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Alexander Tzagoloff

Publications and source records attributed to Alexander Tzagoloff.

At least 19 recordsLinked to original sources
PubMed2006

The metalloprotease encoded by ATP23 has a dual function in processing and assembly of subunit 6 of mitochondrial ATPase.

In the present study we have identified a new metalloprotease encoded by the nuclear ATP23 gene of Saccharomyces cerevisiae that is essential for expression of mitochondrial ATPase (F(1)-F(O) complex). Mutations in ATP23 cause the accumulation of the precursor form of subunit 6 and prevent assembly of F(O). Atp23p is associated with the mitochondrial inner membrane and is conserved from yeast to humans. A mutant harboring proteolytically inactive Atp23p accumulates the subunit 6 precursor but is nonetheless able to assemble a functional ATPase complex. These results indicate that removal of the subunit 6 presequence is not an essential event for ATPase biogenesis and that Atp23p, in addition to its processing activity, must provide another important function in F(O) assembly. The product of the yeast ATP10 gene was previously shown to interact with subunit 6 and to be required for its association with the subunit 9 ring. In this study one extra copy of ATP23 was found to be an effective suppressor of an atp10 null mutant, suggesting an overlap in the functions of Atp23p and Atp10p. Atp23p may, therefore, also be a chaperone, which in conjunction with Atp10p mediates the association of subunit 6 with the subunit 9 ring.

Amino Acid Sequence↗
PubMed2006

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.

5' Untranslated Regions↗
PubMed2006

The Saccharomyces cerevisiae ATP22 gene codes for the mitochondrial ATPase subunit 6-specific translation factor.

Mutations in the Saccharomyces cerevisiae ATP22 gene were previously shown to block assembly of the F0 component of the mitochondrial proton-translocating ATPase. Further inquiries into the function of Atp22p have revealed that it is essential for translation of subunit 6 of the mitochondrial ATPase. The mutant phenotype can be partially rescued by the presence in the same cell of wild-type mitochondrial DNA and a rho- deletion genome in which the 5'-UTR, first exon, and first intron of COX1 are fused to the fourth codon of ATP6. The COX1/ATP6 gene is transcribed and processed to the mature mRNA by splicing of the COX1 intron from the precursor. The hybrid protein translated from the novel mRNA is proteolytically cleaved at the normal site between residues 10 and 11 of the subunit 6 precursor, causing the release of the polypeptide encoded by the COX1 exon. The ability of the rho- suppressor genome to express subunit 6 in an atp22 null mutant constitutes strong evidence that translation of subunit 6 depends on the interaction of Atp22p with the 5'-UTR of the ATP6 mRNA.

5' Untranslated Regions↗
PubMed2005

COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.

In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA.

Amino Acid Sequence↗
PubMed2005

The Saccharomyces cerevisiae COQ10 gene encodes a START domain protein required for function of coenzyme Q in respiration.

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q2. Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q6/mol of protein. We propose that Coq10p is a Q6-binding protein and that in the coq10 mutant Q6 it is not able to act as an electron carrier, possibly because of improper localization.

Amino Acid Sequence↗
PubMed2005

COQ9, a new gene required for the biosynthesis of coenzyme Q in Saccharomyces cerevisiae.

Currently, eight genes are known to be involved in coenzyme Q6 biosynthesis in Saccharomyces cerevisiae. Here, we report a new gene designated COQ9 that is also required for the biosynthesis of this lipoid quinone. The respiratory-deficient pet mutant C92 was found to be deficient in coenzyme Q and to have low mitochondrial NADH-cytochrome c reductase activity, which could be restored by addition of coenzyme Q2. The mutant was used to clone COQ9, corresponding to reading frame YLR201c on chromosome XII. The respiratory defect of C92 is complemented by COQ9 and suppressed by COQ8/ABC1. The latter gene has been shown to be required for coenzyme Q biosynthesis in yeast and bacteria. Suppression by COQ8/ABC1 of C92, but not other coq9 mutants tested, has been related to an increase in the mitochondrial concentration of several enzymes of the pathway. Coq9p may either catalyze a reaction in the coenzyme Q biosynthetic pathway or have a regulatory role similar to that proposed for Coq8p.

Chromatography, High Pressure Liquid↗
PubMed2004

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.

Base Sequence↗
PubMed2004

COX23, a homologue of COX17, is required for cytochrome oxidase assembly.

Deletion of reading frame YHR116W of the Saccharomyces cerevisiae nuclear genome elicits a respiratory deficiency. The encoded product, here named Cox23p, is shown to be required for the expression of cytochrome oxidase. Cox23p is homologous to Cox17p, a water-soluble copper protein previously implicated in the maturation of the Cu(A) center of cytochrome oxidase. The respiratory defect of a cox23 null mutant is rescued by high concentrations of copper in the medium but only when the mutant harbors COX17 on a high copy plasmid. Overexpression of Cox17p by itself is not a sufficient condition to rescue the mutant phenotype. Cox23p, like Cox17p, is detected in the intermembrane space of mitochondria and in the postmitochondrial supernatant fraction, the latter consisting predominantly of cytosolic proteins. Because Cox23p and Cox17p are not part of a complex, the requirement of both for cytochrome oxidase assembly suggests that they function in a common pathway with Cox17p acting downstream of Cox23p.

Amino Acid Sequence↗
PubMed2004

Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase.

The F(0)F(1)-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products. The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits. Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F(0) sector of the ATPase. Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p. This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase. Pulse labeling and chase of mitochondrial translation products in vivo indicate that Atp6p is less stable and more rapidly degraded in an atp10 null mutant than in wild type. Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.

