Asp1, a Conserved 1/3 Inositol Polyphosphate Kinase,Regulates the Dimorphic Switch in Schizosaccharomyces pombe

The ability to undergo dramatic morphological changes in response to extrinsic cues is conserved in fungi. We have used the model yeast Schizosaccharomyces pombe to determine which intracellular signal regulates the dimorphic switch from the single-cell yeast form to the filamentous invasive growth form. The S. pombe Asp1 protein, a member of the conserved Vip1 1/3 inositol polyphosphate kinase family, is a key regulator of the morphological switch via the cAMP protein kinase A (PKA) pathway. Lack of a functional Asp1 kinase domain abolishes invasive growth which is monopolar, while an increase in Asp1-generated inositol pyrophosphates (PP) increases the cellular response. Remarkably, the Asp1 kinase activity encoded by the N-terminal part of the protein is regulated negatively by the C-terminal domain of Asp1, which has homology to acid histidine phosphatases. Thus, the fine tuning of the cellular response to environmental cues is modulated by the same protein. As the Saccharomyces cerevisiae Asp1 ortholog is also required for the dimorphic switch in this yeast, we propose that Vip1 family members have a general role in regulating fungal dimorphism.Eucaryotic cells are able to define and maintain a particular cellular organization and thus cellular morphology by executing programs modulated by internal and external signals. For example, signals generated within a cell are required for the selection of the growth zone after cytokinesis in the fission yeast Schizosaccharomyces pombe or the emergence of the bud in Saccharomyces cerevisiae (37, 44, 81). Cellular morphogenesis is also subject to regulation by a wide variety of external signals, such as growth factors, temperature, hormones, nutrient limitation, and cell-cell or cell-substrate contact (13, 34, 66, 75, 81). Both types of signals will lead to the selection of growth zones accompanied by the reorganization of the cytoskeleton.The ability to alter the growth form in response to environmental conditions is an important virulence-associated trait of pathogenic fungi which helps the pathogen to spread in and survive the host''s defense system (7, 32). Alteration of the growth form in response to extrinsic signals is not limited to pathogenic fungi but is also found in the model yeasts S. cerevisiae and S. pombe, in which it appears to represent a foraging response (1, 24).The regulation of polarized growth and the definition of growth zones have been studied extensively with the fission yeast S. pombe. In this cylindrically shaped organism, cell wall biosynthesis is restricted to one or both cell ends in a cell cycle-regulated manner and to the septum during cytokinesis (38). This mode of growth requires the actin cytoskeleton to direct growth and the microtubule cytoskeleton to define the growth sites (60). In interphase cells, microtubules are organized in antiparallel bundles that are aligned along the long axis of the cell and grow from their plus ends toward the cell tips. Upon contact with the cell end, microtubule growth will first pause and then undergo a catastrophic event and microtubule shrinkage (21). This dynamic behavior of the microtubule plus end is regulated by a disparate, conserved, microtubule plus end group of proteins, called the +TIPs. The +TIP complex containing the EB1 family member Mal3 is required for the delivery of the Tea1-Tea4 complex to the cell tip (6, 11, 27, 45, 77). The latter complex docks at the cell end and recruits proteins required for actin nucleation (46, 76). Thus, the intricate cross talk between the actin and the microtubule cytoskeleton at specific