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R O Poyton

Publications and source records attributed to R O Poyton.

At least 19 recordsLinked to original sources
PubMed1995

Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast cytochrome c.

Subunit V, one of the nuclear-coded subunits of yeast cytochrome c oxidase, has two isoforms, Va and Vb. These alter the in vivo intramolecular rates of electron transfer within the holoenzyme (Waterland, R. A., Basu, A., Chance, B., and Poyton, R. O. (1991) J. Biol. Chem. 266, 4180-4186). The isozyme with Vb has a higher turnover rate and a higher intramolecular transfer rate than the isozyme with Va. To determine how these isoforms affect catalysis, we have examined their effects on the binuclear reaction center and on the interaction between cytochrome c oxidase and the two isoforms, iso-1 and iso-2, of yeast cytochrome c. Infrared spectroscopy of carbon monoxide liganded to heme a3 has revealed a single conformer for the binuclear reaction center in the isozyme with Vb but two discrete conformers in the isozyme with Va. The kinetics of interaction for all four pairwise combinations of isozymes with each subunit V isoform and the two cytochrome c isoforms are biphasic, with high and low affinity electron transfer reactions. In general, the isoforms of cytochrome c and subunit V do not alter the Km but do affect the TNmax. The TNmax for isozymes carrying Vb are higher at both high and low affinity sites for each cytochrome c isoform. Iso-1-cytochrome c supports a higher TNmax than Iso-2-cytochrome c. Surprisingly, the combinatorial effect of both sets of isoforms on TNmax is minimized with the pairs of isoforms (iso-1-cytochrome c and subunit Va or iso-2 and subunit Vb) that are co-expressed in cells. Together, these findings support the conclusion that the subunit V isoforms modulate catalysis and suggest that they do so by affecting the environment or structure of the binuclear reaction center. They also suggest that the coexpression of the two cytochrome c isoforms with two subunit V isoforms serves to minimize differences in electron transfer rates brought about by the subunit V isoforms.

Cytochrome c Group↗
PubMed1992

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

Base Sequence↗
PubMed1992

ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae.

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

Base Sequence↗
PubMed1991

The isoforms of yeast cytochrome c oxidase subunit V alter the in vivo kinetic properties of the holoenzyme.

One of the nuclear-coded subunits of yeast cytochrome c oxidase is specified by a gene family composed of two genes, COX5a and COX5b. These genes are regulated differentially by oxygen and encode isoforms of subunit V, designated Va and Vb, which have only 66% primary sequence identity. Yeast cells require one or the other isoform for a functional cytochrome c oxidase (Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell Biol. 7, 3520-3526). To determine if these isoforms of subunit V alter the catalytic properties of holocytochrome c oxidase, we have analyzed various aspects of cytochrome c oxidase function in intact yeast cells that produce only one type of isoform. From measurements of room temperature turnover numbers and low temperature rates of ligand binding, single turnover cytochrome c oxidation, and internal electron transfer (heme a oxidation), we have found that isozymes which incorporate the Vb isoform have both higher turnover rates and higher rates of heme a oxidation than isozymes which incorporate Va. These findings support the conclusion that the isoforms of subunit V modulate cytochrome c oxidase activity in vivo and suggest that they do so by altering the rates of one or more intramolecular electron transfer reactions.

Carbon Monoxide↗
PubMed1991

Mitochondrial gene expression in Saccharomyces cerevisiae. Proteolysis of nascent chains in isolated yeast mitochondria optimized for protein synthesis.

We demonstrate here that mitochondrial translation products synthesized by isolated yeast mitochondria are subject to rapid proteolysis. The loss of label from mitochondrial peptides synthesized in vitro comes from two distinct pools of peptides: one that is rapidly degraded (t1/2 of minutes) and one that is much more resistant to proteolysis (t1/2 of hours). As the length of the incubation period increases, the percentage of labelled peptides in the rapidly-turning-over pool decreases and cannot be detected after 60 min of incubation. This proteolysis is inhibited by chloramphenicol and is dependent on the presence of ATP. The loss of label during the chase occurs from fully completed translation products. The proteolysis observed here markedly affects measurements of rates of mitochondrial protein synthesis in isolated yeast mitochondria. In earlier work, in which proteolysis was not considered, mitochondrial translation was thought to stop after 20-30 min of incubation. In the present study, by taking proteolysis into account, we demonstrate that the rate of translation in isolated mitochondria is actually constant for nearly 60 min and then decreases to near zero by 80 min of incorporation. These findings have allowed us to devise a procedure for measuring the 'true' rate of translation in isolated mitochondria. In addition, they suggest that mitochondrial translation products which normally assemble with nuclear-encoded gene products into multimeric enzyme complexes are unstable without their nuclear-encoded counterparts.

