Thomas Lisowsky

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins.

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full‐length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.

Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase

The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell. In this study we report the enzymatic activity of this protein as a flavin‐linked sulfhydryl oxidase catalyzing the formation of disulfide bridges. Deletion of the amino‐terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy‐terminal domain of the protein. This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein. The carboxy‐terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human. Thus Erv1p represents the first FAD‐linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.

Dual function of a new nuclear gene for oxidative phosphorylation and vegetative growth in yeast

SummaryA new gene essential for cell viability and indispensable for the biogenesis of a functional respiratory chain in Saccharomyces cerevisiae was isolated by complementing a temperature-sensitive mutant. This conditional nuclear mutation selectively affects oxidative phosphorylation at restrictive temperatures. At the molecular level a severe and complex defect inside mitochondria is observed, with drastically reduced levels of mitochondrial transcripts. Surprisingly a null mutation in this nuclear gene in a haploid yeast strain leads to cell death. Spores containing a disrupted copy of the gene exhibit a severe growth defect and cell division stops irreversibly after 3 to 4 days. It is shown that the null and conditional mutants are indeed allelic. This finding demonstrates a dual function of the gene product in oxidative phosphorylation and vegetative growth. The putative protein product, as deduced from the sequence of the relevant reading frame is characterized by a low molecular weight of approximately 14 kDa, a high content of charged amino acids and a very low codon bias index. A transcript of low abundance and with a length of about 600 nucleotides can be assigned to this gene.

Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase

BACKGROUND Augmenter of Liver Regeneration is an important secondary hepatic growth factor. Augmenter of liver regeneration protein has been shown to control mitochondrial gene expression and the lytic activity of liver-resident Natural Killer cells through the levels of interferon-gamma, but the precise enzymatic function of this protein is unknown. AIMS To define the enzymatic activity of augmenter of liver regeneration protein. The carboxy terminus of augmenter of liver regeneration protein contains a special CXXC motif characteristic for redox proteins and with faint homologies to the redox-active site of sulfhydryl oxidases. Tests were, therefore, carried out to establish whether isolated augmenter of liver regeneration protein can also function in the formation of sulfur bridges. METHODS Purified augmenter of liver regeneration proteins from rat and human were tested in enzyme assays for the ability to introduce disulfide bonds into protein substrates. The isolated proteins were tested for the formation of dimers and the presence of bound FAD was investigated spectroscopically. The function of the conserved CXXC motif was investigated by in vitro mutagenesis experiments and subsequent enzyme assays. RESULTS In this study, we demonstrate that rat and human augmenter of liver regeneration protein are flavin-linked sulfhydryl oxidases that catalyze the formation of disulfide bonds in reduced protein substrates. A flavin moiety is firmly but not covalently attached to the protein. In human cell cultures augmenter of liver regeneration protein is expressed in a long and short form that both exist as covalently linked dimers. The active site of the enzyme is associated with a conserved CXXC motif in the carboxy-terminal domain, that is present in the homologous proteins from yeast to humans and also in the human Q6 growth regulator protein. In vitro mutagenesis of one cysteine residue in the CXXC motif results in loss of enzymatic function and the mutated protein no longer binds FAD. CONCLUSIONS For the first time, these data assign an enzymatic activity to the important hepatic growth factor augmenter of liver regeneration protein. The finding that augmenter of liver regeneration protein acts as a FAD-linked sulfhydryl oxidase is essential to identify the molecular targets inside liver cells and to elucidate the precise role of mammalian augmenter of liver regeneration protein in hepatic cell growth, liver disease and regeneration.

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family.

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of theERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimersin vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl carrier protein.

The nuclear genes (acp-1, ACP 1) encoding the mitochondrial acyl carrier protein were disrupted in Neurospora crassa and Saccharomyces cerevisiae. In N. crassa acp-1 is a peripheral subunit of the respiratory NADH: ubiquinone oxidoreductase (complex I). S. cerevisiae lacks complex I and its ACP1 appears to be located in the mitochondrial matrix. The loss of acp-1 in N. crassa causes two biochemical lesions. Firstly, the peripheral part of complex I is not assembled, and the membrane part is not properly assembled. The respiratory ubiquinol: cytochrome c oxidose (complex IV) are made in normal amounts. Secondly, the lysophospholipid content of mitochondrial membranes is increased four-fold. In S. cerevisiae, the loss of aCP1 leads to a pleiotropic respiratory deficient phenotype.