intracellular locations is necessary for cell cycle-dependent polarized growth of the fission yeast cell.The intense analysis of polarized growth control in single-celled S. pombe makes this yeast an attractive organism for the identification of key regulatory components of the dimorphic switch. S. pombe multicellular invasive growth has been observed for specific strains under specific conditions, such as nitrogen and ammonium limitation and the presence of excess iron (1, 19, 50, 61).Here, we have identified an evolutionarily conserved key regulator of the S. pombe dimorphic switch, the Asp1 protein. Asp1 belongs to the highly conserved family of Vip1 1/3 inositol polyphosphate kinases, which is one of two families that can generate inositol pyrophosphates (PP) (17, 23, 42, 54). The inositol polyphosphate kinase IP6K family, of which the S. cerevisiae Kcs1 protein is a member, is the “classical” family that can phosphorylate inositol hexakisphosphate (IP₆) (70, 71). These enzymes generate a specific PP-IP₅ (IP₇), which has the pyrophosphate at position 5 of the inositol ring (20, 54). The Vip1 family kinase activity was unmasked in an S. cerevisiae strain with KCS1 and DDP1 deleted (54, 83). The latter gene encodes a nudix hydrolase (14, 68). The mammalian and S. cerevisiae Vip1 proteins phosphorylate the 1/3 position of the inositol ring, generating 1/3 diphosphoinositol pentakisphosphate (42). Both enzyme families collaborate to generate IP₈ (17, 23, 42, 54, 57).Two modes of action have been described for the high-energy moiety containing inositol pyrophosphates. First, these molecules can phosphorylate proteins by a nonenzymatic transfer of a phosphate group to specific prephosphorylated serine residues (2, 8, 69). Second, inositol pyrophosphates can regulate protein function by reversible binding to the S. cerevisiae Pho80-Pho85-Pho81 complex (39, 40). This cyclin-cyclin-dependent kinase complex is inactivated by inositol pyrophosphates generated by Vip1 when cells are starved of inorganic phosphate (39, 41, 42).Regulation of phosphate metabolism in S. cerevisiae is one of the few roles specifically attributed to a Vip1 kinase. Further information about the cellular function of this family came from the identification of the S. pombe Vip1 family member Asp1 as a regulator of the actin nucleator Arp2/3 complex (22). The 106-kDa Asp1 cytoplasmic protein, which probably exists as a dimer in vivo, acts as a multicopy suppressor of arp3-c1 mutants (22). Loss of Asp1 results in abnormal cell morphology, defects in polarized growth, and aberrant cortical actin cytoskeleton organization (22).The Vip1 family proteins have a dual domain structure which consists of an N-terminal “rimK”/ATP-grasp superfamily domain found in certain inositol signaling kinases and a C-terminal part with homology to histidine acid phosphatases present in phytase enzymes (28, 53, 54). The N-terminal domain is required and sufficient for Vip1 family kinase activity, and an Asp1 variant with a mutation in a catalytic residue of the kinase domain is unable to suppress mutants of the Arp2/3 complex (17, 23, 54). To date, no function has been described for the C-terminal phosphatase domain, and this domain appears to be catalytically inactive (17, 23, 54).Here we describe a new and conserved role for Vip1 kinases in regulating the dimorphic switch in yeasts. Asp1 kinase activity is essential for cell-cell and cell-substrate adhesion and the ability of S. pombe cells to grow invasively. Interestingly, Asp1 kinase activity is counteracted by the putative phosphatase domain of this protein, a finding that allows us to describe for the first time a function for the C-terminal part of Vip1 proteins.     