Chloramphenicol↗
PubMed1990

Mitochondrial import of cytochrome c oxidase subunit VIIa in Saccharomyces cerevisiae. Identification of sequences required for mitochondrial localization in vivo.

Subunit VIIa of yeast cytochrome c oxidase is a small (59 amino acids) protein of the inner mitochondrial membrane that lacks a cleavable amino-terminal presequence. To identify regions within this polypeptide that are essential for its import, gene fusions were constructed using a leader peptide substitution vector (pLPS) developed in this laboratory (Glaser, S. M., Trueblood, C. E., Dircks, L. K., Poyton, R. O., and Cumsky, M. G. (1988) J. Cell. Biochem. 36, 275-287). In this vector, oligonucleotide sequences encoding all or part of subunit VIIa were fused in-frame with the coding region of mature cytochrome c oxidase subunit Va. The plasmid pLPS is ideal for assaying protein sequences for their ability to direct mitochondrial import in vivo since subunit Va's leader peptide is essential for import and because subunit V is required for cytochrome c oxidase activity and respiration. Strains containing these fusions but lacking both subunit V genes (COX5a and COX5b) were analyzed to determine whether the chimeric protein is directed to mitochondria. Our findings indicate that the amino-terminal 17 amino acids of subunit VIIa are sufficient to localize subunit Va to the mitochondrion and that a 6-amino acid-long region within the amino terminus (Gly8-Arg13) is essential. In addition, some import (approximately 10% of wild type) is observed with the highly charged carboxyl terminus of subunit VIIa, suggesting that the subunit may contain redundancy in its import information.

Amino Acid Sequence↗
PubMed1990

Two group I mitochondrial introns in the cob-box and coxI genes require the same MRS1/PET157 nuclear gene product for splicing.

We have studied the role of the product of the nuclear gene PET157 in mitochondrial pre-mRNA splicing. Cytoduction experiments show that a mitochondrial genome deleted for the three introns bI3, aI5 and aI6 is able to suppress the pet157-1 mutation: the strain recovers respiratory competency indicating that the product of the PET157 gene is only required for mitochondrial pre-mRNA splicing. Characterization of the high molecular weight pre-mRNAs which accumulate in the pet157 mutant demonstrate that the product of the PET157 gene is required for the excision of two group I introns bI3 and aI6 (corresponding to aI5 beta) located in the cob-box and coxI genes respectively. Furthermore, the pet157 mutant strain accumulates the bI3 maturase in the form of a polypeptide of 50K (p50) previously observed in mitochondrial mutants defective in the excision of bI3. We have shown by restriction analysis and allelism tests that the pet157-1 mutation is allelic to the nuclear mrs1 mutation, previously described as specifically blocking the excision of bI3. Finally, revertants obtained by the deletion of bI3 or aI6 from the mitochondrial DNA were isolated from the MRS1 disrupted allele, confirming the involvement of the product of the MRS1/PET157 gene in the excision of the two introns bI3 and aI6.

Alleles↗
PubMed1990

Release of two Saccharomyces cerevisiae cytochrome genes, COX6 and CYC1, from glucose repression requires the SNF1 and SSN6 gene products.

We show here that SNF1 and SSN6 are required for derepression of the glucose-repressible yeast genes COX6 and CYC1, which encode the mitochondrial proteins cytochrome c oxidase subunit VI and iso-1-cytochrome c, respectively. In an snf1 mutant genetic background, the transcription of both COX6 and CYC1 continued to be repressed after cells were shifted into derepressing media. In an ssn6 mutant genetic background, both COX6 and CYC1 were expressed constitutively at high levels in repressing media. SSN6 acted epistatically to SNF1 in the regulation of both cytochrome genes. These findings are similar to previous findings on the effects of SNF1 and SSN6 on SUC2 expression in Saccharomyces cerevisiae and are consistent with a model proposing that SNF1 exerts its effect through SSN6 on COX6 and CYC1.

