Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR)

A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoR^Sl) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and Cu^I-CsoR^Sl to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoR^Sl reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P₁-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ΔcsoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon.     

Essential Role for Vacuolar Acidification in Candida albicans Virulence

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V₀ subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn²⁺ toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.     

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.     

Pattern of phosphoproteins during cell differentiation in Streptomyces collinus

Transition from vegetative cells to aerial mycelium and spores of Streptomyces collinus is accompanied by changes in the pattern of proteins phosphorylated. Preparation from spores exhibits lower phosphorylation activity than those of vegetative cells and aerial mycelium. Phosphorylation of proteins from aerial mycelium was markedly stimulated by the presence of Mn²⁺. Our data indicate that phosphorylation of proteins on Ser/Thr residues is involved in transition of vegetative cells to aerial mycelium.     

A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.     

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR^Sl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR^Sl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR^Sl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoR^Sl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control.     

Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.     

Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface

The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (DeltardlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the DeltardlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.     

Nucleoside diphosphate kinase-1 regulates hyphal development via the transcriptional regulation of catalase in Neurospora crassa

A nucleoside diphosphate kinase-1-disrupted (ndk-1^RIP-1) mutant was observed to be defective in aerial hyphal and conidial development. In this study, two types of hyphae, fine and thick, were observed in wild-type (Wt) strains. However, only fine-type hyphae were observed in the ndk-1^RIP-1 mutants. The ndk-1^RIP-1 mutants were stimulated by oxidative stress and constitutively expressed an antioxidant enzyme catalase (CAT)-3. Furthermore the ndk-1^RIP-1 mutants could form thick hyphae by oxidative stress and a disruption of cat-3. These results suggest that the loss of thick hyphae in the ndk-1^RIP-1 mutants may be caused by the over-expression of cat-3.     

Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1 ⁺ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1 ⁺ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.     

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.     

Mycophenolic Acid Production by Penicillium brevicompactum on Solid Media

When grown on Czapek-Dox agar, Penicillium brevicompactum produced mycophenolic acid after a vegetative mycelium had been formed and as aerial hyphae were developing. Nutrients were still plenteous in the agar when the synthesis began. If aerial hyphal development was prevented by placing a dialysis membrane over the growing fungus, no mycophenolic acid was produced. When the dialysis membrane was peeled back and, as a consequence, production of aerial hyphae began, mycophenolic acid biosynthesis was observed. We concluded that mycophenolic acid was produced only by P. brevicompactum colonies that possessed an aerial mycelium.     

NsdD Is a Key Repressor of Asexual Development in Aspergillus nidulans

Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (−) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (−) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.     

How yield relates to ash content, ��13C and ��18O in maize grown under different water regimes

Background and Aims

Stable isotopes have proved a valuable phenotyping tool when breeding for yield potential and drought adaptation; however, the cost and technical skills involved in isotope analysis limit its large-scale application in breeding programmes. This is particularly so for Δ¹⁸O despite the potential relevance of this trait in C₄ crops. The accumulation of minerals (measured as ash content) has been proposed as an inexpensive way to evaluate drought adaptation and yield in C₃ cereals, but little is known of the usefulness of this measure in C₄ cereals such as maize (Zea mays). The present study investigates how yield relates to ash content, Δ¹³C and Δ¹⁸O, and evaluates the use of ash content as an alternative or complementary criterion to stable isotopes in assessing yield potential and drought resistance in maize.

Methods

A set of tropical maize hybrids developed by CIMMYT were subjected to different water availabilities, in order to induce water stress during the reproductive stages under field conditions. Ash content and Δ¹³C were determined in leaves and kernels. In addition, Δ¹⁸O was measured in kernels.

Key Results

Water regime significantly affected yield, ash content and stable isotopes. The results revealed a close relationship between ash content in leaves and the traits informing about plant water status. Ash content in kernels appeared to reflect differences in sink–source balance. Genotypic variation in grain yield was mainly explained by the combination of ash content and Δ¹⁸O, whilst Δ¹³C did not explain a significant percentage of such variation.

Conclusions

Ash content in leaves and kernels proved a useful alternative or complementary criterion to Δ¹⁸O in kernels for assessing yield performance in maize grown under drought conditions.     

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.