Genomic characterization of POS5, the Saccharomyces cerevisiae mitochondrial NADH kinase

Disruption of the Saccharomyces cerevisiae mitochondrial NADH kinase POS5 increases the mitochondrial mutation rate 50-fold. Whereas most multicellular eukaryotic genomes have one NADH kinase gene, the yeast genome contains three distinct genes encoding NAD/H kinase activity. To determine if all three genes are essential for viability we constructed combinations of gene knockouts. We show that only the pos5Deltautr1Delta combination is synthetically lethal, demonstrating an essential overlapping function, and showing that NAD/H kinase activity is essential for eukaryotic viability. The single human NAD/H kinase gene can rescue the lethality of the double knockout in yeast, demonstrating that the single human gene can fill the various functions provided by the three yeast genes. The human NAD/H kinase gene harbors very common sequence variants, but all of these equally complement the synthetic lethality in yeast, illustrating that each of these are functionally wild-type. To understand the molecular mechanism of the mitochondrial genome instability of pos5 mutation we performed gene expression analysis on the pos5Delta. The pos5Delta resulted in an increase in expression of most of the iron transport genes including key genes involved in iron-sulfur cluster assembly. Decreased expression occurred in many genes involved in the electron transport chain. We show that the pos5Delta expression pattern is similar to the frataxin homolog knockout (yfh1Delta), the yeast model for Friedreich's ataxia. These combined data show that the POS5 NAD/H kinase is an important protein required for a variety of essential cellular pathways and that deficient iron-sulfur cluster assembly may play a critical role in the mitochondrial mutator phenotype observed in the pos5Delta.     

The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis

Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis.     

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 pos5Δ 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 pos5Δ, sod1Δ and sod2Δ cells, implying that cells infer stress levels from mitochondrial activity rather than sensing reactive oxygen species directly. Additionally, pos5Δ, 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 pos5Δ expression profile is quite similar to the hap1Δ expression profile previously reported, which may indicate a shared mechanism.     

Characterization of a Saccharomyces cerevisiae gene that encodes a mitochondrial phosphate transporter-like protein

The mitochondrial phosphate transporter of Saccharomyces cerevisiae, encoded by MIR1 (YJR077C) gene, shows divergence among the transporters in various eukaryotes. We have characterized another gene, YER053C, that appeared to encode an orthologous mitochondrial phosphate transporter of yeast. The predicted amino acid sequence of the YER053C protein is much more similar to that of mitochondrial phosphate transporters of other species than that of MIR1. RNA gel blot analysis indicated that, like the MIR1 promoter, the YER053C promoter is functional and that its activity varies according to aeration. An MIR1 gene null mutant did not grow on glycerol medium, whereas a YER053C null mutant grew well on the medium, suggesting that the YER053C gene is not essential for the mitochondrial function. YER053C also did not support the growth of the MIR1 null mutant on glycerol. The MIR1 and YER053C proteins were expressed in Escherichia coli and then reconstituted into liposomes. Unlike the proteoliposomes of MIR1, those of YER053C did not exhibit significant phosphate transport activity. Unexpectedly, it was shown that YER053C is localized in vacuoles, not mitochondria, by immunological electron microscopy. These results suggest that, during evolution, yeast lost the function and/or mitochondrial targeting of YER053C and then recruited an atypical MIR1 as the only transporter.     

Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3' to 5' DNA helicase

The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene. Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway. The Mph1 protein contains the seven conserved motifs of the superfamily 2 (SF2) family of nucleic acid unwinding enzymes. Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination. To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity. We show that Mph1 has a robust ATPase function that requires single-stranded DNA for activation. Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1. We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3' to 5' polarity with respect to the DNA strand on which this protein translocates. The DNA helicase activity of Mph1 is enhanced by the heterotrimeric single-stranded DNA binding protein replication protein A. These results, thus, establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance.     

Characterization of NADH kinase from Saccharomyces cerevisiae

At least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mitochondria, and NAD+-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria. The identity of NAD+ kinase detected in the two fractions remains equivocal. NADH kinase was highly purified 1,041-fold from the mitochondrial fraction. The Km values for NADH and ATP were 105 microM and 2.1 mM, respectively. The relative molecular mass was estimated to be 160,000 by means of molecular sieve chromatography. From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.     

