Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae.

H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins. Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae. However, no H2A.F/Z variant has been reported in this budding yeast species. We sought to distinguish among three explanations for these observations: (i) that S. cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S. cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule. Repeated attempts to clone an S. cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2. To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1. Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function. The Tetrahymena H2A genes confer a cold-sensitive phenotype. Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins. Proteins from S. cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies. These studies make it likely that S. cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog. A hypothesis is presented relating the absence of H2A.F/Z in S. cerevisiae to its function in other organisms.     

Caspase-dependent apoptosis in yeast

Damaging environment, certain intracellular defects or heterologous expression of pro-apoptotic genes induce death in yeast cells exhibiting typical markers of apoptosis. In mammals, apoptosis can be directed by the activation of groups of proteases, called caspases, that cleave specific substrates and trigger cell death. In addition, in plants, fungi, Dictyostelium and metazoa, paracaspases and metacaspases have been identified that share some homologies with caspases but showing different substrate specificity. In the yeast Saccharomyces cerevisiae, a gene (MCA1/YCA1) has been identified coding for a metacaspase involved in the induction of cell death. Metacaspases are not biochemical, but sequence and functional homologes of caspases, as deletion of them rescues entirely different death scenarios. In this review we will summarize the current knowledge in S. cerevisiae on apoptotic processes, induced by internal and external triggers, which are dependent on the metacaspase gene YCA1.     

Ubiquitin-independent mechanisms of mouse ornithine decarboxylase degradation are conserved between mammalian and fungal cells

The polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is degraded by the 26 S proteasome via a ubiquitin-independent pathway in mammalian cells. Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1 (AZ1). Mouse ODC (mODC) that is expressed in the yeast Saccharomyces cerevisiae is also rapidly degraded by the proteasome of that organism. We have now carried out in vivo and in vitro studies to determine whether S. cerevisiae proteasomes recognize mODC degradation signals. Mutations of mODC that stabilized the protein in animal cells also did so in the fungus. Moreover, the mODC degradation signal was able to destabilize a GFP or Ura3 reporter in GFP-mODC and Ura3-mODC fusion proteins. Co-expression of AZ1 accelerated mODC degradation 2-3-fold in yeast cells. The degradation of both mODC and the endogenous yeast ODC (yODC) was unaffected in S. cerevisiae mutants with various defects in ubiquitin metabolism, and ubiquitinylated forms of mODC were not detected in yeast cells. In addition, recombinant mODC was degraded in an ATP-dependent manner by affinity-purified yeast 26 S proteasomes in the absence of ubiquitin. Degradation by purified yeast proteasomes was sensitive to mutations that stabilized mODC in vivo, but was not accelerated by recombinant AZ1. These studies demonstrate that cell constituents required for mODC degradation are conserved between animals and fungi, and that both mammalian and fungal ODC are subject to proteasome-mediated proteolysis by ubiquitin-independent mechanisms.     

The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene.

The product of the yeast SNP1 gene has high homology to two domains of the metazoan U1 snRNP protein 70K, which binds to stem/loop I of the U1 RNA. However, the absence of other domains conserved in metazoan 70K and the minimal effect of yeast U1 RNA stem/loop I deletion make the assignment of SNP1 as yeast 70K less clear. To address this question, we have expressed the SNP1 gene as a fusion protein in E. coli and developed a gel shift assay for U1 RNA binding. We show here that the product of the yeast SNP1 gene binds directly and specifically to the first 47 nucleotides of yeast U1 RNA, which include the stem/loop 1 structure. We therefore conclude that the SNP1 gene product is the yeast 70K homolog. This is the first yeast protein to be identified as a homolog of a metazoan snRNP protein.     

Maintenance of cohesin at centromeres after meiosis I in budding yeast requires a kinetochore-associated protein related to MEI-S332

