Characterization of the atypical Ppz/Hal3 phosphatase system from the pathogenic fungus Cryptococcus neoformans

Ppz Ser/Thr protein phosphatases (PPases) are found only in fungi and have been proposed as potential antifungal targets. In Saccharomyces cerevisiae Ppz1 (ScPpz1) is involved in regulation of monovalent cation homeostasis. ScPpz1 is inhibited by two regulatory proteins, Hal3 and Vhs3, which have moonlighting properties, contributing to the formation of an unusual heterotrimeric PPC decarboxylase (PPCDC) complex crucial for CoA biosynthesis. Here we report the functional characterization of CnPpz1 (CNAG_03673) and two possible Hal3‐like proteins, CnHal3a (CNAG_00909) and CnHal3b (CNAG_07348) from the pathogenic fungus Cryptococcus neoformans. Deletion of CnPpz1 or CnHal3b led to phenotypes unrelated to those observed in the equivalent S. cerevisiae mutants, and the CnHal3b‐deficient strain was less virulent. CnPpz1 is a functional PPase and partially replaced endogenous ScPpz1. Both CnHal3a and CnHal3b interact with ScPpz1 and CnPpz1 in vitro but do not inhibit their phosphatase activity. Consistently, when expressed in S. cerevisiae, they poorly reproduced the Ppz1‐regulatory properties of ScHal3. In contrast, both proteins were functional monogenic PPCDCs. The CnHal3b isoform was crystallized and, for the first time, the 3D‐structure of a fungal PPCDC elucidated. Therefore, our work provides the foundations for understanding the regulation and functional role of the Ppz1‐Hal3 system in this important pathogenic fungus.     

Functional mapping of the disparate activities of the yeast moonlighting protein Hal3

The Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric PPCDC (phosphopantothenoylcysteine decarboxylase), the third enzyme of the CoA biosynthetic pathway. In the present study we initiated the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core [PD (PPCDC domain)] is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 NtD (N-terminal domain) is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3's heteromeric PPCDC activity. Both the NtD and the acidic CtD (C-terminal domain) also appear to be important for Hal3's Ppz1 regulatory function, although our results indicate that the CtD fulfils the key role in this regard. Finally, we show that the introduction of two key asparagine and cysteine residues, essential for monofunctional PPCDC activity but absent in Hal3, is not sufficient to convert it into such a homomeric PPCDC, and that additional modifications of Hal3's PD aimed at increasing its resemblance to known PPCDCs also fails to introduce this activity. This suggests that Hal3 has undergone significant evolutionary drift from ancestral PPCDC proteins. Taken together, our work highlights specific structural determinants that could be exploited for full understanding of Hal3's cellular functions.     

Functional characterization of the yeast Ppz1 phosphatase inhibitory subunit Hal3: a mutagenesis study

Saccharomyces cerevisiae Hal3 is a conserved protein that binds the carboxyl-terminal catalytic domain of the PP1c (protein phosphatase 1)-related phosphatase Ppz1 and potently inhibits its activity, thus modulating all of the characterized functions so far of the phosphatase. It is unknown how Hal3 binds to Ppz1 and inhibits its activity. Although it contains a putative protein phosphatase 1c binding-like sequence (263KLHVLF268), mutagenesis analysis suggests that this motif is not required for Ppz1 binding and inhibition. The mutation of the conserved His378 (possibly involved in dehydrogenase catalytic activity) did not impair Hal3 functions or Ppz1 binding. Random mutagenesis of the 228 residue-conserved central region of Hal3 followed by a loss-of-function screen allowed the identification of nine residues important for Ppz1-related Hal3 functions. Seven of these residues cluster in a relatively small region spanning from amino acid 446 to 480. Several mutations affected Ppz1 binding and inhibition in vitro, whereas changes in Glu460 and Val462 did not alter binding but resulted in Hal3 versions unable to inhibit the phosphatase. Therefore, there are independent Hal3 structural elements required for Ppz1 binding and inhibition. S. cerevisiae encodes a protein (Vhs3) structurally related to Hal3. Recent evidence suggests that both mutations are synthetically lethal. Surprisingly, versions of Hal3 carrying mutations that strongly affected Ppz1 binding or inhibitory capacity were able to complement lethality. In contrast, the mutation of His378 did not. This finding suggests that Hal3 may have both Ppz1-dependent and independent functions involving different structural elements.     

Functional characterization of the Saccharomyces cerevisiae VHS3 gene: a regulatory subunit of the Ppz1 protein phosphatase with novel, phosphatase-unrelated functions

The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G(1)/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His(459), equivalent to the supposedly functionally relevant His(90) in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions.     

