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(A ugmenter of L iver R egeneration) 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 © 1999 John Wiley & Sons, Ltd.     

Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein l19

A gene designated RPL19A has been identified in the region downstream from the 3′-end of the Saccharomyces cerevisiae MIS1 gene encoding the mitochondrial C₁-tetrahydrofolate synthase. The gene codes for the yeast ribosomal protein YL19 which exhibits 57·5% identity with the mammalian ribosomal protein L19. RPL19A is one of two functional copies of the YL19 gene located on chromosome II. The disruption of RPL19A has no effect on the growth of the yeast. The RPL19A gene contains an intron located near the 5′-end. The 5′-flanking region contains one similar and one complete UAS_rpg upstream activating sequence. RPL19A was also found to be adjacent to the chromosome II AAC3 gene, encoding the mitochondrial ADP/ATP carrier protein. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^tm/EMBL data bank with the accession number Z36751.     

DNA sequence analysis of a 17 kb fragment of yeast chromosome XI physically localizes the MRB1 gene and reveals eight new open reading frames,including a homologue of the KIN1/KIN2 and SNF1 protein kinases

We report in this paper the sequence of a part of chromosome XI of Saccharomyces cerevisiae. This 17 kbp nucleotide sequence represents the right half of cosmid pUKG151 and contains nine open reading frames, YKL453, 450, 449, 448, 445, 443, 442, 441 and the 5′ part of YKL440. YKL440 was previously identified as the MBR1 gene and plays a role in mitochondrial biogenesis. YKL443 is a homologue of the yeast serine-rich protein (SRP1), while YKL453 presents strong homologies with the KIN1/KIN2/SNF1 kinase family. It must be pointed out that the size of this gene is well above average for yeast.     

DNA sequence analysis of the YCN2 region of chromosome XI in Saccharomyces cerevisiae

A 6·8 kbp DNA fragment localized to the left arm of chromosome XI from Saccharomyces cerevisiae was sequenced and analysed (EMBL accession no. X69765). Two genes involved in protein phosphatase activity were identified: YCN2 and an open reading frame encoding a protein that shares 46% amino acid identity with the sds22⁺ protein from Schizosaccharomyces pombe. A comparison of the genomic YCN2 sequence with the published cDNA sequence suggests the presence of an intron near the 5′ end of the gene. Further sequence analysis suggests the presence of three additional genes near YCN2: a mitochondrial acyl-carrier protein, a gene encoding a putative hydrophobic protein, and a new gene coding for a tRNA^Leu (UAA) isoacceptor located near a delta sequence.     

The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene

The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin structures in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.     

Isolation and expression of the gene encoding mitochondrial ADP/ATP carrier (AAC) from the pathogenic yeast Candida parapsilosis

A gene homologous to Saccharomyces cerevisiae AAC genes coding for mitochondrial ADP/ATP carriers has been cloned from the pathogenic yeast Candida parapsilosis. A probe obtained by PCR amplification from C. parapsilosis DNA, using primers derived from the conserved transmembrane region of yeast ADP/ATP carriers, was used for screening of the C. parapsilosis genomic library. The cloned gene was sequenced and found to encode a polypeptide of 303 amino acids that shows homology with other yeast and fungal mitochondrial ADP/ATP carriers. The gene was designated CpAAC1 and was able to complement the growth phenotypes of S. cerevisiae double deletion mutant (Δaac2; Δaac3). The expression of the CpAAC1 gene was reduced under semi‐anaerobic conditions and it was affected at normal aerobic conditions by the nature of carbon sources used for growth. Hybridization experiments indicate that C. parapsilosis possesses a single gene encoding a mitochondrial ADP/ATP carrier. The GenBank Accession No. for the C. parapsilosis CpAAC1 gene is AF085429. Copyright © 1999 John Wiley & Sons, Ltd.     

Identification of peroxisomal membrane ghosts with an epitope-tagged integral membrane protein in yeast mutants lacking peroxisomes

Many yeast peroxisome biogenesis mutants have been isolate in which peroxisomes appear to be completely absent. Introduction of a wild-type copy of the defective gene causes the reappearance of peroxisomes, despite the fact that new peroxisomes are thought to form only from pre-existing peroxisomes. This apparent paradox has been explained for similar human mutant cell lines (from patients with Zellweger syndrome) by the discovery of peroxisomal membrane ghosts in the mutant cells (Santos, M. J., T. Imanaka, H. Shio, G. M. Small and P. B. Lazarow. 1988. Science 239 , 1536–1538). Introduction of a wild-type gene is thought to restore to the ghosts the ability to import matrix proteins, and thus lead to the refilling of the peroxisomes. It is vitally important to our understanding of peroxisome biogenesis to determine whether the yeast mutants contain ghosts. We have solved this problem by introducing an epitope-tagged version of Pas3p, a peroxisome integral membrane protein (that is essential for peroxisome biogenesis). Nucleotides encoding a nine amino acid HA epitope were added to the PAS3 gene immediately before the stop codon. The tagged gene (PAS3_HA) was inserted in the genome, replacing the wild-type gene at its normal locus. It was fully functional (the cells assembled peroxisomes normally and grew on oleic acid) but the expression level was too low to detect the protein with monoclonal antibody 12CA5. PAS3_HA was expressed in greater quantity from an episomal plasmid with the CUP1 promoter. The gene product, Pas3p_HA, was detected by immunogold labelling on the membranes of individual and clustered peroxisomes; the clusters appeared as large spots in immunofluorescence. PAS3_HA was similarly expressed in peroxisome biogenesis mutants peb2 and peb4, which lack morphologically recognizable peroxisomes. Gold-labelled membranes were clearly visible in both mutants: in peb2 the labelled membrane vesicles were generally much smaller than those in peb4, which resembled normal peroxisomes in size.     

Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co‐expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5′‐gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast‐enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast‐enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon‐optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest. Copyright © 2009 John Wiley & Sons, Ltd.     

Functional differences among the six Saccharomyces cerevisiae tRNATrp genes

The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNA^Trp which are scattered on five chromosomes. Other, non-functional tDNA^Trp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNAas well as a 34-nucleotide intervening sequence, are identical. However, the 5′ and 3′ flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNA^Trp to search for functional differences among these genes. Previous studies with one tDNA^Trp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNA^Trp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 μ vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNA^Trp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5′-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3′-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5′-flanking sequences of the tRNA^Trp genes. © 1997 John Wiley & Sons, Ltd.     

Boron-dependent regulation of translation through AUGUAA sequence in yeast

Under high boron (B) conditions, nodulin 26-like intrinsic protein 5;1 (NIP5;1) mRNA, a boric acid channel, is destabilized to avoid excess B entry into roots of Arabidopsis thaliana. In this regulation, the minimum upstream open reading frame (uORF), AUGUAA, in its 5′-untranslated region (5′-UTR) is essential, and high B enhances ribosome stalling at AUGUAA and leads to suppression of translation and mRNA degradation. This B-dependent AUGUAA-mediated regulation occurs also in animal transient expression and reticulocyte lysate translation systems. Thus, uncovering the ubiquitousness of B-dependent unique regulation is important to reveal the evolution of translational regulation. In the present study, we examined the regulation in Saccharomyces cerevisiae. Reporter assay showed that in yeast, carrying ATGTAA in 5′-UTR of NIP5;1 upstream of the reporter gene, the relative reporter activities were reduced significantly under high B conditions compared with control, whereas deletion of ATGTAA abolished such responses. This suggests that AUGUAA mediates B-dependent regulation of translation in Saccharomyces cerevisiae. Moreover, the deletion of ATGTAA resulted in up to 10-fold increase in general reporter activities indicating the suppression effect of AUGUAA on translation of the main ORF. Interestingly, mRNA level of the reporter gene was not affected by B in both yeast cells with and without AUGUAA. This finding reveals that in yeast, unlike the case in plants, mRNA degradation is not associated with AUGUAA regulation. Together, results suggest that B-dependent AUGUAA-mediated translational regulation is common among eukaryotes.     

Structural comparison of the Pichia pastoris alcohol oxidase genes

In methylotrophic yeasts, alcohol oxidase is the first enzyme in the methanol-utilization pathway. The genome of one such yeast, Pichia pastoris, contains two alcohol oxidase genes, AOX1 and AOX2. Sequence analysis indicated that each gene encodes a similar protein of 663 amino acids. The protein-coding regions of the genes were 92% and 97% homologous at the nucleotide and predicted amino acid sequence levels, respectively. In contrast to homology observed within the protein-coding portions of the AOX genes, no homology was found in either the 5′ or 3′ non-coding regions. Although alcohol oxidase is found in peroxisomes of P. pastoris, the AOX amino acid sequences did not contain a peptide sequence similar to the peroxisomal transport sequence found at the C-terminus of some peroxisomally located proteins in higher eukaryotes.     

Nucleotide sequence analysis of a 32,500 bp region of the right arm of Saccharomyces cerevisiae chromosome IV

We have sequenced a region containing 32.5 kb of the right arm of chromosome IV of Saccharomyces cerevisiae. Twenty open reading frames (ORFs) greater than 100 amino acids could be identified in this region. Six ORFs correspond to known yeast genes, including DOA4, UBC5 and UBC3, the gene products of which are involved in ubiquitin metabolism. UBC5 is preceded by the two tRNA genes tRNA-Arg2 and tRNA-Asp. Six genes were discovered with homologies to non-yeast genes or with homologies to other yeast ORFs. One of these could be identified as ribosomal protein gene RPS13. The putative function of eight ORFs remains unclear because comparison to different DNA or protein databases revealed no significant patterns. The sequence from cosmid 2F21 was obtained entirely by a combined subcloning and walking primer strategy, and has been deposited in the EMBL data library under Accession Number X84162.