BACKGROUND: The halving of chromosome number that occurs during meiosis depends on three factors. First, homologs must pair and recombine. Second, sister centromeres must attach to microtubules that emanate from the same spindle pole, which ensures that homologous maternal and paternal pairs can be pulled in opposite directions (called homolog biorientation). Third, cohesion between sister centromeres must persist after the first meiotic division to enable their biorientation at the second. RESULTS: A screen performed in fission yeast to identify meiotic chromosome missegregation mutants has identified a conserved protein called Sgo1 that is required to maintain sister chromatid cohesion after the first meiotic division. We describe here an orthologous protein in the budding yeast S. cerevisiae (Sc), which has not only meiotic but also mitotic chromosome segregation functions. Deletion of Sc SGO1 not only causes frequent homolog nondisjunction at meiosis I but also random segregation of sister centromeres at meiosis II. Meiotic cohesion fails to persist at centromeres after the first meiotic division, and sister centromeres frequently separate precociously. Sgo1 is a kinetochore-associated protein whose abundance declines at anaphase I but, nevertheless, persists on chromatin until anaphase II. CONCLUSIONS: The finding that Sgo1 is localized to the centromere at the time of the first division suggests that it may play a direct role in preventing the removal of centromeric cohesin. The similarity in sequence composition, chromosomal location, and mutant phenotypes of sgo1 mutants in two distant yeasts with that of MEI-S332 in Drosophila suggests that these proteins define an orthologous family conserved in most eukaryotic lineages.     

Transfer RNA methytransferases and their corresponding modifications in budding yeast and humans: activities, predications, and potential roles in human health

Throughout the kingdoms of life, transfer RNA (tRNA) undergoes over 100 enzyme-catalyzed, methyl-based modifications. Although a majority of the methylations are conserved from bacteria to mammals, the functions of a number of these modifications are unknown. Many of the proteins responsible for tRNA methylation, named tRNA methyltransferases (Trms), have been characterized in Saccharomyces cerevisiae. In contrast, only a few human Trms have been characterized. A BLAST search for human homologs of each S. cerevisiae Trm revealed a total of 34 human proteins matching our search criteria for an S. cerevisiae Trm homolog candidate. We have compiled a database cataloging basic information about each human and yeast Trm. Every S. cerevisiae Trm has at least one human homolog, while several Trms have multiple candidates. A search of cancer cell versus normal cell mRNA expression studies submitted to Oncomine found that 30 of the homolog genes display a significant change in mRNA expression levels in at least one data set. While 6 of the 34 human homolog candidates have confirmed tRNA methylation activity, the other candidates remain uncharacterized. We believe that our database will serve as a resource for investigating the role of human Trms in cellular stress signaling.     

PDK1 homologs activate the Pkc1-mitogen-activated protein kinase pathway in yeast

PDK1 (phosphoinositide-dependent kinase 1) is a mammalian growth factor-regulated serine/threonine kinase. Using a genetic selection based on a mutant form of the yeast MAP kinase kinase Ste7, we isolated a gene, PKH2, encoding a structurally and functionally conserved yeast homolog of PDK1. Yeast cells lacking both PKH2 and PKH1, encoding another PDK1 homolog, were nonviable, indicating that Pkh1 and Pkh2 share an essential function. A temperature-sensitive mutant, pkh1(D398G) pkh2, was phenotypically similar to mutants defective in the Pkc1-mitogen-activated protein kinase (MAPK) pathway. Genetic epistasis analyses, the phosphorylation of Pkc1 by Pkh2 in vitro, and reduced Pkc1 activity in the pkh1(D398G) pkh2 mutant indicate that Pkh functions upstream of Pkc1. The Pkh2 phosphorylation site in Pkc1 (Thr-983) is part of a conserved PDK1 target motif and essential for Pkc1 function. Thus, the yeast PDK1 homologs activate Pkc1 and the Pkc1-effector MAPK pathway.     

Functional complementation of an oxa1− yeast mutation identifies an Arabidopsis thaliana cDNA involved in the assembly of respiratory complexes

The nuclear gene OXA1 is essential for respiratory growth in yeast. It codes for a chaperon-like protein, and has pleiotropic effects on the assembly of cytochrome c oxidase and ATP synthase of the mitochondrial respiratory chain. To study respiratory complex formation in plants, we have cloned a homolog of the yeast oxa1⁻ in Arabidopsis thaliana , OXA1At , by functional complementation of a yeast oxa1⁻ mutant. OXA1At is a single copy gene and appears to be constitutively expressed in A. thaliana . Although OXA1At encodes a protein sharing only 30% amino acid identity with the yeast Oxa1 protein, hydrophobic domains likely corresponding to trans -membrane domains are strictly conserved. Cytochrome spectra and measurements of respiratory activities show that replacement of the yeast Oxa1 protein with the A. thaliana homolog leads to correct assembly and activity of cytochrome c oxidase, but to partial restoration of ATPase activity. Our results suggest that the Oxa1At protein is essential for the respiratory complex assembly in A. thaliana , and that genes involved in mitochondrial multiprotein complex formation can be conserved between plants and other organisms.     

