Yng2p-dependent NuA4 histone H4 acetylation activity is required for mitotic and meiotic progression.

In all eukaryotes, multisubunit histone acetyltransferase (HAT) complexes acetylate the highly conserved lysine residues in the amino-terminal tails of core histones to regulate chromatin structure and gene expression. One such complex in yeast, NuA4, specifically acetylates nucleosome-associated histone H4. Recent studies have revealed that NuA4 comprises at least 11 subunits, including Yng2p, a yeast homolog of the candidate human tumor suppressor gene, ING1. Consistent with prior data, we find that cells lacking Yng2p are deficient for NuA4 activity and are temperature-sensitive. Furthermore, we show that the NuA4 complex is present in the absence of Yng2p, suggesting that Yng2p functions to maintain or activate NuA4 HAT activity. Sporulation of diploid yng2 mutant cells reveals a defect in meiotic progression, whereas synchronized yng2 mutant cells display a mitotic delay. Surprisingly, genome-wide expression analysis revealed little change from wild type. Nocodazole arrest and release relieves the mitotic defects, suggesting that Yng2p may have a critical function prior to or during metaphase. Rather than a uniform decrease in acetylated forms of histone H4, we find striking cell-to-cell heterogeneity in the loss of acetylated histone H4 in yng2 mutant cells. Treating yng2 mutants with the histone deacetylase inhibitor trichostatin A suppressed the mitotic delay and restored global histone H4 acetylation, arguing that reduced H4 acetylation may underlie the cell cycle delay.     

The Yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone H3

The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.     

Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase

Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity. We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase.     

Rad23 provides a link between the Png1 deglycosylating enzyme and the 26 S proteasome in yeast

In addition to a role in DNA repair events in yeast, several lines of evidence indicate that the Rad23 protein (Rad23p) may regulate the activity of the 26 S proteasome. We report evidence that a de-N-glycosylating enzyme, Png1p, may be involved in the proteasomal degradation pathway via its binding to Rad23p. Interaction of Rad23p and Png1p was first detected by two-hybrid screening, and this interaction in vivo was confirmed by biochemical analyses. The Png1p-Rad23p complex was shown to be distinct from the well established DNA repair complex, Rad4p-Rad23p. We propose a model in which Rad23p functions as an escort protein to link the 26 S proteasome with proteins such as Rad4p or Png1p to regulate their cellular activities.     

Bob1, a Gim5/MM-1/Pfd5 homolog, interacts with the MAP kinase kinase Byr1 to regulate sexual differentiation in the fission yeast, Schizosaccharomyces pombe

The MAPKK Byr1 is an essential component of a Ras-dependent MAPK module required for sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Here we describe the genetic and molecular characterization of a highly conserved protein, Bob1, which was identified from a two-hybrid screen for Byr1-interacting proteins. Byrl and Bobl proteins coprecipitate from S. pombe cell lysates, and both proteins localize to the tips and septa of S. pombe cells. S. pombe bob1 null (bob1delta) mutants lack obvious growth defects but exhibit a significant mating deficiency, which can be suppressed by overexpression of Byrl. Overexpression of Bob1 also leads to inhibition of mating in S. pombe, and this defect is likewise suppressed by Byrl overexpression. Bob1 is highly homologous in structure to the mammalian MM-1/Pfd5 and budding yeast Gim5/Pfd5-Sc proteins, which have been implicated as regulators of actin and tubulins. Similar to budding yeast gim5/pfd5-Sc mutants, S. pombe bob1delta cells have cytoskeletal defects, as judged by hypersensitivity to cytoskeletal disrupting drugs. byr1delta mutants do not share this characteristic with bob1delta mutants, and byr1delta bob1delta mutants are not significantly more sensitive to cytoskeletal disrupting drugs than cells carrying only the bob1delta mutation. Taken together, our results suggest that Bob1 has Byr1-related function(s) required for proper mating response of S. pombe cells and Byrl-independent function(s) required for normal cytoskeletal control. We show that the human MM-1/Pfd5 protein can substitute for its counterpart in fission yeast, providing evidence that the functions of Bob1-related proteins have been highly conserved through evolution. Our results lead us to propose that Bob1-related proteins may play diverse roles in eukaryotic organisms.     

The Birt-Hogg-Dube and tuberous sclerosis complex homologs have opposing roles in amino acid homeostasis in Schizosaccharomyces pombe

Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Deltatsc1 and Deltatsc2, were up-regulated in Deltabhd, and levels of specific intracellular amino acids known to be low in Deltatsc1 and Deltatsc2 were elevated in Deltabhd. This "opposite" profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1(+) dramatically increased permease expression levels in Deltabhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Deltabhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.     

Mdt1/ASCIZ: A new DNA damage response protein family

DNA damage response pathways are crucial for genome stability and prevention of cancer, and are overall remarkably conserved from yeast to mammals. Two novel DNA damage response proteins, yeast Mdt1 (Modifier of DNA damage tolerance 1) and human ASCIZ (ATM/ATR-substrate Chk2-interacting Zn2+-finger protein), were recently identified based on their interactions with the N-terminal FHA domains of the conserved checkpoint kinases Rad53 and Chk2, respectively, and ASCIZ was subsequently re-isolated as an ATM-interacting protein (ATMIN). Mdt1 and ASCIZ share remarkable sequence similarity (36% highly conserved residues, 17% identity) and extended SQ/TQ cluster domains (SCDs) typical of DNA damage response proteins. However, despite their structural similarities and conserved interactions with the checkpoint machinery, the two proteins seem to respond to different DNA lesions: the strongest phenotypes of ASCIZ deficiency are increased sensitivity to DNA base damaging agents and altered immunoglobulin gene diversification following enzyme-induced base damage in B lymphocytes, whereas absence of Mdt1 leads to hypersensitivity to 3'-blocked DNA double-strand breaks and inefficient recombinational maintenance of telomeres. The Mdt1/ASCIZ family may function as structurally related scaffolds that facilitate efficient DNA repair, albeit with diverged lesion specificity.     

Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase

Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1(+): Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.     

The fission yeast Schizosaccharomyces pombe has two distinct tRNase Z(L)s encoded by two different genes and differentially targeted to the nucleus and mitochondria

tRNase Z is the endonuclease that is involved in tRNA 3'-end maturation by removal of the 3'-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3'-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.     

Characterization of multicopy suppressor genes that complement a defect in the Wis1-Sty1 MAP kinase cascade involved in stress responses in Schizosaccharomyces pombe

The Wis1-Sty1 mitogen-activated protein (MAP) kinase cascade is one of the major signaling systems involved in a wide range of stress responses in Schizosaccharomyces pombe. It is known that Deltawis1 and Deltasty1 mutants exhibit highly pleiotropic phenotypes, including a phenotype of temperature sensitivity for growth. In this study, we screened multicopy suppressor genes that allow both the Deltawis1 and Deltasty1 mutants to grow simultaneously at a non-permissive temperature, 37 degrees C. Two such multicopy suppressors were cloned and characterized as sds23(+) and hxk2(+) genes. The former is known to specify a protein that functions as a multicopy suppressor for mutations of the PP1 protein phosphatase and the 20S cyclosome/anaphase-promoting complex (APC), and the latter encodes hexokinase 2. It was revealed that the multicopy sds231 gene restored a defect in the mating efficiency caused by the Deltawis1 and Deltasty1 mutations, whereas the multicopy hxk2(+) gene suppressed a phenotype of heat-shock sensitivity for growth of these mutant cells. These findings are discussed with special reference to the Wis1-Sty1 MAP kinase signaling pathway in S. pombe.     

The small subunit of the splicing factor U2AF is conserved in fission yeast.

The human splicing factor U2 auxiliary factor (hsU2AF) is comprised of two interacting subunits of 65 and 35 kDa. Previously we identified the Schizosaccharomyces pombe homolog, spU2AF59, of the human large subunit. We have screened a fission yeast cDNA library in search of proteins that interact with spU2AF59 using the yeast two-hybrid system and have identified a homolog of the hsU2AF35 subunit. The S. pombe U2AF small subunit is a single copy gene that encodes a protein which shares 55% amino acid identity and 17% similarity with the human small subunit. Unlike the human protein, the yeast protein lacks an arginine/serine-rich region. The predicted molecular mass of the spU2AF small subunit is 23 kDa. The region of spU2AF59 that interacts with spU2AF23 is similar to the region in which the human small and large subunits interact.     

Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.