some Streptomyces spp. probably because of the repression of pheA; pheA encodes prephenate dehydratase, which is located at the 3′ terminus of the putative operon structure. Overall, the evidence indicates that Sco is crucial for the utilization of copper under a low‐copper condition and for the activation of the multiple Cu²⁺‐containing oxidases that play divergent roles in the complex physiology of Streptomyces.     

PrrC, a Sco homologue from Rhodobacter sphaeroides, possesses thiol-disulfide oxidoreductase activity

PrrC is a Sco homologue in Rhodobacter sphaeroides that is associated with PrrBA, a two-component signal transduction system that induces photosynthesis gene expression in response to a decrease in oxygen tension. Although Sco proteins have been shown to bind copper the observation that they are structurally-related to thioredoxins suggested that they might possess thiol-disulfide oxidoreductase activity. Our results show that PrrC reduces Cu(2+) to Cu(+) and possesses disulfide reductase activity. These results indicate that some bacterial Sco proteins may have biochemical properties that are distinct from those of mitochondrial Sco proteins.     

Deletion of the mitochondrial Pim1/Lon protease in yeast results in accelerated aging and impairment of the proteasome

The Saccharomyces cerevisiae homolog of the ATP-dependent Lon protease, Pim1p, is essential for mitochondrial protein quality control, DNA maintenance, and respiration. Here, we demonstrate that Pim1p activity declines in aging cells and that Pim1p deficiency shortens the replicative life span of yeast mother cells. This accelerated aging of pim1Δ cells is accompanied by elevated cytosolic levels of oxidized and aggregated proteins, as well as reduced proteasome activity. Overproduction of Hsp104p greatly diminishes aggregation of oxidized cytosolic proteins, rescues proteasome activity, and restores life span of pim1Δ cells to near wild-type levels. Our results show that defects in mitochondrial protein quality control have global intracellular effects leading to the increased generation of misfolded proteins and cytosolic protein aggregates, which are linked to a decline in replicative potential.