Blotting, Northern↗
PubMed1990

Overexpression of a leaderless form of yeast cytochrome c oxidase subunit Va circumvents the requirement for a leader peptide in mitochondrial import.

Subunit Va of Saccharomyces cerevisiae cytochrome c oxidase is a nucleus-encoded mitochondrial protein that is derived from a precursor with a 20-residue leader peptide. We previously reported that this leader peptide is required for import of subunit Va into mitochondria in vivo (S. M. Glaser, C. E. Trueblood, L. K. Dircks, R. O. Poyton, and M. G. Cumsky, J. Cell. Biochem. 36:275-278, 1988). Here we show that overproduction of a leaderless form of subunit Va circumvents the leader peptide requirement for import into mitochondria in vivo.

Cell Nucleus↗
PubMed1989

Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5 beta from pre-mRNA for cytochrome c oxidase subunit I.

The nuclear PET54 gene of Saccharomyces cerevisiae was cloned and a pet54::LEU2 gene disruption strain was constructed. Analysis of the phenotype of this strain revealed a defect in expression of two mitochondrial genes: COX1, which encodes cytochrome c oxidase subunit I, and COX3, which encodes cytochrome c oxidase subunit III. The defect in COX1 gene expression in the pet54 mutant was shown to be the result of inefficient excision of COX1 intron aI5 beta. Two lines of evidence indicate that inefficient excision of intron aI5 beta is the sole defect in COX1 gene expression. First, a pet54::LEU2 cytoductant bearing the 'short' mitochondrial genome that lacks both COX1 introns aI5 alpha and aI5 beta is defective only in COX3 gene expression and not in COX1 mRNA splicing or mRNA translation. Second, Northern analysis of COX1 transcipts from the pet54 mutant showed that a 3.8 kb COX1 transcript containing unexcised intron aI5 beta and lacking intron aI5 alpha is accumulated while the amount of 2.2 kb mature COX1 mRNA is diminished. In an effort to relate the role of the PET54 gene product in splicing of COX1 pre-mRNA to the previously characterized role for PET54 in translation of mitochondrial COX3 mRNA, the sequence of the PET54-responsive portion of the COX3 5' untranslated leader region was compared to the COX1 intron aI5 beta sequence. Two blocks of RNA sequence present in COX3 have similar counterparts within intron aI5 beta of COX1. The possibility that the PET54 protein binds to one or the other of these blocks of RNA sequence and the potential consequences of this interaction are discussed.

Base Sequence↗
PubMed1989

Isolation and characterization of a yeast strain carrying a mutation in the mitochondrial promoter for COX2.

Subunit II of cytochrome c oxidase is a mitochondrially encoded protein required for cellular respiration. A respiration-deficient yeast strain has been isolated and shown genetically to carry a mutation in the subunit II structural gene COX2. The respiration-deficient strain produces no subunit II polypeptide and is missing two major transcripts of the subunit II gene. The mutation, first mapped by rho- recombinational rescue to the 5' end of the gene, has been localized precisely, by DNA sequencing, 58 nucleotides upstream of the COX2 ATG initiation codon. The mutant strain carries a single nucleotide change (A to T) relative to the wild type sequence. This mutation occurs in a sequence with substantial homology to a previously identified yeast mitochondrial promoter consensus sequence. These results provide the first in vivo evidence for the importance of the consensus sequence for promoter function.

Base Sequence↗
PubMed1989

A role for membrane potential in the biogenesis of cytochrome c oxidase subunit II, a mitochondrial gene product.