ERV1 is involved in the cell division cycle and the maintenance of mitochondrial genomes in Saccharomyces cerevisiae

In former studies it was found that the ERV1 gene is essential for cell viability and for the biogenesis of functional mitochondria. A temperature-sensitive nuclear mutant exhibits a severe reduction in all the mitochondrial transcripts. Elimination of the gene leads to growth arrest after a few cell divisions. The putative gene product bears the characteristics of a regulatory factor since it has low expression rate and a high content of charged amino acids. In this study it is further verified that the ERV1 gene alone is responsible for the observed cellular and mitochondrial defects. The 5′ region of the gene is analysed by DNA deletions and complementation studies. Expression of the gene under the control of the GAL1-10 promoter in a disruption strain of ERV1 allows a more detailed specification of its influence on mitochondrial and cellular functions. Immediate and complete loss of mitochondrial genomes is observed after the promoter has been shut off, whereas the yeast cells are still able to grow for a limited time under these conditions. Analysis of the cells by in-vivo DNA flurorescence demonstrates a specific arrest in the cell-division cycle as the terminal phenotype. To further characterize the temperature-sensitive allele of ERV1 the mutated gene has been isolated and sequenced. A single point mutation which leads to the exchange of a single amino acid is found in the reading frame.

A nuclear gene essential for mitochondrial replication suppresses a defect of mitochondrial transcription in Saccharomyces cerevisiae

SummaryA genomic DNA fragment from yeast was isolated by transforming a temperature sensitive pet mutant. This mutant, pet-ts 798, has previously been characterized by its altered mitochondrial transcription apparatus. Subcloning and DNA sequencing of the genomic DNA fragment identified a reading frame responsible for the restoration of the pet-ts phenotype. The reading frame of 1023 bp is transcribed as an RNA of about 1100 nucleotides. The putative protein of 40 kDa possesses a hydrophobic aminoterminus and acidic and basic domains characteristic of recently described transcriptional activators. The inactivation of the functional gene by the introduction of an insertion fragment into the reading frame, leads to a stable pet phenotype. Further analysis of this mutant created by gene disruption makes clear that the respiratory defect is caused by the complete loss of mitochondrial DNA. Experimental evidence is given that the cloned gene acts as an intergenic suppressor of the mutant pet-ts798. Therefore, the isolated gene represents a new factor involved in the regulation of mitochondrial replication and transcription.

A mutant for the yeast scERV1 gene displays a new defect in mitochondrial morphology and distribution

The yeast scERV1 gene is the best characterized representative of a new gene family found in different lower and higher eukaryotes. The gene product is essential for the yeast cell and has a complex influence on different aspects of mitochondrial biogenesis. The homologous mammalian ALR(Augmenter of Liver Regeneration) genes from man, mouse and rat are important at different developmental stages of the organism as, for example, in spermatogenesis and liver regeneration. In this study the influence of scERV1 on the morphology of mitochondria and its submitochondrial localization are investigated. A temperature‐sensitive mutant of the gene was stained with a mitochondria‐specific dye and fluorescence was inspected at the permissive and restrictive temperature. A new phenotype for morphological defects of mitochondria was identified. Already at the permissive temperature mitochondrial vesicles accumulate at defined positions in the cell. After shift to the restrictive temperature, morphological changes, and finally complete loss of mitochondrial structures, are observed. Ultrastructural studies confirm these findings and demonstrate the loss of the mitochondrial inner membrane and at the final stage a drastic reduction or complete absence of mitochondria from the cell. GFP fusion experiments with the scERV1 gene and subcellular localization by fractionation experiments identify the gene product inside mitoplasts and the cytosol. Re‐investigation of the mutant phenotype demonstrates that after longer incubation of the mutant at the restrictive temperature an irreversible defect of the cells, even on glucose complete medium, is found that is in accordance with a complete loss or irreversible damage of mitochondria. Copyright

Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization.

The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle. Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration. A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis. The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals. The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man. In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species. This finding leads to the problem whether the amino termini have comparable or divergent functions in different species. In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein. Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells. In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria. Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells. Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.