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ΔBbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.Mycoinsecticides are considered promising biological control agents and alternatives or supplements to chemical pesticides (15). However, the dearth of physiological, genetic, and molecular knowledge of entomopathogenic fungi has retarded their widespread application.For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13, 14, 18, 20, 29, 49, 50, 51, 52, 53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle. M. anisopliae and B. bassiana produce hydrophobic spores which contact and adhere to the insect cuticle (12). Once attached, the conidium germinates and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via cuticle-degrading enzymes (11, 19, 46) as well as mechanical pressure (24, 53). Hyphae proliferate within the hemocoel, emerge from inside the insect, and subsequently conidiate on the cadaver (15). However, much remains to be elucidated regarding the mechanisms of insect fungal pathogenesis.To obtain detailed knowledge of the mechanisms of fungal pathogenesis, a pool of B. bassiana transfer DNA (T-DNA) insertional mutants had been generated through an Agrobacterium-mediated-transformation method (21). A mutant, designated T12, characterized by the presence of more conidia, was isolated, and its flanking sequence was obtained by T-DNA tagging. The flanking fragment contained an open reading frame (ORF), which corresponded to a gene termed JEN1, encoding a transporter of carboxylates (http://www.ncbi.nlm.nih.gov/Blast.cgi). Organic acid transportation is important for the metabolism of almost all cells of multicellular organisms and unicellular microorganisms (17, 25, 26). Transport across the plasma membrane is the first step in the metabolism of these substrates, which may affect many aspects of the organism, including regulation of energy metabolism (9, 34) and acid-base equilibrium status (10).JEN1p has been identified in several fungal species, e.g., Saccharomyces cerevisiae, Candida albicans, and Kluyveromyces lactis (9, 35, 45), which is a lactate/pyruvate symporter (1, 9, 34). The enzyme imports lactate or some short-chain monocarboxylates across the plasma membrane into cells. Then, the lactate is stereo-specifically oxidized to pyruvate. This reaction is performed by ferricytochrome c oxidoreductase in mitochondria (23, 33) and is tightly connected to the respiratory chain (34). JEN1 was induced by lactic, pyruvic, acetic, and propionic acids and repressed by glucose (2, 9, 35, 45). Nevertheless, for entomopathogenic fungi, the characterization of JEN1p has not been investigated, and its role in infection is still a mystery.For this paper, we studied the functions of a putative carboxylate transport gene, JEN1, in B. bassiana (BbJEN1). Our results demonstrated that BbJEN1 is involved in conidiation of B. bassiana and that the gene is a new factor influencing virulence in entomopathogenic fungi.     

Neurofibromin Homologs Ira1 and Ira2 Affect Glycerophosphoinositol Production and Transport in Saccharomyces cerevisiae

Saccharomyces cerevisiae produces extracellular glycerophosphoinositol through phospholipase-mediated turnover of phosphatidylinositol and transports glycerophosphoinositol into the cell upon nutrient limitation. A screening identified the RAS GTPase-activating proteins Ira1 and Ira2 as required for utilization of glycerophosphoinositol as the sole phosphate source, but the RAS/cyclic AMP pathway does not appear to be involved in the growth phenotype. Ira1 and Ira2 affect both the production and transport of glycerophosphoinositol.Membrane phospholipids are continually synthesized and degraded as cells grow and respond to environmental conditions. A major pathway of phosphatidylinositol (PI) turnover in Saccharomyces cerevisiae is its deacylation to produce extracellular glycerophosphoinositol (GroPIns) (3). Plb3, an enzyme with phospholipase B (PLB)/lysophospholipase activity, is thought to be primarily responsible for the production of extracellular GroPIns, with Plb1 playing a lesser role (11, 12, 13). GroPIns is transported into the cell by the Git1 permease (17). GIT1 expression is upregulated by phosphate limitation and inositol limitation. In fact, GroPIns can act as the cell''s sole source of both inositol (17) and phosphate (1).A screening for gene products involved in the process by which GroPIns enters the cellular metabolism identified Ira1 and Ira2, yeast homologs of the mammalian protein neurofibromin. Alterations in NF1, the gene encoding neurofibromin, are associated with the pathogenesis of neurofibromatosis type 1, an autosomal dominant genetic disease (4, 5, 25). Ira1 and Ira2 and neurofibromin function as RAS GTPase-activating proteins (RAS GAPs). S. cerevisiae Ras1 and Ras2 activate adenylate cyclase to modulate cyclic AMP (cAMP) levels. The binding of cAMP to the regulatory subunits of protein kinase A (Bcy1) results in dissociation and activation of the catalytic subunits (Tpk1 to Tpk3). Ira1 and Ira2 inactivate RAS and thereby downregulate the pathway (18, 19). Hydrolysis of cAMP by the phosphodiesterases encoded by PDE1 and PDE2 also downregulate the pathway (7, 20, 23). The RAS/cAMP pathway responds to nutrient signals to modulate fundamental cellular processes, including stress resistance, metabolism, and cell proliferation (7, 20, 21).     