Mitochondrial NADH kinase, Pos5p, is required for efficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the mitochondrial inner membrane readily allows transport of cytosolic NAD(+), but not NADPH, to the matrix. Pos5p is the only known NADH kinase in the mitochondrial matrix. The enzyme phosphorylates NADH to NADPH and is the major source of NADPH in the matrix. The importance of mitochondrial NADPH for cellular physiology is underscored by the phenotypes of the Δpos5 mutant, characterized by oxidative stress sensitivity and iron-sulfur (Fe-S) cluster deficiency. Fe-S clusters are essential cofactors of proteins such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S] in mitochondria. Intact mitochondria isolated from wild-type yeast can synthesize these clusters and insert them into the corresponding apoproteins. Here, we show that this process of Fe-S cluster biogenesis in wild-type mitochondria is greatly stimulated and kinetically favored by the addition of NAD(+) or NADH in a dose-dependent manner, probably via transport into mitochondria and subsequent conversion into NADPH. Unlike wild-type mitochondria, Δpos5 mitochondria cannot efficiently synthesize Fe-S clusters on endogenous aconitase or imported ferredoxin, although cluster biogenesis in isolated Δpos5 mitochondria is restored to a significant extent by a small amount of imported Pos5p. Interestingly, Fe-S cluster biogenesis in wild-type mitochondria is further enhanced by overexpression of Pos5p. The effects of Pos5p on Fe-S cluster generation in mitochondria indicate that one or more steps in the biosynthetic process require NADPH. The role of mitochondrial NADPH in Fe-S cluster biogenesis appears to be distinct from its function in anti-oxidant defense.     

A novel NADH kinase is the mitochondrial source of NADPH in Saccharomyces cerevisiae

Mitochondria require NADPH for anti-oxidant protection and for specific biosynthetic pathways. However, the sources of mitochondrial NADPH and the mechanisms of maintaining mitochondrial redox balance are not well understood. We show here that in Saccharomyces cerevisiae, mitochondrial NADPH is largely provided by the product of the POS5 gene. We identified POS5 in a S.cerevisiae genetic screen for hyperoxia-sensitive mutants, or cells that cannot survive in 100% oxygen. POS5 encodes a protein that is homologous to NAD(+) and NADH kinases, and we show here that recombinant Pos5p has NADH kinase activity. Pos5p is localized to the mitochondrial matrix of yeast and appears to be important for several NADPH-requiring processes in the mitochondria, including resistance to a broad range of oxidative stress conditions, arginine biosynthesis and mitochondrial iron homeostasis. Pos5p represents the first member of the NAD(H) kinase family that has been identified as an important anti-oxidant factor and key source of the cellular reductant NADPH.     

The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function.

In eukaryotes, the posttranslational conjugation of ubiquitin to various cellular proteins marks them for degradation. Interestingly, several proteins have been reported to contain ubiquitin-like (ub-like) domains that are in fact specified by the DNA coding sequences of the proteins. The biological role of the ub-like domain in these proteins is not known; however, it has been proposed that this domain functions as a degradation signal rendering the proteins unstable. Here, we report that the product of the Saccharomyces cerevisiae RAD23 gene, which is involved in excision repair of UV-damaged DNA, bears a ub-like domain at its amino terminus. This finding has presented an opportunity to define the functional significance of this domain. We show that deletion of the ub-like domain impairs the DNA repair function of RAD23 and that this domain can be functionally substituted by the authentic ubiquitin sequence. Surprisingly, RAD23 is highly stable, and the studies reported herein indicate that its ub-like domain does not mediate protein degradation. Thus, in RAD23 at least, the ub-like domain affects protein function in a nonproteolytic manner.     

The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase.

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.     

A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal beta-keto-reductase.

A number of Saccharomyces cerevisiae membrane-bound oxidoreductases were examined for potential roles in microsomal fatty acid elongation, by assaying heterologous elongating activities in individual deletion mutants. One yeast gene, YBR159w, was identified as being required for activity of both the Caenorhabditis elegans elongase PEA1 (F56H11.4) and the Arabidopsis thaliana elongase FAE1. Ybr159p shows some limited homology to human steroid dehydrogenases and is a member of the short-chain alcohol dehydrogenase superfamily. Disruption of YBR159w is not lethal, in contrast to previous reports, although the mutants are slow growing and display high temperature sensitivity. Both Ybr159p and an Arabidopsis homologue were shown to restore heterologous elongase activities when expressed in ybr159Delta mutants. Biochemical characterization of microsomal preparations from ybr159Delta cells revealed a primary perturbation in beta-ketoacyl reduction, confirming the assignment of YBR159w as encoding a component of the microsomal elongase.     

The PRP31 gene encodes a novel protein required for pre-mRNA splicing in Saccharomyces cerevisiae.