Subunit II of yeast cytochrome c oxidase is synthesized on mitochondrial ribosomes as a precursor containing a transient NH2-terminal presequence and is inserted into the mitochondrial inner membrane from the matrix side. Using an optimized in vitro mitochondrial translation system (McKee, E.E., and Poyton, R. O. (1984) J. Biol. Chem. 259, 9320-9331), we have examined the requirement for an electrochemical potential (delta mu H+) across the inner mitochondrial membrane during subunit II biogenesis. When mitochondrial gene products are synthesized under conditions that prevent formation of a normal delta mu H+, accumulation of unprocessed subunit II (pre-II) occurs. The majority of pre-II generated in this way is inserted into the lipid bilayer, as judged by resistance to extraction with 0.1 M Na2CO3. Therefore, it appears that a delta mu H+ is required for the normal biogenesis of subunit II, and that the delta mu H+ is required for a function other than the insertion of pre-II into the lipid bilayer of the inner membrane.

DNA, Mitochondrial↗
PubMed1989

Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene.

The COX6 gene encodes subunit VI of cytochrome c oxidase. Previously, this gene and its mRNAs were characterized, and its expression has been shown to be subject to glucose repression/derepression. In this study we have examined the effects of heme and the HAP1 (CYP1) and HAP2 genes on the expression of COX6. By quantitating COX6 RNA levels and assaying beta-galactosidase activity in yeast cells carrying COX6-lacZ fusion genes, we have found that COX6 is regulated positively by heme and HAP2, but is unaffected by HAP1. Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon. These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2. The other three, CYC1, COX4, and COX5a, encode iso-1-cytochrome c, cytochrome c oxidase subunit IV, and an isolog, Va, of cytochrome c oxidase subunit V, respectively. Thus, it appears that the biogenesis of two interacting proteins, cytochrome c and cytochrome c oxidase, in the mitochondrial respiratory chain, are under the control of common factors.

Blotting, Northern↗
PubMed1989

Organization and expression of the COX6 genetic locus in Saccharomyces cerevisiae: multiple mRNAs with different 3' termini are transcribed from COX6 and regulated differentially.

COX6 and its surrounding genetic locus have been characterized for the yeast Saccharomyces cerevisiae. Flanking genes are found closely spaced upstream and downstream of COX6. The upstream gene and COX6 are transcribed from opposite strands and are separated by no more than 300 bp. COX6 is transcribed into three different size classes of mRNA (1000b, 830b, and 700b) differing in length in their 3' untranslated regions. All three classes of mRNAs are found on polysomes and, hence, are most likely translated. The different COX6 mRNAs vary in abundance during growth in rich media and are affected differentially as cells are shifted into media containing high or low glucose concentrations. The largest mRNA is much more susceptible to glucose repression/derepression than are the two smaller mRNAs, whereas the smallest RNA is preferentially accumulated during growth in rich media. These findings demonstrate that COX6 mRNAs with different 3'-termini are either synthesized differentially or differ in stability and suggest the existence of a complex system regulating COX6 expression.

Amino Acid Sequence↗
PubMed1989

Identification of an upstream activation sequence and other cis-acting elements required for transcription of COX6 from Saccharomyces cerevisiae.

Transcription of Saccharomyces cerevisiae COX6, the nuclear gene for subunit VI of cytochrome c oxidase, is activated in heme-proficient cells, requires the HAP2 gene, and is subject to glucose repression. In this study, by deletion mutagenesis of the COX6 promoter, we identified two regions that are important for transcription. The first was an upstream activation site, UAS6. It was found to be contained within an 84-base-pair (bp) sequence, between bp -256 and -340 of the COX6 translational initiation codon, and to contain sequences required for activation by heme and HAP2 and for release from glucose repression. When located upstream of a CYC1-lacZ fusion gene, deleted for both of its UASs, this segment functioned as a UAS element. Although UAS6 could promote expression in either orientation, it showed a marked orientation dependence in its response to HAP2 and the carbon source. The second region lay between bp -255 and -91. It contained two of the three major 5' termini of COX6 mRNAs and a putative TATA box. Deletion analysis of this region demonstrated that the putative TATA box is not required for transcription and that this region is separable into two redundant domains.

Chromosome Deletion↗
PubMed1988

Functional analysis of mitochondrial protein import in yeast.

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.

Amino Acid Sequence↗
PubMed1988

Identification of REO1, a gene involved in negative regulation of COX5b and ANB1 in aerobically grown Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.

Electron Transport Complex IV↗