Roles of Hop1 and Mek1 in Meiotic Chromosome Pairing and Recombination Partner Choice in Schizosaccharomyces pombe

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.Sexual reproduction in eukaryotes involves formation of haploid gametes from diploid cells by one round of DNA replication, pairing of the homologous chromosomes, and recombination and then by the two meiotic divisions (53). In fungi the gametes differentiate into haploid spores, which germinate to form vegetative cells. Crossover (CO) formation between homologous chromosomes and DNA repair processes between sister chromatids are required for spore viability (10, 55, 58).In vegetative cells homologous recombination (HR) is important for repair of DNA damage and stalled replication forks, with the sister chromatid as the preferred partner (28). Many of the enzymes involved in mitotic HR also contribute to meiotic recombination. In addition, meiosis-specific cytological structures and enzymes enhance recombination frequency (meiotic induction) and shift partner preference from sister chromatids to homologous chromosomes (3, 47, 64, 74). In detail the steps of HR vary between different types of sequence organization (allelic versus sister versus ectopic), between different types of DNA damage, between meiotic and mitotic cells, and between species (10, 55, 58).Meiotic recombination, including CO formation, is initiated by DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae and other eukaryotes, DSBs are formed by Spo11. Many cofactors are required (29). The Schizosaccharomyces pombe homolog is Rec12, also requiring auxiliary factors whose elimination leads to loss of meiotic DSB formation (12). The 5′ single-strand ends at DSBs are processed by nucleases. In S. cerevisiae the MRX complex made up by the proteins Rad50, Mre11, and Xrs2 is required for this resection, as well as for DSB formation. The corresponding MRN complex of S. pombe (Rad50, Rad32, and Nbs1) is not required for DSB formation but is essential for DSB repair (43, 72). Deletion of rad50, rad32, or ctp1 (homologous to SAE2/COM1 in S. cerevisiae and CtIP in humans) leads to very low spore viability. These proteins are also essential for DSB processing (23, 24, 32, 43, 60, 62).Free DNA 3′ ends at DSBs are recruited for invasion of a sister or homologous chromatid by the strand transfer proteins Rad51 and Dmc1, again involving many accessory proteins (16). This results in the central intermediates of HR: heteroduplex DNA consisting of single strands originating from different chromatids and Holliday junctions (HJs). In S. cerevisiae HJs form preferably between homologs with a two- to sixfold excess over intersister HJs (64). Surprisingly, meiotic HJs form with about a fourfold excess between sisters in S. pombe (11). Eventually the intermediates are resolved into crossover (CO) and noncrossover (NCO) events. COs show exchange of the flanking sequences of the two chromatids involved and usually carry a patch of conversion (unilateral transfer of DNA sequences from one chromatid to its interacting partner) near the DSB site. NCOs are conversion events without associated COs (22). In S. pombe loss of core HR functions leads to very low spore viability: deletion of rad51 but not of dmc1 (20), double mutation of rad54 and rdh54 (7), inactivation of the endonuclease activity encoded by mus81 and eme1 (5, 52), and combined deletion of rad22 and rti1 (homologs of RAD52 of S. cerevisiae). But, differently from the other core functions, Rad22 and Rti1 are not required for CO and NCO (50).Early in meiotic prophase of many eukaryotes, axial elements (called lateral elements in later stages) form along sister chromatids, and pairing of homologous chromosomes is initiated, leading to juxtaposition of the homologous chromosomes along their whole length in the synaptonemal complex (SC) (54). In S. pombe no SC is formed, but linear elements (LEs), resembling axial elements of other eukaryotes, are formed. LEs do not form continuously along the chromosomes (1) but load the proteins Rec10, Hop1, and Mek1 (36, 44, 57), which are homologs of, or at least related, to the S. cerevisiae proteins Red1, Hop1, and Mek1, respectively, localizing to axial/lateral elements (2, 