The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon shift to the non- permissive temperature. The wild-type PRP31 gene was cloned and shown to be essential for cell viability. The PRP31 gene is predicted to encode a 60 kDa polypeptide. No similarities with other known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino acid sequence. A PRP31 allele bearing a triple repeat of the hemagglutinin epitope has been generated. The tagged protein is functional in vivo and a single polypeptide species of the predicted size was detected by Western analysis with proteins from yeast cell extracts. Functional Prp31p is required for the processing of pre-mRNA species both in vivo and in vitro, indicating that the protein is directly involved in the splicing pathway.     

The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase

We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3 Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ  :: CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1 , which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a Δ cit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a Δ cit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.     

过表达NADH激酶基因对酿酒酵母乙醇发酵的影响

甘油是酿酒酵母乙醇代谢途径中的主要副产物,降低甘油生成,可以提高乙醇的产率和原料的利用率。以工业酒精酵母单倍体S1(MATa)为研究对象,构建了一个4.5 kb左右的基因敲除突变盒gpd2Δ::PGK1PT-POS5-HyBR,利用醋酸锂转化法转入S1,得到重组菌S3(gpd2Δ::PGK1PT-POS5-HyBR),使得工业酒精酵母在敲除GPD2的同时整合过表达了NADH激酶基因POS5。结果表明,在150 g/L的葡萄糖摇瓶发酵实验中,重组菌S3在不影响菌株生理特性的条件下,乙醇得率(g ethanol/g glucose)比原始菌株S1提高了8%,甘油得率(g glycerol/g glucose)降低了33.64%。本研究证明过表达NADH激酶基因可降低乙醇发酵中副产物甘油的生成并提高乙醇得率。     

Cardiolipin is not required to maintain mitochondrial DNA stability or cell viability for Saccharomyces cerevisiae grown at elevated temperatures

In eukaryotic cells, the phospholipid cardiolipin (CL) is primarily found in the inner mitochondrial membrane. Saccharomyces cerevisiae mutants, unable to synthesize CL because of a null allele of the CRD1 gene (encodes CL synthase), have been reported with different phenotypes. Some mutants, when grown on a nonfermentable carbon source at elevated temperatures, exhibit mitochondrial DNA instability, loss of viability, and significant defects in several functions that rely on the mitochondrial energy transducing system (ETS). These mutants also lack the immediate precursor to CL, phosphatidylglycerol (PG), when grown on glucose as a carbon source. Other mutants show reduced growth efficiency on a nonfermentable carbon source but much milder phenotypes associated with growth at elevated temperatures and increased levels of PG when grown on glucose. We present evidence that mitochondrial DNA instability, loss of viability, and defects in the ETS exhibited at elevated temperatures by some mutants are caused by the reduced expression of the PET56 gene in the presence of the his3 Delta 200 allele and not the lack of CL alone. We also found that PG is present and elevated in all crd1 Delta strains when grown on glucose. A supermolecular complex between complex III and complex IV of the mitochondrial ETS detected in wild type cells was missing in all of the above crd1 Delta cells. The level of components of the ETS was also reduced in crd1 Delta cells grown at elevated temperatures because of reduced gene expression and not reduced stability. These results suggest that all phenotypes reported for cells carrying the his3 Delta 200 allele and lacking CL should be re-evaluated.     

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.     

The ubiquitin ligase Rsp5 is required for ribosome stability in Saccharomyces cerevisiae

Rsp5p is a conserved HECT-domain ubiquitin ligase with diverse roles in cellular physiology. Here we report a previously unknown role of Rsp5p in facilitating the stability of the cytoplasmic ribosome pool in budding yeast. Yeast strains carrying temperature-sensitive mutations in RSP5 showed a progressive decline in levels of 18S and 25S rRNAs and accumulation of rRNA decay fragments when cells grown in rich medium were shifted to restrictive temperature. This was accompanied by a decreased number of translating ribosomes and the appearance of ribosomal subunits with an abnormally low sedimentation rate in polysome analysis. Abrogating Rsp5p function affected stability of other tested noncoding RNA species (tRNA and snoRNA), but to a lower extent than that of rRNA, and also inhibited processing of rRNA and tRNA precursors, in agreement with previous studies. The breakdown of cellular ribosomes was not affected by deletion of key genes involved in autophagy, previously implicated in ribosome turnover upon starvation. Our results suggest that functional Rsp5p is required to maintain the integrity of cytoplasmic ribosomes under rich nutrient conditions.