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.

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.

Frequency distribution histograms for the rapid analysis of data.

The mean and standard error are good representations for the response of a population to an experimental parameter and are frequently used for this purpose. Frequency distribution histograms show, in addition, responses of individuals in the population. Both the statistics and a visual display of the distribution of the responses can be obtained easily using a microcomputer and available programs. The type of distribution shown by the histogram may suggest different mechanisms to be tested.

Effect of positively charged local anesthetics on a membrane-bound phosphatase in Acholeplasma laidlawii.

The plasma membrane p-nitrophenylphosphatase activity of Acholeplasma laidlawii was stimulated by the spin-labeled local anesthetic 2-[N-methyl-N-(2,2,6,6-tetramethylpiperidinooxyl)]ethyl p-hexyloxybenzoate, abbreviated as C6SL, and its methylated quaternary analog, C6SLMeI. The tertiary amine C6SL (at a concentration of 5 X 10(-5) M) was more potent at pH 6.5 than at pH 7.7. In contrast, the permanently-charged C6SLMeI was equally potent, independently of pH. These results suggest that cationic forms of the anesthetics are responsible for stimulating the enzyme. Electron spin resonance studies of C6SL- and C6SLMeI-labeled membranes showed that these anesthetics in their cationic forms interacted electrostatically with components of the Acholeplasma membrane. For C6SL, this interaction was pH dependent and correlated with the pH dependency of the anesthetic-induced enzyme stimulation in the Acholeplasma membranes. Further, studies using 5-doxylstearic acid labels and non-spin-labeled anesthetics at various pH values showed that the membrane-fluidizing effect of anesthetics was not correlated with anesthetic-induced pNPPase stimulation. Our observations are consistent with the hypothesis that electrostatic interactions between cationic local anesthetics and anionic membrane components may lead to functional changes mediated by membrane proteins.

Genetic recombination in sexual crosses of phycomyces.

Sexual crosses between strains of Phycomyces blakesleeanus , involving three auxotrophic and one color marker and yielding a high proportion of zygospore germination, are described. Samples of 20-40 germ spores from 311 individual fertile germ sporangia originating from five two-factor and three three-factor crosses were characterized. The results show: (1) absence of any contribution of apogamic nuclei to the progeny, (2) confirmation of Burgeff's conjecture that the germ spores of any germ sporangium in most cases derive from one meiosis. In a cross involving two allelic markers the analysis of 175 pooled germ sporangia suggests an intragenic recombination frequency of 0.6%. All other factor combinations tested are unlinked. The bulk of the germ spores are homokaryotic. However, a small portion (4%) are heterokaryotic with respect to mating type.

Absorption and screening in Phycomyces.

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mmicro. As in mycelia, beta-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in beta-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of beta-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress beta-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mmicro was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mmicro. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole.