Hyperactive glycogen synthase mutants of Saccharomyces cerevisiae suppress the glc7-1 protein phosphatase mutant

A yeast glc7-1 mutant expressing a variant of protein phosphatase type 1 fails to accumulate glycogen. This defect is associated with hyperphosphorylated and inactive glycogen synthase, consistent with Glc7p acting directly to dephosphorylate and activate glycogen synthase. To characterize the glycogen synthesis defect of this mutant in more detail, we isolated 26 pseudorevertants of the glc7-1 mutant. All pseudoreversion events were due to missense mutations in GSY2, the gene encoding the major isoform of glycogen synthase. A majority of the mutations responsible for the suppression were in the 3' end of the gene, corresponding to the phosphorylated COOH terminus of Gsy2p. Phosphorylation of the mutant proteins was reduced, suggesting that they are poor substrates for glycogen synthase kinases. Suppressor mutations outside this domain did not decrease the phosphorylation of the resulting proteins, indicating that these proteins are immune to the regulatory effects of phosphorylation. Since no growth defect has been observed for strains with altered glycogen levels, the relative levels of fitness of GSY2 mutants that fail to accumulate glycogen and that hyperaccumulate glycogen were assayed by cocultivation experiments. A wild-type strain outcompeted both hypo- and hyperaccumulating strains, suggesting that glycogen levels contribute substantially to the fitness of yeast.     

Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae

The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients.     

A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity

In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg²⁺ ions and an apparent K_m of 3.6×10⁻⁵ M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation.     

Identification of CBS2 as a mitochondrial protein in Saccharomyces cerevisiae

Summary The nuclear genome encoded yeast protein CBS2 is required for translational activation of mitochondrial cytochrome b RNA. Genetic studies have shown that the target sequence of the CBS2 protein is the 5 untranslated leader sequence of cytochrome b RNA. Here we report on the intracellular localization of CBS2. CBS2 protein, expressed in Escherichia coli and prepared from inclusion bodies, was used as an antigen to raise a polyclonal rabbit antiserum. Affinity-purified CBS2 antibodies detect a 45 kDa protein in mitochondrial lysates of wild-type cells, which is absent in a strain in which the CBS2 gene has been deleted. The protein is overexpressed in mitochondrial extracts of a transformant carrying the CBS2 gene on a high copy number plasmid, but undetectable in the post-mitochondrial supernatant. Intramitochondrial localization of CBS2 was verified by in vitro import of CBS2 protein that had been synthesized in a reticulocyte lysate programmed with CBS2 mRNA transcribed in vitro. Mitochondrial import of CBS2 is not accompanied by any detectable proteolytic processing.     

Purification and characterization of glycogen synthase from a glycogen-deficient strain of Saccharomyces cerevisiae

Chromatography of wild-type yeast extracts on DEAE-cellulose columns resolves two populations of glycogen synthase I (glucose-6-P-independent) and D (glucose-6-P-dependent) (Huang, K. P., Cabib, E. (1974) J. Biol. Chem. 249, 3851-3857). Extracts from a glycogen-deficient mutant strain, 22R1 (glc7), yielded only the D form of glycogen synthase. Glycogen synthase D purified from either wild-type yeast or from this glycogen-deficient mutant displayed two polypeptides with molecular masses of 76 and 83 kDa on sodium dodecyl sulfate-gel electrophoresis in a protein ratio of about 4:1. Phosphate analysis showed that glycogen synthase D from either strain of yeast contained approximately 3 phosphates/subunit. The 76- and 83-kDa bands of the mutant strain copurified through a variety of procedures including nondenaturing gel electrophoresis. These two polypeptides showed immunological cross-reactivity and similar peptide maps indicating that they are structurally related. The relative amounts of these two forms remained constant during purification and storage of the enzyme and after treatment with cAMP-dependent protein kinase or with protein phosphatases. The two polypeptides were phosphorylated to similar extent in vitro by the catalytic subunit of mammalian cyclic AMP-dependent protein kinase. Phosphorylation of the enzyme in the presence of labeled ATP followed by tryptic digestion and reversed phase high performance liquid chromatography yielded two labeled peptides from each of the 76- and 83-kDa subunits. Treatment of wild-type yeast with Li+ increased the glycogen synthase activity, measured in the absence of glucose-6-P, by approximately 2-fold, whereas similar treatment of the glc7 mutant had no effect. The results of this study indicate that the GLC7 gene is involved in a pathway that regulates the phosphorylation state of glycogen synthase.     

Protein phosphatase 2Bw and protein phosphatase Z are Saccharomyces cerevisiae enzymes.

cDNAs encoding three protein phosphatases, termed PP2Bw (Da Cruz e Silva, E.F. and Cohen, P.T.W. (1989) Biochim. Biophys. Acta 1009, 293-296), PPZ1 and PPZ2 that have been isolated from a Clontech 'rabbit brain' library are shown to be Saccharomyces cerevisiae clones. PPZ1 and PPZ2 are two novel yeast phosphatases showing 93% amino acid sequence identity to one another. PPZ1 shows approx. 60% sequence identity to S. cerevisiae or mammalian PP1 and approx. 40% identity to S. cerevisiae or mammalian PP2A. These and other observations suggest that the two isoforms of PPZ have functions distinct from those of PP1.     

Regulation of the cell cycle by protein phosphatase 2A in Saccharomyces cerevisiae.

Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G(2)/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G(1)/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic.     

Identification and characterization of a novel RanGTP-binding protein in the yeast Saccharomyces cerevisiae

The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1,2 ) and nuclear envelope formation (3,4 ) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle.     

The Saccharomyces cerevisiae type 2A protein phosphatase Pph22p is biochemically different from mammalian PP2A.

The Saccharomyces cerevisiae type 2A protein phosphatase (PP2A) Pph22p differs from the catalytic subunits of PP2A (PP2Ac) present in mammals, plants and Schizosaccharomyces pombe by a unique N-terminal extension of approximately 70 amino acids. We have overexpressed S. cerevisiae Pph22p and its N-terminal deletion mutant Delta N-Pph22p in the GS115 strain of Pichia pastoris and purified these enzymes to apparent homogeneity. Similar to other heterologous systems used to overexpress PP2Ac, a low yield of an active enzyme was obtained. The recombinant enzymes designed with an 8 x His-tag at their N-terminus were purified by ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on Ni2+-nitrilotriacetic acid agarose. Comparison of biochemical properties of purified Pph22p and Delta N-Pph22p with purified human 8 x His PP2Ac identified similarities and differences between these two enzymes. Both enzymes displayed similar specific activities with 32P-labelled phosphorylase a as substrate. Furthermore, selected inhibitors and metal ions affected their activities to the same extend. In contrast to the mammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not Delta N-Pph22p, interacted strongly with protamine. Also with regard to the effects of protamine and polylysine on phosphatase activity Pph22p, but not Delta N-Pph22p, behaved similarly to the PP2Ac-PR65 dimer, indicating a regulatory role for the N-terminal extension of Pph22p. The N-terminal extension appears also responsible for interactions with phospholipids. Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents. These properties make the S. cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far.     

Crystallization and preliminary X-ray investigation of phosphoribosylaminoimidazolesuccinocarboxamide synthase from the yeast Saccharomyces cerevisiae.

Crystals of phosphoribosylaminoimidazolesuccinocarboxamide synthase (EC 6.3.2.6) from the yeast Saccharomyces cerevisiae were grown by the vapor diffusion hanging-drop technique, using ammonium sulfate as the precipitant. The crystals had dimensions up to 1.2 mm. X-ray diffraction experiments indicated a space group of P2(1)2(1)2(1) and unit cell parameters of a = 62.3 A, b = 63.5 A and c = 80.9 A, with one molecule in the asymmetric unit. Native data have been collected to 2.5 A resolution.     

Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme.

The yeast glycogen branching enzyme (EC 2.4.1.18) is shown to be induced in batch culture simultaneously with the onset of intracellular glycogen accumulation. The branching enzyme structural gene (GLC3) has been cloned. Its predicted amino acid sequence is very similar to procaryotic branching enzymes. Northern analysis indicates that GLC3 mRNA abundance increases in late exponential growth phase coincident with glycogen accumulation. Disruption of the branching enzyme structural gene establishes that branching enzyme activity is an absolute requirement for maximal glycogen synthesis.     