Protein complex Ppz1p/Hal3p and the efficiency of nonsense suppression in yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

It is known that the efficiency of nonsense suppression in yeasts is controlled both genetically and epigenetically. Since many components of translation machinery are represented by phosphoproteins, the efficiency depends, in particular, on the activity of kinases and phosphatases that include the Ppz1p/Hal3p complex. It contains Ppz1p phosphatase, which is a catalytic subunit, and Hal3p that negatively regulates its function. The aim of this work was to study the mechanisms which relate the activity of Ppz1p/Hal3p complex to nonsense suppression efficiency. In this study, we used a genetic approach implicating the analysis of nonsense suppression phenotype of the strains overexpressing HAL3 or PPZ1 genes and also bearing deletions or mutant alleles of genes, which presumably could participate in the manifestation of these overexpressions. We have shown that Hal3p inhibits not only Ppz1p but also the homologous phosphatase Ppz2p. Our data indicate that Ppz2p is also involved in the control of nonsense suppression efficiency. In the course of search for Ppz1p target protein, it was shown that Ppz1p dephosphorylates at least two proteins involved in translation. Moreover, Ppz1p affects the efficiency of nonsense suppression not only due to its phosphatase activity but also due to another mechanism triggered by its interaction with Hsp70 chaperones.     

Centromere localization and function of Mis18 requires Yippee‐like domain‐mediated oligomerization

Mis18 is a key regulator responsible for the centromere localization of the CENP‐A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP‐A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N‐terminal Yippee‐like domain, is crucial for its centromere localization and function. The crystal structure of the N‐terminal Yippee‐like domain reveals a fold containing a cradle‐shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N‐terminal Yippee‐like domain forms a homodimer in vitro and in vivo, full‐length Mis18, including the C‐terminal α‐helical domain, forms a homotetramer in vitro. We also show that the Yippee‐like domains of human Mis18α/Mis18β interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation.     

Cloning by functional complementation, and inactivation, of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^– mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.     

Human and Mouse Homologs of theSchizosaccharomyces pombe rad17+Cell Cycle Checkpoint Control Gene

TheSchizosaccharomyces pombe rad17⁺cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human (RAD17Sp) and mouse (mRAD17Sp) homologs of theS. pombeRad17 (Rad17Sp) protein. The humanRAD17Spopen reading frame (ORF) encodes a protein of 681 amino acids; themRAD17SpORF codes for a protein of 688 amino acids. ThemRAD17Spmessenger is highly expressed in the testis as a single 3-kb mRNA species. The human RAD17Sp and mRAD17Sp proteins are 24% identical and 46% similar to theS.pombeRad17Sp protein. Sequence homology was also noted with theSaccharomyces cerevisiaeRad24Sc (which is the structural counterpart ofS.pombeRad17Sp) and structurally related polypeptides fromCaenorhabditis elegans, Arabidopsis thaliana, Pyrococcus horikoshii,andDrosophila melanogaster.The degree of conservation between the mammalian RAD17Sp proteins and those of the other species is consistent with the evolutionary distance between the species, indicating that these proteins are most likely true counterparts. In addition, homology was found between the Rad17Sp homologs and proteins identified as components of mammalian replication factor C (RF-C)/activator 1, especially in several highly conserved RF-C-like domains including a “Walker A” motif. Using FISH and analysis of a panel of rodent–human cell hybrids, the humanRAD17Spgene (HGMW-approved symbolRAD17could be localized on human chromosome 5q13–q14, a region implicated in the etiology of small cell lung carcinoma, non-small-cell lung carcinoma, duodenal adenocarcinoma, and head and neck squamous cell carcinoma. Our results suggest that the structure and function of the checkpoint “rad” genes in the G2/M checkpoint pathway are evolutionary conserved between yeast and higher eukaryotes.     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Cloning by functional complementation,and inactivation,of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^? mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

The Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe

SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro. To investigate the in vivo function of SpCCE1 we made an Spcce1::ura4 ⁺ insertion mutant strain. This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination. In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S. pombe. In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation. A similar function can probably be attributed to SpCCE1 in S. pombe, since the majority of mitochondrial DNA from the Spcce1::ura4 ⁺ strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions. Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4 ⁺ strain. Possible explanations are discussed. Received: 28 October 1999 / Accepted: 28 March 2000     

Isolation of the ribosomal protein gene S6 from Schizosaccharomyces pombe

Summary We screened a Schizosaccharomyces pombe genomic library using the ribosomal protein gene SI0 from Saccharomyces cerevisiae as a probe. Hybrid-selected translation of the positive clones revealed a ribosomal protein of S. pombe which is probably equivalent to the ribosomal protein SI0 from S. cerevisiae.     