67). Hop1 carries a HORMA domain, also present in proteins associating with axial elements and regulating the progress of recombination in higher eukaryotes: Arabidopsis thaliana (61), Caenorhabditis elegans (9, 41), and mammals (18).In S. cerevisiae localization of Hop1 and Mek1 (meiosis-specific protein kinase) to axial elements is dependent on Red1 (2, 67). Mutation of the three S. cerevisiae genes results in reduction of DSB formation, CO and conversion frequencies, and spore viability (26, 31, 59). Direct comparison of unequal sister chromatid recombination (USCR) frequencies in an assay excluding the scoring of intrachromatid recombination (ICR) revealed no increase in the hop1 null mutant but about fourfold increases in the red1 and mek1 null mutants (69). The S. cerevisiae Hop1, Red1, and Mek1 proteins are involved in biasing meiotic DSB repair to occur between homologous chromosomes rather than between sister chromatids (47). Activated Mek1 kinase is required for the inhibition of sister chromatid-mediated DSB repair by Rad51, when the DMC1 gene is deleted and the meiotic recombination checkpoint is activated (4, 27, 38, 47). For Mek1 activation, phosphorylation of Hop1 by the Mec1/Tel1 kinases is also required (6).Less is known about the S. pombe proteins. Hop1 of S. pombe was identified as a nonsignificant hit by sequence comparison with full-length S. cerevisiae Hop1 and contains an N-terminal HORMA domain and a central zinc finger motif like Hop1 in S. cerevisiae. In addition they share a short homology block toward the C terminus (36). The Mek1 protein of S. pombe shares 34% identity and 54% similarity with its S. cerevisiae counterpart along the whole sequence. It contains an FHA domain in the N-terminal part like the other members of its family of checkpoint kinases and is involved in regulation of the meiotic cell cycle (57). Hop1 and Mek1 are strongly expressed in meiosis but not expressed or only slightly expressed in vegetative cells (42, 57). In prophase both proteins localize to LEs as defined by colocalization with the LE component Rec10 (36). Deletion of the distant RED1 homolog rec10 abolishes LE formation (36, 44) and strongly reduces meiotic recombination (17, 70). Rec10, but not Hop1 and Mek1, is required for localization of Rec7 (a distant homolog of S. cerevisiae Rec114) to meiotic chromosomes (34). Rec7 and Rec10 are required for Rec12 activity (12, 29).Obtaining information on the functions of Hop1 and Mek1 in S. pombe was the aim of the work presented here, especially on their possible roles in homolog versus sister discrimination for DSB repair. Deletion mutants have been studied with respect to spore viability and the frequencies of CO and conversion. They have also been assessed for genetic recombination events between sister chromatids in the known PS1 assay (63) and the newly developed VL1 assay (for details, see Fig. ​Fig.3).3). Physical analysis of DSB formation and repair has been performed in meiotic time course experiments. It is proposed that S. pombe Hop1 and Mek1 are promoting interactions between homologous chromosomes rather than inhibiting interactions between sister chromatids.Open in a separate windowFIG. 3.PS1 and VL1 assay systems for intrachromosomal recombination. Strains with constructs carrying repeated DNA sequences have been assayed for prototroph formation either by intrachromatid recombination (ICR, yielding prototrophs only in PS1) or by unequal sister chromatid recombination (USCR, in PS1 and VL1). Crosses of the constructs were performed with strains carrying a deletion of the ade6 gene to exclude other homologous recombination events. (A) The PS1 assay involves copies of the ade6 gene inactivated by either the hot spot mutation M26 or the mutation 469. The repeated sequences are separated by the ura4⁺ marker (63). ICR (left) or USCR (right) between the repeated sequences can lead to formation of adenine prototrophs that have lost the ura4⁺ marker by crossover (CO) or single-strand annealing (SSA) events. Adenine prototrophs maintaining the ura4⁺ marker can derive from noncrossover (NCO) events. Both types of pairing may lead to CO or NCO products. (B) The newly constructed VL1 assay (see the supplemental material) involves