Identification of A1 protein as the fourth member of 13 kDa-type acidic ribosomal protein family in yeast Saccharomyces cerevisiae

The identity of protein A1 predicted by a cDNA clone from yeast Saccharomyces cerevisiae which has common carboxyl-terminus to 13 kDa-type acidic ribosomal proteins has been examined. The unique gene for A1 was isolated using the cDNA clone and found to possess two boxes similar to upstream activation sequences for ribosomal protein genes (UASrpg) in the 5'-flanking region. The in vitro-translation product directed by hybrid-selected mRNA with A1 cDNA comigrated with a minor component of split proteins from ribosome by electrofocusing. In addition, the mRNA level for A1 was found to be lower than other two major acidic ribosomal proteins suggesting that A1 is the fourth member of the protein family so far identified which is expressed at relatively low level.     

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.     

Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle

Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This enzyme is a collection of varied heterotrimeric complexes, each composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit. To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of the PP2A subunits of Saccharomyces cerevisiae. We found the following: the level of each subunit remained constant throughout the cell cycle; there is at least 10 times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding. Using green fluorescent protein-tagged forms of each subunit, we monitored the sites of significant accumulation of each protein throughout the cell cycle. The two regulatory subunits displayed distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck, and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Although Rts1p and Tpd3p required heterotrimer formation to achieve normal localization, Cdc55p achieved its normal localization in the absence of either an A or C subunit.     

Phosphorylated fructose-1,6-bisphosphatase dephosphorylating protein phosphatase from Saccharomyces cerevisiae

Phosphorylation of fructose-1,6-bisphosphatase with cyclic AMP-dependent protein kinase from yeast is accompanied by a 50% decrease in the catalytic activity (Pohlig, G. and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Using reactivation of phoshorylated fructose-1,6-bisphosphatase as assay, a protein phosphatase was about 2,000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Upon incubation with phosphorylated fructose-1,6-bisphosphatase the purified protein phosphatase not only reverses the 50% inactivation caused by phosphorylation, but also the previously observed change in the pH optimum and in the ratio of activity with Mg2+ or Mn2+. The phosphatase is strongly inhibited by heparin and fluoride. L-Carnitine, orthophosphate, pyrophosphate, and succinate inhibit to 50% at concentrations from 1 to 10 mM. The molecular mass of the native phosphatase was found to be 180,000 Da. Sodium dodecyl sulfate-gel electrophoresis suggested four subunits with a molecular mass of 45,000 Da each. Half-maximal activity was observed with 5 mM Mg2+ or Mn2+, the pH optimum of activity was found at pH 7. Using polyclonal antibodies, disappearance of 32P-labeled fructose-1,6-bisphosphatase and concomitant liberation of the expected amount of inorganic [32P] phosphate was demonstrated.     

A particulate form of alkaline phosphatase in the yeast,Saccharomyces cerevisiae

A new form of alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) has been identified in the yeast Saccharomyces cerevisiae. Utilizing either synthetic or natural substrates, the enzyme exhibited a broad pH activity curve with maximum activity between 8.5 and 9.0. The enzyme was nonspecific with respect to substrate, attacking a variety of compounds containing phosphomonoester linkages, but has no detectable activity against polyphosphate, pyrophosphate or phosphodiester linkages. The enzyme exhibited an apparent K_m of 0.25 mM with respect to p-nitrophenyl phosphate, 0.38 mM with respect to α-naphthyl phosphate, and 1.0 mM with respect to 5′ AMP. The enzyme is regulated in a constitutive manner and its activity does not increase during phosphate starvation or sporulation, as does the repressible alkaline phosphatase. The enzyme is tightly bound to a particulate fraction of the cell, tentatively identified as the tonoplast membrane. It is not solubilized by treatment with high concentrations of NaCl, KH₂PO₄ or chaotropic agents. Triton X-100 (0.1%) solubilizes 12% of the particulate activity. This enzyme is differentiated from the other alkaline phosphatases found in yeast by its chromatographic elution from DEAE-cellulose, kinetic parameters, heat stability and pH stability, as well as its particulate nature. This particulate alkaline phosphatase was found in every strain examined. It has a significantly lower specific activity in the phoH mutant and a higher activity in the acid phosphatase constitutive mutant A137.