Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as ''overflow metabolism'' or ''the Crabtree effect.'' The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.alternative oxidase ͉ Crabtree effect ͉ NADH oxidase ͉ redox metabolism R edox homeostasis is a fundamental requirement for sustained metabolism and growth in all biological systems. The intracellular redox potential is primarily determined by the NADH/NAD ratio and to a lesser extent by the NADPH/NADP ratio. In Saccharomyces cerevisiae, Ͼ200 reactions involve these cofactors spread over a large spectrum of cellular functions (1). Because NADH is a highly connected metabolite in the metabolic network (1), any change in the NADH/NAD ratio leads to widespread changes in metabolism (2). NADH is generated primarily in the cytosol by glycolysis and in the mitochondria by the tricarboxylic acid (TCA) cycle. Because the NADH/NAD redox couple cannot traverse the mitochondrial membrane in S. cerevisiae and other eukaryotic cells (3), distinct mechanisms oxidize NADH to NAD in the cytosol and mitochondria. Cytosolic NADH is oxidized by two external (cytosolic) mitochondrial membrane-bound NADH dehydrogenases encoded by NDE1 and NDE2 genes with catalytic sites facing the cytosol (4). Additionally, glycerol-3-phosphate dehydrogenases (encoded by GPD1 and GPD2) oxidize cytosolic NADH with concomitant glycerol formation when the NADH formation rate surpasses its oxidation rate (5). Mitochondrial NADH is oxidized by one internal mitochondrial membrane-bound NADH dehydrogena...

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.

Expression of the Saccharomyces cerevisiae mitochondrial COX1 locus, which contains several introns and is co‐transcribed with the downstream genes AAP1, OLI2 and ENS2, is controlled by at least 18 nuclear‐encoded proteins. The PET309 gene, encoding one of these proteins, was cloned, sequenced and shown to contain an open reading frame of 965 codons. Isonuclear PET309+ and delta pet309::URA3 strains carrying mitochondrial genomes that differ in the number of COX1 introns, were generated. Analysis of RNA species from these strains demonstrated an inverse relationship between the number of introns present in the precursor RNA and the amount of COX1 and AAP1/OLI2/ENS2 RNAs accumulated in a pet309 mutant. Hence, PET309 plays a role either in transcription of intron‐containing primary transcripts from the COX1‐AAP1‐OLI2‐ENS2 transcription unit or in stabilization of primary transcripts. PET309 is also required in translation of COX1 mRNA. A mitochondrial bypass suppressor of the pet309 deletion mutation was isolated, and shown to consist of a DNA rearrangement at the COX1 locus, such that the 5′ untranslated leader region (UTR) of the COB gene was fused to COX1 at nucleotide −174 of its 5′ UTR. This result suggests that Pet309p acts through the COX1 5′ UTR to activate initiation of translation of the COX1 coding region.

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We have identified two chromosomal genes of Escherichia coli K12 that are required for the expression of conjugative plasmid functions in the presence of normal plasmid DNA. Hfr cells with mutations in both of these genes are resistant to donor-specific bacteriophage and defective as conjugal donors. These characteristics can be attributed to the inability of mutant Hfr cells to elaborate F-pili, surface organelles required both for conjugal donor ability and for sensitivity to donor-specific bacteriophages. Mutant cels are also defective in surface exclusion, the property of donor cells to act as poor conjugal recipients. This defect can be attributed in part to a reduction in the amount of the F-plasmid traTgene product in the outer membrane of mutant cells; this protein is one of two plasmid gene products required for the full expression of surface exclusion. We have designated the chromosomal genes identified by these mutations as cpxA and cpxB; the mnemonic cpx signifying conjugative plasmid expression.

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase. In a pet309 null mutant, there is a defect both in accumulation of COX1 pre-RNA, if it contains introns, and in translation of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4031Ϫ4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c-myc protein. A monoclonal antibody against the c-myc epitope detected a 98-kDa protein in mitochondria of yeast cells that expressed the PET309Ϫc-myc fusion protein from a high copy number plasmid. This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional analyses of mitochondrial subfractions demonstrated that the PET309Ϫc-myc fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane). These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane. How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed.Keywords : Saccharomyces cerevisiae; yeast; mitochondria; translation ; RNA processing.A novel aspect of mitochondrial gene expression is the con-[1, 4], may substitute for the function performed by IF3 in other prokaryotic-like translation systems. trol of mitochondrial RNA translation by gene-specific factors encoded by nuclear genes (for review, see [1Ϫ3]). In SaccharoMitochondrial RNA processing is also controlled by genespecific factors encoded by nuclear genes (for review, see [2, myces cerevisiae, it is likely that each mitochondrial mRNA employs specific factors required for translation initiation. These 3]). Several mitochondrial genes in S. cerevisiae contain introns may act in addition to general translation initiation factors, or which are thought to be spliced by ribozyme mechanisms faciliinstead of them. A nuclear gene for mitochondrial translation tated by protein factors (RNA chaperones) that facilitate proper initiation factor 2 (IF2) has been found, but a clear homologue folding of the intron ribozymes. Both mitochondrial-encoded of prokaryotic translation initiation factor 3 (IF3) has not been maturases and nuclear-encoded proteins required for mitofound, despite the availability of the complete DNA sequence chondrial RNA splicing have been identified. Additionally, of the S. cerevisiae genome. The function of yeast gene-specific nuclear-encoded trans-acting proteins required for mitochondrial mitochondrial translation factors such as Pet122p or Cbs2p, RNA 5′ or 3′ end processing or mRNA stability...

Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species. Evidence is presented here for two additional catalase isozymes in H. capsulatum. Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts. Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated. The transcriptional expression of the H. capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA. Results at the transcript levels for these genes are shown for three conditions : cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H 2 O 2 ) and carbon source (glucose vs glycerol). Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress. A phylogenetic analysis of presently available catalase sequences and intron residences was done. The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes.

The nuclear genes PET117 and PET191 are required for the assembly of active cytochrome c oxidase in S. cerevisiae, yet their gene products are not subunits of the final assembled cytochrome c oxidase complex. Plasmids bearing PET117 or PET191 were isolated by their ability to complement the pet117-1 or pet191-1 mutations, respectively. By restriction mapping, subcloning, and deletion analysis of yeast DNA fragments that complement these mutations, the PET117 and PET191 genes were localized to smaller regions of DNA, which were then sequenced from both strands. The PET117 open reading frame is of 107 codons and the PET191 open reading frame is of 108 codons. Neither the PET191 nor PET117 DNA sequences have been reported previously, and the derived amino-acid sequences of the PET191 and PET117 open reading frames exhibit no significant primary amino-acid sequence similarity to other protein sequences available in the NBRF data base, or from translated Genbank sequences. By hybridization of PET117 or PET191 probes first to a chromosome blot and next to a library of physically mapped fragments of yeast genomic DNA, the map locations of the PET191 and PET117 genes were determined. PET117 is located on chromosome V near the HIS1 gene and PET191 is located on chromosome X near the CYC1 gene.