Structure and function of Pet100p, a molecular chaperone required for the assembly of cytochrome c oxidase in Saccharomyces cerevisiae.

The assembly of cytochrome c oxidase in the inner mitochondrial membranes of eukaryotic cells requires the protein products of a large number of nuclear genes. In yeast, some of these act globally and affect the assembly of several respiratory-chain protein complexes, whereas others act in a cytochrome c oxidase-specific fashion. Many of these yeast proteins have human counterparts, which when mutated lead to energy-related diseases. One of these proteins, Pet100p, is a novel molecular chaperone that functions to incorporate a subcomplex containing cytochrome c oxidase subunits VII, VIIa and VIII into holo-(cytochrome c oxidase). Here we report the topological disposition of Pet100p in the inner mitochondrial membrane and show that its C-terminal domain is essential for its function as a cytochrome c oxidase-specific 'assembly facilitator'.

Effects of anoxia and the mitochondrion on expression of aerobic nuclear COX genes in yeast: evidence for a signaling pathway from the mitochondrial genome to the nucleus.

Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes. Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated. Recently, it has been reported that induction of some hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process. In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeast aerobic nuclear COX genes. We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclear cox mutants and mitochondrial rho(0) and mit(-) mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions. This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase. We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another. These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclear COX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.

Mitochondrial-nuclear crosstalk is involved in oxygen-regulated gene expression in yeast.

The expression of several oxygen-regulated nuclear genes in the yeast Saccharomyces cerevisiae is affected by the mitochondrion. Recent evidence suggests two levels of mitochondrial involvement. On the one hand, mitochondrial respiratory function is essential for the anoxic induction of some hypoxic genes. On the other hand, the mitochondrial genome itself functions independently of its respiratory function, in the optimal expression of some aerobic genes. These findings suggest that the mitochondrion release at least two types of 'signals' that function in the expression of oxygen-regulated genes.

Models for oxygen sensing in yeast: implications for oxygen-regulated gene expression in higher eucaryotes.

Adaptation to changes in oxygen tension in cells, tissues, and organisms depends on changes in the level of expression of a large and diverse set of proteins. It is likely that most cells and tissues possess an oxygen sensing apparatus and signal transduction pathways for regulating expression of oxygen-responsive genes. Although progress has been made in understanding the transcriptional machinery involved in oxygen-regulated gene expression of eucaryotic genes the underlying mechanism(s) of oxygen sensing and the signaling pathways that connect oxygen sensor(s) to the transcription machinery of eucaryotes are still poorly understood. The yeast Saccharomyces cerevisiae is ideal for addressing these problems. Indeed, it is well-suited for broadly based studies on oxygen sensing at the cellular level because it lends itself well to genetic and biochemical studies and because its genome has been completely sequenced. This review focuses on oxygen-regulated gene expression and current models for oxygen sensing in this yeast and then considers their applicability for understanding oxygen sensing in mammals.

Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic genes.

Oxygen availability affects the transcription of a number of genes in nearly all organisms. Although the molecular mechanisms for sensing oxygen are not precisely known, heme is thought to play a pivotal role. Here, we address the possibility that oxygen sensing in yeast, as in mammals, involves a redox-sensitive hemoprotein. We have found that carbon monoxide (CO) completely blocks the anoxia-induced expression of two hypoxic genes, OLE1 and CYC7, partially blocks the induction of a third gene, COX5b, and has no effect on the expression of other hypoxic or aerobic genes. In addition, transition metals (Co and Ni) induce the expression of OLE1 and CYC7 in a concentration-dependent manner under aerobic conditions. These findings suggest that the redox state of an oxygen-binding hemoprotein is involved in controlling the expression of at least two hypoxic yeast genes. By using mutants deficient in each of the two major yeast CO-binding hemoproteins (cytochrome c oxidase and flavohemoglobin), respiratory inhibitors, and cob1 and rho0 mutants, we have found that the respiratory chain is involved in the anoxic induction of these two genes and that cytochrome c oxidase is likely the hemoprotein "sensor." Our findings also indicate that there are at least two classes of hypoxic genes in yeast (CO sensitive and CO insensitive) and imply that multiple pathways/mechanisms are involved in modulating the expression of hypoxic yeast genes.

