Transcriptional response to mitochondrial NADH kinase deficiency in Saccharomyces cerevisiae.

Yeast cells lacking the mitochondrial NADH kinase encoded by POS5 display increased sensitivity to hydrogen peroxide, a slow-growth phenotype, reduced mitochondrial function and increased levels of mitochondrial protein oxidation and mtDNA mutations. Here we examined gene expression in pos5Delta cells, comparing these data to those from cells containing deletions of superoxide dismutase-encoding genes SOD1 or SOD2. Surprisingly, stress-response genes were down-regulated in pos5Delta, sod1Delta and sod2Delta cells, implying that cells infer stress levels from mitochondrial activity rather than sensing reactive oxygen species directly. Additionally, pos5Delta, but not sod1 or sod2, cells displayed an anaerobic expression profile, indicating a defect in oxygen sensing that is specific to pos5, and is not a general stress-response. Finally, the pos5Delta expression profile is quite similar to the hap1Delta expression profile previously reported, which may indicate a shared mechanism.

Construction and application of a protein and genetic interaction network (yeast interactome).

Cytoscape is a bioinformatic data analysis and visualization platform that is well-suited to the analysis of gene expression data. To facilitate the analysis of yeast microarray data using Cytoscape, we constructed an interaction network (interactome) using the curated interaction data available from the Saccharomyces Genome Database (www.yeastgenome.org) and the database of yeast transcription factors at YEASTRACT (www.yeastract.com). These data were formatted and imported into Cytoscape using semi-automated methods, including Linux-based scripts, that simplified the process while minimizing the introduction of processing errors. The methods described for the construction of this yeast interactome are generally applicable to the construction of any interactome. Using Cytoscape, we illustrate the use of this interactome through the analysis of expression data from a recent yeast diauxic shift experiment. We also report and briefly describe the complex associations among transcription factors that result in the regulation of thousands of genes through coordinated changes in expression of dozens of transcription factors. These cells are thus able to sensitively regulate cellular metabolism in response to changes in genetic or environmental conditions through relatively small changes in the expression of large numbers of genes, affecting the entire yeast metabolome.

Mitochondrial and nuclear DNA defects in Saccharomyces cerevisiae with mutations in DNA polymerase gamma associated with progressive external ophthalmoplegia.

A number of nuclear mutations have been identified in a variety of mitochondrial diseases including progressive external ophthalmoplegia (PEO), Alpers syndrome and other neuromuscular and oxidative phosphorylation defects. More than 50 mutations have been identified in POLG, which encodes the human mitochondrial DNA (mtDNA) polymerase gamma, PEO and Alpers patients. To rapidly characterize the effects of these mutations, we have developed a versatile system that enables the consequences of homologous mutations, introduced in situ into the yeast mtDNA polymerase gene MIP1, to be evaluated in vivo in haploid and diploid cells. Overall, distinct phenotypes for expression of each of the mip1-PEO mutations were observed, including respiration-defective cells with decreased viability, dominant-negative mutant polymerases, elevated levels of mitochondrial and nuclear DNA damage and chromosomal mutations. Mutations in the polymerase domain caused the most severe phenotype accompanied by loss of mtDNA and cell viability, whereas the mutation in the exonuclease domain showed mild dominance with loss of mtDNA. Interestingly, the linker region mutation caused elevated mitochondrial and nuclear DNA damage. The cellular processes contributing to these observations in the mutant yeast cells are potentially relevant to understanding the pathologies observed in human mitochondrial disease patients.

POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA.

In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD(+), with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8(m) reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases.