Lacour T, Achstetter T, Dumas B
J Biol Chem. 1998 Sep 11;273(37):23984–92
The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namelyadrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adrenodoxinreductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide,cytochrome c reduction, and, more importantly, in vitro 11beta-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent Km values were determined to be 0.5, 0.6, and 0.1 microM for NADPH, NADH, and bovineadrenodoxin, respectively. These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential foryeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.
Hernando Y, Carter AT, Parr A, Hove-Jensen B, Schweizer M
J Biol Chem. 1999 Apr 30;274(18):12480–7
The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphatesynthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate,enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosylpyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es).
Launhardt H, Munder T
Molecular and General Genetics MGG. Springer; 2000;264(3):317–24
In the post-genome sequencing era the functional analysis of newly discovered proteins becomes more and more important. In this report we describe a genetic approach to the post-translational regulation of protein function in Saccharomyces cerevisiae by creating conditional lethal mutants. The yeast ORFs YDL139c, YDL147w, ERG3 and ERG11 were tagged with sequences encoding the hormone-binding domains ofmammalian steroid receptors by PCR-mediated, targeted integration into the yeast genome. We found that the function of the chimeric proteins is regulated in a hormone-dependent way. This technique provides another important tool for the functional analysis of the yeast proteome.
Wang SW, Toda T, MacCallum R, Harris AL, Norbury C
Mol Cell Biol. 2000 May 1;20(9):3234–44
The S-M checkpoint is an intracellular signaling pathway that ensures that mitosis is not initiated in cells undergoing DNA replication. We identified cid1, a novel fission yeast gene, through its ability when overexpressed to confer specific resistance to a combination of hydroxyurea, which inhibits DNA replication, and caffeine, which overrides the S-M checkpoint. Cid1 overexpression also partially suppressed the hydroxyurea sensitivity characteristic of DNA polymerase delta mutants and mutants defective in the "checkpoint Rad" pathway. Cid1 is a member of a family of putative nucleotidyltransferases including budding yeast Trf4 and Trf5, and mutation of amino acid residues predicted to be essential for this activity resulted in loss of Cid1 function in vivo. Two additional Cid1-like proteins play similar but nonredundant checkpoint-signaling roles in fission yeast. Cells lacking Cid1 were found to be viable but specifically sensitive to the combination of hydroxyurea and caffeine and to be S-M checkpoint defective in the absence of Cds1. Genetic data suggest that Cid1 acts in association with Crb2/Rhp9 and through the checkpoint-signaling kinase Chk1 to inhibit unscheduled mitosis specifically when DNA polymerase delta or epsilon is inhibited.
Zheng L, Campbell M, Murphy J, Lam S, Xu JR
Mol Plant Microbe Interact. 2000 Jul;13(7):724–32
In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium.bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated thatBMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.
Kaufman BA, Newman SM, Hallberg RL, Slaughter CA, Perlman PS, Butow RA
Proc Natl Acad Sci USA. 2000 Jul 5;97(14):7772–7
The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNAorganization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected approximately 20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associatedproteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2Delta mutation increases the petite-inducing phenotype of an abf2Delta mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of rho(+) mtDNA.
O'Sullivan JM, Mihr MJ, Santos MA, Tuite MF
Yeast. Wiley Online Library; 2001;18(4):313–22
A number of Candida species translate the standard leucine-CUG codon as serine using a novel ser-tRNA(CAG). This tRNA, which has an unusual anticodon stem-loop structure, has been implicated in the evolution of this codon reassignment. However, such a sense codon reassignment might also require a change in the specificity of the cognate aminoacyl tRNA-synthetase, in this case the ser-tRNA synthetase. Here we describe the cloning and sequence analysis of the C. albicans seryl aminoacyl-tRNA synthetase (CaSerRS) gene (CaSES1). The predicted CaSerRS sequence shows a significant level of amino acid identity to SerRs from other organisms and fully complements a S. cerevisiae SerRS null strain without any apparent defect in growth rate. This suggests that the SerRS recognizes and charges S. cerevisiae ser-tRNAs with similar efficiency to that of the S. cerevisiae SerRS. Using an antibody raised against CaSerRS, we also demonstrate the presence of SerRS in a range of Candida spp. showing CUG codon reassignment. We conclude that the key element in CUG reassigment in Candida spp. is the tRNA that decodes the CUG codon rather than a SerRS structural change. The nucleotide sequence of the CaSES1 gene has been deposited at GenBank under Accession No. AF290915.