Structure/function of oxygen-regulated isoforms in cytochrome c oxidase.

Eukaryotic cytochrome c oxidases are complex oligomeric membrane proteins composed of subunit polypeptides encoded by both nuclear and mitochondrial genomes. While the mitochondrially encoded subunits are encoded by unique genes, some of the nuclear-encoded subunits are encoded by multigene families. The isoforms produced by these multigene families are tissue-specific and/or developmentally regulated in mammals and environmentally regulated in lower eukaryotes. Isoforms for one of the subunits, V, in the yeast Saccharomyces cerevisiae and one of the subunits, VII, in the slime mold Dictyostelium discoideum are regulated differentially by oxygen concentration. Extensive studies with the yeast subunit V isoforms have revealed that the genes for these proteins are switched on or off at very low oxygen concentrations (0.5-1 micromol l-1 O2) and that they affect the catalytic properties of holocytochrome c oxidase differentially. By altering an internal step in electron transfer between heme a and the binuclear reaction center (composed of heme a3 and CuB), the 'hypoxic' isoform, Vb, enhances the catalytic constant three- to fourfold relative to the 'aerobic' isoform, Va. Modeling studies suggest that this occurs by an interaction between transmembrane helix VII of subunit I and the transmembrane helix in subunit V. The inverse regulation of these two isoforms allows cells to assemble different types of holoenzyme isoenzymes in response to oxygen concentration. Oxygen also regulates the level of transcription of the genes for the other nuclear-coded subunits of yeast cytochrome c oxidase and affects the level of two of the mitochondrially encoded subunits (I and II) post-transcriptionally. Thus, the level of cytochrome c oxidase activity that is produced at different oxygen tensions in yeast is determined in part by the number of holoenzyme molecules that are assembled and in part by the oxygen-regulated isoforms of subunit V. The possibility that this type of control exists in other organisms is considered.

Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast.

The budding yeast Saccharomyces cerevisiae is a facultative aerobe that responds to changes in oxygen availability (and carbon source) by initiating a biochemically complex program that ensures that energy demands are met under two different physiological states: aerobic growth, supported by oxidative and fermentative pathways, and anaerobic growth, supported solely by fermentative processes. This program includes the differential expression of a large number of genes, many of which are involved in the direct utilization of oxygen. Research over the past decade has defined many of the cis-sites and trans-acting factors that control the transcription of these oxygen-responsive genes. However, the manner in which oxygen is sensed and the subsequent steps involved in the transduction of this signal have not been precisely determined. Heme is known to play a pivotal role in the expression of these genes, acting as a positive modulator for the transcription of the aerobic genes and as a negative modulator for the transcription of the hypoxic genes. Consequently, cellular concentrations of heme, whose biosynthesis is oxygen-dependent, are thought to provide a gauge of oxygen availability and dictate which set of genes will be transcribed. But the precise role of heme in oxygen sensing and the transcriptional regulation of oxygen-responsive genes is presently unclear. Here, we provide an overview of the transcriptional regulation of oxygen-responsive genes, address the functional roles that heme and hemoproteins may play in this regulation, and discuss possible mechanisms of oxygen sensing in this simple eukaryotic organism.

Neither respiration nor cytochrome c oxidase affects mitochondrial morphology in Saccharomyces cerevisiae.

Previous studies have reported that mitochondrial morphology and volume in yeast cells are linked to cellular respiratory capacity. These studies revealed that mitochondrial morphology in glucose-repressed or anaerobically grown cells, which lack or have reduced levels of respiration, is different from that in fully respiring cells. Although both oxygen deprivation and glucose repression decrease the levels of respiratory chain proteins, they decrease the expression of many non-mitochondrial proteins as well, making it difficult to determine whether it is a defect in respiration or something else that effects mitochondrial morphology. To determine whether mitochondrial morphology is dependent on respiration per se, we used a strain with a null mutation in PET100, a nuclear gene that is specifically required for the assembly of cytochrome c oxidase. Although this strain lacks respiration, the mitochondrial morphology and volumes are both comparable to those found in its respiration-proficient parent. These findings indicate that respiration is not involved in the establishment or maintenance of yeast mitochondrial morphology, and that the previously observed effects of oxygen availability and glucose repression on mitochondrial morphology are not exerted through the respiratory chain. By applying the principle of symmorphosis to these findings, we conclude that the shape and size of the mitochondrial reticulum found in respiring yeast cells is maintained for reasons other than respiration.