different truncations of the ade6 gene separated by the hyg^R marker (also called hphMX6), conferring hygromycin resistance. The left truncation carries a 3′ portion of ade6; the right truncation carries a 5′ portion of ade6. While the gray parts of the truncations are not overlapping, the white sections of 500-bp length are of almost identical sequence, allowing for homologous pairing. CO and SSA products resulting from ICR retain only the central portion of ade6 and remain auxotrophic. Adenine prototrophic CO and NCO products resulting from USCR both retain hygromycin resistance. Note that NCO events may arise through loop formation of one sister chromatid and pairing with a single block (500 bp) of the repeated ade6 sequence (39).     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Protein Phosphatase Type 1-Interacting Protein Ysw1 Is Involved in Proper Septin Organization and Prospore Membrane Formation during Sporulation

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1Δ mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1Δ cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.Diploid cells of Saccharomyces cerevisiae subjected to nitrogen limitation in the presence of a nonfermentable carbon source undergo the developmental process of sporulation (14, 23, 35). Four nuclei produced by two rounds of nuclear division, meiosis I and II, are encapsulated by newly formed double-membrane structures, called prospore membranes, and are finally packaged into spores covered with layered spore walls (35).In this process, prospore membrane formation is one of the most dynamic events. Early in meiosis II, the cytoplasmic surface of the meiotic spindle pole body (SPB) is modified by the recruitment of sporulation-specific protein complex that acts as a site of vesicle recruitment (2, 22, 39). Post-Golgi secretory vesicles dock to the surface of the SPBs and fuse with each other, generating prospore membranes (33, 34). The prospore membranes then grow to engulf daughter nuclei through a series of stages that are categorized by the membranes'' appearance in the fluorescence microscope (12). Initially, the membranes appear as small horseshoes that enlarge to become small round membrane structures. The prospore membranes then extend into a tube-like shape, engulfing the nucleus, as well as some cytosol and organelles (12). After this extension, prospore membrane undergoes a rapid change to a mature round form. This rounding of the membrane is coordinated with membrane closure (12). Spore wall materials are then deposited into the luminal space created by closure of the prospore membrane (9).In addition to the meiotic plaque of the SPB, two protein complexes are associated with the prospore membrane as it forms. One is the leading edge protein complex, which exists at the lip of the prospore membranes and consists of three components: Ssp1, Ady3, and Don1 (27, 30, 38). Ssp1 is the most important of the three and is required for proper extension of the prospore membrane (30). The second complex is a sporulation-specific septin structure. The septins are a family of cytoskeletal proteins, which form filaments (18, 50). Septins are conserved from yeast to mammals. They were originally found and have been extensively studied in S. cerevisiae. In vegetatively growing S. cerevisiae cells, five septin proteins—Cdc3, Cdc10, Cdc11, Cdc12, and Shs1—form a ring at the bud neck that serves as a scaffold for many additional proteins, as well as a barrier to diffusion of proteins between the mother and the bud (19, 29, 50). In sporulating cells, the set of septin proteins is changed. Cdc3 and Cdc10, along with two sporulation-specific septins, Spr3 and Spr28, form a pair of parallel bars or sheets associated with each prospore membrane (11, 15, 29). Although deletion of sporulation-specific septins has only modest effects on sporulation (11, 15), their specific localization suggests that they have some function during prospore membrane formation. Septin organization in vegetatively growing cells is regulated by phosphorylation and dephosphorylation of septin components and septin-associated proteins (29). In sporulating cells, a sporulation-specific protein phosphatase type 1 (PP1) complex