O'Sullivan JM, Mihr MJ, Santos M, Tuite MF
Gene. 2001 Sep 5;275(1):133-40
In a number of Candida species the 'universal' leucine codon CUG is decoded as serine. To help understand the evolution of such a codonreassignment we have analyzed the Candida albicans leucyl-tRNA synthetase (CaLeuRS) gene (CaCDC60). The predicted CaLeuRS sequence shows a significant level of amino acid identity to LeuRS from other organisms. A mitochondrial LeuRS (ScNAM2) homologue, which shared low identity with the CaLeuRS, was also identified in C. albicans. Antigenically-related LeuRSs were identified in a range of Candida species decoding the CUG codon as both serine and leucine, using an antibody raised against the N-terminal 15 amino acids of the CaLeuRS. Complementation experiments demonstrated that the CaLeuRS was able to functionally complement a Saccharomyces cerevisiae cdc60::kanMX null mutation. We conclude that there is no alteration in tRNA recognition and aminoacylation by the C. albicans LeuRS, which argues against it having a role in codonreassignment. The nucleotide sequences of the CaCDC60 and CaNAM2 genes were deposited at GenBank under Accession numbers AF293346 and AF352020, respectively.
Wang SW, Goodwin A, Hickson ID, Norbury CJ
Nucleic Acids Res. 2001 Jul 15;29(14):2963–72
In the budding yeast Saccharomyces cerevisiae the Srs2/RadH DNA helicase promotes survival after ultraviolet (UV) irradiation, and has been implicated in DNA repair, recombination and checkpoint signalling following DNA damage. A second helicase, Sgs1, is the S.cerevisiae homologue of the human BLM and WRN proteins, which are defective in cancer predisposition and/or premature ageing syndromes. Saccharomyces cerevisiae cells lacking both Srs2 and Sgs1 exhibit a severe growth defect. We have identified an Srs2 orthologue in the fission yeast Schizosaccharomycespombe, and have investigated its role in responses to UV irradiation and inhibition of DNA replication. Deletion of fission yeast srs2 caused spontaneous hyper-recombination and UV sensitivity, and simultaneous deletion of the SGS1 homologue rqh1 caused a severe growth defect reminiscent of that seen in the equivalent S.cerevisiae mutant. However, unlike in budding yeast, inactivation of the homologous recombination pathway did not suppress this growth defect. Indeed, the homologous recombination pathway was required for maintenance of normal fission yeast viability in the absence of Srs2, and loss of homologous recombination and loss of Srs2 contributed additively to UV sensitivity. We conclude thatSrs2 plays related, but not identical, roles in the two yeast species.
Georgakopoulos T, Koutroubas G, Vakonakis I, Tzermia M, Prokova V, Voutsina A, Alexandraki D
Yeast. John Wiley & Sons, Ltd; 2001 Sep 15;18(12):1155–71
Saccharomyces cerevisiae YFR021w, YGR223c and YPL100w are paralogous ORFs of unknown function. Phenotypic analysis of overexpression, single-, double- and triple-ORF deletion strains under various growth conditions indicated mitochondria-related functions for all three ORFs. Two-hybrid screens of a yeast genomic library identified potentially interacting proteins for the three ORFs. Among these, the transcriptional activator Rtg3p interacted with both Yfr021wp and Ypl100wp and both ORF single deletions reduced the constitutive expression of the RTG-regulated CIT2 and DLD3 genes and caused typical retrograde response of CIT2 and DLD3 under growth conditions requiring functional mitochondria, indicating thatYFR021w and YPL100w are also involved in unidentified mitochondrial functions. Ptr3p, a component of the amino acid sensor Ssy1p/Ptr3p, was also found as a two-hybrid interactant of Yfr021wp. Of the three single-ORF deletions, ypl100w Delta exhibited ptr3 Delta-similar phenotypes. These findings, combined with the fact that RTG-dependent expression is modulated by specific amino acids, suggested possible relations of Yfr021wp and Ypl100wp to amino acid signalling pathways. Under most conditions examined, the effects of the single- and double-ORF deletions indicated thatYFR021w, YPL100w and YGR223c are not parts of the same pathway. We found no unique phenotype attributed to the deletion of YGR223c. However, its function interferes with the function of the other two ORFs, as revealed by the effects of double- and triple-ORF deletions.