A fermentor system for regulating oxygen at low concentrations in cultures of Saccharomyces cerevisiae.

The growth of yeast cells to high densities at low, but constant, oxygen concentrations is difficult because the cells themselves respire oxygen; hence, as cell mass increases, so does oxygen consumption. To circumvent this problem, we have designed a system consisting of a computer-controlled gas flow train that adjusts oxygen concentration in the gas flow to match cellular demand. It does this by using a proportional-integral-differential algorithm in conjunction with a three-way valve to mix two gases, adjusting their proportions to maintain the desired oxygen concentration. By modeling yeast cell yields at intermediate to low oxygen concentrations, we have found that cellular respiration declines with oxygen concentration, most likely because of a decrease in the expression of genes for respiratory proteins. These lowered rates of oxygen consumption, together with the gas flow system described here, allow the growth of yeast cells to high densities at low oxygen concentrations. This system can also be used to grow cells at any desired oxygen concentration and for regulated shifts between oxygen concentrations.

Effects of oxygen concentration on the expression of cytochrome c and cytochrome c oxidase genes in yeast.

Oxygen is an important environmental regulator for the transcription of several genes in Saccharomyces cerevisiae, but it is not yet clear how this yeast or other eukaryotes actually sense oxygen. To begin to address this we have examined the effects of oxygen concentration on the expression of several nuclear genes (CYC1, CYC7, COX4, COX5a, COX5b, COX6, COX7, COX8, and COX9) for proteins of the terminal portion of the respiratory chain. COX5b and CYC7 are hypoxic genes; the rest are aerobic genes. We have found that the level of expression of these genes is determined by oxygen concentration per se and not merely the presence or absence of oxygen and that each of these genes has a low oxygen threshold (0. 5-1 microM O2) for expression. For some aerobic genes (COX4, COX5a, COX7, COX8, and COX9) there is a gradual decline in expression between 200 microM O2 (air) and their oxygen threshold. Below this threshold expression drops precipitously. For others (COX5a and CYC1) the level of expression is nearly constant between 200 microM O2 and their threshold and then drops off. The hypoxic genes COX5b and CYC7 are not expressed until the oxygen concentration is below 0.5 microM O2. These studies have also revealed that COX5a and CYC1, the genes for the aerobic isoforms of cytochrome c oxidase subunit V and cytochrome c, and COX5b and CYC7, the genes for the hypoxic isoforms of cytochrome c oxidase subunit V and cytochrome c, are coexpressed at a variety of oxygen concentrations and switch on or off at extremely low oxygen concentrations. By shifting cells from one oxygen concentration to another we have found that aerobic genes are induced faster than hypoxic genes and that transcripts from both types of gene are turned over quickly. These findings have important implications for cytochrome c oxidase function and biogenesis and for models of oxygen sensing in yeast.

REO1 and ROX1 are alleles of the same gene which encodes a transcriptional repressor of hypoxic genes in Saccharomyces cerevisiae.

Both the REO1 and ROX1 genes are thought to encode heme-dependent, transcriptional repressors of hypoxic genes in Saccharomyces cerevisiae. However, genetic complementation studies have yielded conflicting results about whether these are the same or different genes. Because of the central importance of these repressors, which control the expression of nearly all known hypoxic genes in yeast, we have sought to resolve this confusion by comparing the phenotypes of reo1 and rox1 mutants using Northern-blot analyses, performing additional complementation studies, and sequencing the ROX1 gene in reo1 strains. Northern-blot analyses of a reo1 strain show wild-type expression of the aerobic genes examined, but de-repression of the Rox1-regulated, hypoxic genes. Aerobic transcript levels of these hypoxic genes were also de-repressed in a diploid strain created by mating a rox1 disrupted strain with a reo1 strain, indicating that genetic complementation did not occur between these two strains. Sequence analyses of ROX1 in reo1 strains reveal a frame-shift mutation in the 5'-end of its coding region, resulting in a nonsense codon in the sixth position. Taken together, these results provide compelling evidence that reo1 is an allele of ROX1.