The protein complex Ppz1p/Hal3p and nonsense suppression efficiency in the yeast Saccharomyces cerevisiae

It is known that nonsense suppression efficiency in yeast is controlled both genetically and epigenetically. As many components of translation machinery are represented by phosphoproteins, it depends, in particular, on the activity of kinases and phosphatases. The Ppz1p/Hal3p complex is among them. In this complex, the Ppz1p phosphatase is a catalytic subunit and Hal3p negatively regulates its function. The aim of this work was to study mechanisms which relate the activity of Ppz1p/Hal3p complex to nonsense suppression efficiency. In this study we used a genetic approach consisting of the analysis of nonsense suppression phenotype of strains over-expressing HAL3 or PPZ1 genes and also bearing deletions or mutant alleles of genes which presumably could participate in the manifestation of these over-expressions. We have shown that Hal3p inhibits not only Ppz1p, but also the homologous phosphatase Ppz2p. Our data indicate that Ppz2p is also involved in the control of nonsense suppression efficiency. In the course of search for Ppz1p target protein, it was shown that Ppz1p dephosphorylates at least two proteins participating in translation. Moreover, Ppz1p affects nonsense suppression efficiency not only due to its phosphatase activity but also due to another mechanism triggered by its interaction with Hsp70 chaperones.     

Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast

Summary Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation. The level of cAMP inS. pombe cells changed during the transition from exponential growth to stationary phase. It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium. A decrease of approximately 50% was observed in either case, suggesting thatS. pombe cells contain less cAMP when they initiate sexual development.S. pombe cells that expressed the catalytic domain ofSaccharomyces cerevisiae adenylyl cyclase from theS. pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis. These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development inS. pombe. Thepde1 gene, which encodes a protein homologous toS. cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level. Disruption ofpde1 madeS. pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo.     

The <Emphasis Type="Italic">dds20</Emphasis><Superscript>+</Superscript> Gene Controls a Novel Rad51<Superscript>Sp</Superscript>-Dependent Pathway of Recombinational Repair in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Repair of DNA double-strand break (DSB) is an evolutionary conserved Rad51-mediated mechanism. In yeasts, Rad51 paralogs, Saccharomyces cerevisiae Rad55-Rad57 and Schizosaccharomyces pombe Rhp55-Rhp57 are mediators of the nucleoprotein Rad51 filament formation. As shown in this work, a novel Rad51^Sp-dependent pathway of DSB repair acts in S. pombe parallel to the pathway mediated by Rad51 paralogs. A new gene dds20 ⁺ that controls this pathway was identified. The overexpression of dds20 ⁺ partially suppresses defects of mutant rhp55Δ in DNA repair. Cells of dds20Δ manifest hypersensitivity to a variety of genotoxins. Epistatic analysis revealed that dds20 ⁺ is a gene of the recombinational repair group. The role of Dds20 in repair of spontaneous damages occurring in the process of replication and mating-type switching remains unclear. The results obtained suggest that Dds20 has functions beyond the mitotic S phase. The Dds20 protein physically interacts with Rhp51(Rad51^Sp). Dds20 is assumed to operate at early recombinational stages and to play a specific role in the Rad51 protein filament assembly differing from that of Rad51 paralogs.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 736–745.Original Russian Text Copyright © 2005 by Salakhova, Savchenko, Khasanov, Chepurnaya, Korolev, Bashkirov.     

Fission Yeast Sec3 Bridges the Exocyst Complex to the Actin Cytoskeleton

The exocyst complex tethers post‐Golgi secretory vesicles to the plasma membrane prior to docking and fusion. In this study, we identify Sec3, the missing component of the Schizosaccharomyces pombe exocyst complex (SpSec3). SpSec3 shares many properties with its orthologs, and its mutants are rescued by human Sec3/EXOC1. Although involved in exocytosis, SpSec3 does not appear to mark the site of exocyst complex assembly at the plasma membrane. It does, however, mark the sites of actin cytoskeleton recruitment and controls the organization of all three yeast actin structures: the actin cables, endocytic actin patches and actomyosin ring. Specifically, SpSec3 physically interacts with For3 and sec3 mutants have no actin cables as a result of a failure to polarize this nucleating formin. SpSec3 also interacts with actin patch components and sec3 mutants have depolarized actin patches of reduced endocytic capacity. Finally, the constriction and disassembly of the cytokinetic actomyosin ring is compromised in these sec3 mutant cells. We propose that a role of SpSec3 is to spatially couple actin machineries and their independently polarized regulators. As a consequence of its dual role in secretion and actin organization, Sec3 appears as a major co‐ordinator of cell morphology in fission yeast .     

Conserved and unique features of the fission yeast core Atg1 complex

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.