Calcium homeostasis and signaling in yeast cells and cardiac myocytes

Calcium ions are the most ubiquitous and versatile signaling molecules in eukaryotic cells. Calcium homeostasis and signaling systems are crucial for both the normal growth of the budding yeast Saccharomyces cerevisiae and the intricate working of the mammalian heart. In this paper, we make a detailed comparison between the calcium homeostasis/signaling networks in yeast cells and those in mammalian cardiac myocytes. This comparison covers not only the components, structure and function of the networks but also includes existing knowledge on the measured and simulated network dynamics using mathematical models. Surprisingly, most of the factors known in the yeast calcium homeostasis/signaling network are conserved and operate similarly in mammalian cells, including cardiac myocytes. Moreover, the budding yeast S. cerevisiae is a simple organism that affords powerful genetic and genomic tools. Thus, exploring and understanding the calcium homeostasis/signaling system in yeast can provide a shortcut to help understand calcium homeostasis/signaling systems in mammalian cardiac myocytes. In turn, this knowledge can be used to help treat relevant human diseases such as pathological cardiac hypertrophy and heart failure.     

Functional redundancies, distinct localizations and interactions among three fission yeast homologs of centromere protein-B

Several members of protein families that are conserved in higher eukaryotes are known to play a role in centromere function in the fission yeast Schizosaccharomyces pombe, including two homologs of the mammalian centromere protein CENP-B, Abp1p and Cbh1p. Here we characterize a third S. pombe CENP-B homolog, Cbh2p (CENP-B homolog 2). cbh2Delta strains exhibited a modest elevation in minichromosome loss, similar to cbh1Delta or abp1Delta strains. cbh2Delta cbh1Delta strains showed little difference in growth or minichromosome loss rate when compared to single deletion strains. In contrast, cbh2Delta abp1Delta strains displayed dramatic morphological and chromosome segregation defects, as well as enhancement of the slow-growth phenotype of abp1Delta strains, indicating partial functional redundancy between these proteins. Both cbh2Delta abp1Delta and cbh1Delta abp1Delta strains also showed strongly enhanced sensitivity to a microtubule-destabilizing drug, consistent with a mitotic function for these proteins. Cbh2p was localized to the central core and core-associated repeat regions of centromeric heterochromatin, but not at several other centromeric and arm locations tested. Thus, like its mammalian counterpart, Cbh2p appeared to be localized exclusively to a portion of centromeric heterochromatin. In contrast, Abp1p was detected in both centromeric heterochromatin and in chromatin at two of three replication origins tested. Cbh2p and Abp1p homodimerized in the budding yeast two-hybrid assay, but did not interact with each other. These results suggest that indirect cooperation between different CENP-B-like DNA binding proteins with partially overlapping chromatin distributions helps to establish a functional centromere.     

Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions.     

Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin.

The actin cytoskeleton is a fundamental component of eukaryotic cells, with both structural and motile roles. Actin and many of the actin-binding proteins found in different cell types are highly conserved, showing considerable similarity in both primary structure and biochemical properties. To make detailed comparisons between homologous proteins, it is necessary to know whether the various proteins are functionally, as well as structurally, conserved. Fimbrin is an example of a cytoskeletal component that, as shown by sequence determinations and biochemical characterizations, is conserved between organisms as diverse as Saccharomyces cerevisiae and humans. In this study, we examined whether the human homolog can substitute for the yeast protein in vivo. We report here that two isoforms of human fimbrin, also referred to as T- and L-plastin, can both substitute in vivo for yeast fimbrin, also known as Sac6p, whereas a third isoform, I-fimbrin (or I-plastin), cannot. We demonstrate that the human T- and L-fimbrins, in addition to complementing the temperature-sensitive growth defect of the sac6 null mutant, restore both normal cytoskeletal organization and cell shape to the mutant cells. In addition, we show that human T- and L-fimbrins can complement a sporulation defect caused by the sac6 null mutation. These findings indicate that there is a high degree of functional conservation in the cytoskeleton, even between organisms as diverse as S. cerevisiae and humans.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.