Function and expression of flavohemoglobin in Saccharomyces cerevisiae. Evidence for a role in the oxidative stress response.

We have studied the function and expression of the flavohemoglobin (YHb) in the yeast Saccharomyces cerevisiae. This protein is a member of a family of flavohemoproteins, which contain both heme and flavin binding domains and which are capable of transferring electrons from NADPH to heme iron. Normally, actively respiring yeast cells have very low levels of the flavohemoglobin. However, its intracellular levels are greatly increased in cells in which the mitochondrial electron transport chain has been compromised by either mutation or inhibitors of respiration. The expression of the flavohemoglobin gene, YHB1, of S. cerevisiae is sensitive to oxygen. Expression is optimal in hyperoxic conditions or in air and is reduced under hypoxic and anaerobic conditions. The expression of YHB1 in aerobic cells is enhanced in the presence of antimycin A, in thiol oxidants, or in strains that lack superoxide dismutase. All three conditions lead to the accumulation of reactive oxygen species and promote oxidative stress. To study the function of flavohemoglobin in vivo, we created a null mutation in the chromosomal copy of YHB1. The deletion of the flavohemoglobin gene in these cells does not affect growth in either rhoo or rho+ genetic backgrounds. In addition, a rho+ strain carrying a yhb1(-) deletion has normal levels of both cyanide-sensitive and cyanide-insensitive respiration, indicating that the flavohemoglobin does not function as a terminal oxidase and is not required for the function or expression of the alternative oxidase system in S. cerevisiae. Cells that carry a yhb1(-)deletion are sensitive to conditions that promote oxidative stress. This finding is consistent with the observation that conditions that promote oxidative stress also enhance expression of YHB1. Together, these findings suggest that YHb plays a role in the oxidative stress response in yeast.

Cloning and characterization of PET100, a gene required for the assembly of yeast cytochrome c oxidase.

The biogenesis of cytochrome c oxidase in Saccharomyces cerevisiae requires a protein encoded by the nuclear gene, PET100. Cells carrying a recessive mutation (pet100-1) in PET100 are respiratory deficient and have reduced levels of cytochrome c oxidase activity. The PET100 gene has been cloned by complementation of pet100-1, sequenced and disrupted. PET100 is located adjacent to the PDC2 gene on chromosome IV and contains an open reading frame of 333 base pairs. The PET100 protein contains a possible membrane-spanning segment and a putative mitochondrial import sequence at its NH2 terminus. A strain carrying a null mutation in PET100 lacks cytochrome c oxidase activity and assembled cytochromes a and a3, but the other respiratory chain carriers are present. The respiratory-deficient phenotype of this strain is not rescued by added hemin or heme A. These findings indicate that the mutation is specific for cytochrome c oxidase and does not affect the biosynthesis of heme A. In addition, mitochondria from the strain carrying a null mutation in PET100 contain each of the subunit polypeptides of cytochrome c oxidase. Together, these findings suggest that PET100p is not required for the synthesis or localization of cytochrome c oxidase subunits to mitochondria, but is required at a later step in their assembly into an active holoenzyme.

Oxygen sensing and molecular adaptation to hypoxia.

This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

Crosstalk between nuclear and mitochondrial genomes.

This review focuses on molecular mechanisms that underlie the communication between the nuclear and mitochondrial genomes in eukaryotic cells. These genomes interact in at least two ways. First, they contribute essential subunit polypeptides to important mitochondrial proteins; second, they collaborate in the synthesis and assembly of these proteins. The first type of interaction is important for the regulation of oxidative energy production. Isoforms of the nuclear-coded subunits of cytochrome c oxidase affect the catalytic functions of its mitochondrially coded subunits. These isoforms are differentially regulated by environmental and developmental signals and probably allow tissues to adjust their energy production to different energy demands. The second type of interaction requires the bidirectional flow of information between the nucleus and the mitochondrion. Communication from the nucleus to the mitochondrion makes use of proteins that are translated in the cytosol and imported by the mitochondrion. Communication from the mitochondrion to the nucleus involves metabolic signals and one or more signal transduction pathways that function across the inner mitochondrial membrane. An understanding of both types of interaction is important for an understanding of OXPHOS diseases and aging.

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.