Adenosine Triphosphatases↗
PubMed2004

Aep3p stabilizes the mitochondrial bicistronic mRNA encoding subunits 6 and 8 of the H+-translocating ATP synthase of Saccharomyces cerevisiae.

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to approximately 35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.

Amino Acid Motifs↗
PubMed2003

The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis.

Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis.

Amino Acid Motifs↗
PubMed2003

MTG1 codes for a conserved protein required for mitochondrial translation.

The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence. A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria. Mtg1p is a member of a family of GTPases with largely unknown functions. The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis. Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants. This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action. The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs. More likely, Mtg1p functions in assembly of the large ribosomal subunit. This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.

Cloning, Molecular↗
PubMed2003

ATP22, a nuclear gene required for expression of the F0 sector of mitochondrial ATPase in Saccharomyces cerevisiae.

Expression of the mitochondrial proton-translocating ATPase of Saccharomyces cerevisiae has been shown to depend on chaperones that target the F1 and F0 sectors of this inner membrane complex. Here we report a new gene, designated ATP22 (reading frame YDR350C on chromosome IV), that provides an essential function in the assembly of F0. ATP22 was cloned by transformation of C208/L2, a strain previously assigned to complementation group G99 of a collection of respiration-defective nuclear pet mutants. C208/L2 and the other atp22 mutants have oligomycin-insensitive F1-ATPase, suggesting that the lesion is confined to F0. This is supported by the sedimentation properties of the mutant ATPase and results of immunochemical analysis of F0 subunit polypeptides. Northern analysis of ATPase transcripts and in vivo pulse labeling of the mitochondrial translation products in the mutant indicate normal expression of subunits 6, 8, and 9, the three mitochondrial gene products of F0. Atp22p therefore functions at a post-translational stage in assembly of F0. Localization studies indicate Atp22p to be a component of the mitochondrial inner membrane. Protease protection experiments further indicate that Atp22p faces the matrix side of the membrane where most of the ATPase proteins are located and assembled.

Adenosine Triphosphatases↗
PubMed2003

Yeast dihydroxybutanone phosphate synthase, an enzyme of the riboflavin biosynthetic pathway, has a second unrelated function in expression of mitochondrial respiration.

aE280/U1 is a pet mutant of Saccharomyces cerevisiae partially deficient in cytochromes a, a3, and cytochrome b. The ability of this mutant to respire is restored by RIB3, a gene previously shown to code for 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP synthase), an enzyme of the riboflavin biosynthetic pathway. The sequences of RIB3 from wild type and aE280/U1 indicated a single base change resulting in an A137T substitution. The alanine 137 is a conserved residue located in a cavity on the surface of the protein distant from the active site and from the subunit interaction domain involved in homodimer formation. The respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1) growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin; 2) the levels of flavin nucleotides are not significantly different in the mutant and wild type. We proposed that in addition to its known function in riboflavin synthesis, RIB3 also functions in expression of mitochondrial respiration. Restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol also supported this conclusion. An antibody against the N-terminal half of DHBP synthase was used to study its subcellular distribution. Most of the protein was localized in the cytosolic fraction, but a small fraction was detected in the mitochondrial intermembrane space.

Cell Compartmentation↗
PubMed2003

Cytochrome oxidase assembly does not require catalytically active cytochrome C.

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

Alleles↗
PubMed2002

COX16 encodes a novel protein required for the assembly of cytochrome oxidase in Saccharomyces cerevisiae.

We have characterized Cox16p, a new cytochrome oxidase (COX) assembly factor. This protein is encoded by COX16, corresponding to the previously uncharacterized open reading frame YJL003w of the yeast genome. COX16 was identified in studies of COX-deficient mutants previously assigned to complementation group G22 of a collection of yeast pet mutants. To determine its location, Cox16p was tagged with a Myc epitope at the C terminus. The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is detected solely in mitochondria. Cox16p-myc is an integral component of the mitochondrial inner membrane, with its C terminus exposed to the intermembrane space. Cox16 homologues are found in both the human and murine genomes, although human COX16 does not complement the yeast mutant. Cox16p does not appear to be involved in maturation of subunit 2, copper recruitment, or heme A biosynthesis. Cox16p is thus a new protein in the growing family of eukaryotic COX assembly factors for which there are as yet no specific functions known. Like other recently described nuclear gene products involved in expression of cytochrome oxidase, COX16 is a candidate for screening in inherited human COX deficiencies.

Amino Acid Sequence↗
PubMed2002

Characterization of COX19, a widely distributed gene required for expression of mitochondrial cytochrome oxidase.

COX19, a nuclear gene of Saccharomyces cerevisiae, was cloned by transformation of a respiratory-deficient mutant from complementation group G188 of a pet mutant collection. The gene codes for an 11-kDa protein (Cox19p) required for expression of cytochrome oxidase. Because cox19 mutants are able to synthesize the mitochondrial and nuclear gene products of cytochrome oxidase, Cox19p probably functions post-translationally during assembly of the enzyme. Cox19p is present in the cytoplasm and mitochondria, where it exists as a soluble intermembrane protein. This dual location is similar to what was previously reported for Cox17p, a low molecular weight copper protein thought to be required for maturation of the CuA center of subunit 2 of cytochrome oxidase. The similarity in their subcellular distribution, combined with the presence of four cysteines in Cox19p that align with a subset of the cysteines in Cox17p, suggests that like the latter, Cox19p may function in metal transport to mitochondria.

Amino Acid Sequence↗