Gip1-Glc7 is required for the formation of septin structures (46), although whether this phosphatase acts directly on the septin proteins is unknown.The PP1 catalytic subunit is highly conserved in eukaryotes and is involved in a variety of cellular processes (8, 44). In S. cerevisiae it is encoded by an essential gene, GLC7, and functions in glycogen synthesis, glucose repression, chromosome segregation, cell wall organization, endocytosis, mating, and sporulation (3, 17, 24, 42, 44, 47, 53). The specificity of this enzyme is determined by targeting subunits. GIP1 was originally isolated in a two-hybrid screen by using GLC7 as a bait, and this interaction was confirmed by coimmunoprecipitation of the two proteins (48). GIP1 is a sporulation-specific gene required for sporulation. Further analysis revealed that Gip1 and Glc7 colocalize with septins during sporulation and are required for both septin organization and spore wall formation (46). The specific targets or cofactors of this PP1 complex are unknown.To elucidate the role of Gip1-Glc7 phosphatase, we screened for high-copy suppressors of a temperature-sensitive allele of gip1 and isolated YSW1. Ysw1 interacts with Gip1 and colocalizes with septins similar to Gip1. Furthermore, a ysw1Δ mutant displays aberrant septin structures and prospore membrane extension. These results suggest that Ysw1 may function with Gip1-Glc7 to regulate proper septin organization and prospore membrane formation.     

Roles of the Glyoxylate and Methylcitrate Cycles in Sexual Development and Virulence in the Cereal Pathogen Gibberella zeae

The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (ΔGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the ΔGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.During the infection process, pathogenic fungi usually encounter nutrient deprivation in the host before gaining access to sufficient nutrients for successful colonization of the living tissue. To cope with a nutrient-limited environment, fungal pathogens seem to rely mostly on fatty acid metabolism for both energy supply and biosynthesis of essential molecules (29). The ability of fungi to use fatty acids as a carbon source for growth is based on the glyoxylate cycle. Fungal pathogens have been proposed to employ the glyoxylate bypass for the use of acetyl coenzyme A (CoA) units produced by the β-oxidation of even-chain-length fatty acids, probably available from host cell membranes or the lipid reservoir inside the fungal spore (7, 12, 20, 27, 28, 41, 44, 46). Recent studies suggest that the glyoxylate pathway plays an important role in fungal virulence toward both plant and animal hosts (12, 20, 27, 44, 46). The key enzymes of the glyoxylate pathway, such as isocitrate lyase (ICL), which catalyzes the cleavage of isocitrate to glyoxylate and succinate, and malate synthase, which mediates the condensation of acetyl-CoA and glyoxylate into malate, are strongly induced within the host (16, 27, 41, 44). Moreover, disruption of genes encoding either of these enzymes causes severely reduced virulence of fungal phytopathogens, including Leptosphaeria maculans (20), Magnaporthe grisea (46), Stagonospora nodorum (44), and Colletotrichum lagenarium (2), and the animal pathogen Candida albicans (27). In contrast, these glyoxylate cycle enzymes have been known to be dispensable in invasive aspergillosis caused by Aspergillus fumigatus (38, 43).During fatty acid and amino acid catabolism by fungi, propionyl-CoA can be generated along with acetyl-CoA, particularly from the breakdown of odd-chain-length fatty acids or of the amino acids valine, isoleucine, and methionine (14). Therefore, fungal pathogens may need to use or remove propionyl-CoA during the infection process because it is toxic to fungi. In fungi, propionyl-CoA is metabolized via the methylcitrate cycle, in which propionyl-CoA is oxidized to pyruvate in four enzymatic steps (4, 5, 6, 19, 30, 31, 40, 49, 50). Recently, the importance of the methylcitrate cycle in fungal virulence was demonstrated in A. fumigatus: a mutant defective in methylcitrate synthase, the first enzyme of this cycle, displayed attenuated virulence in mice and insects (19, 31). However, the role of methylisocitrate lyase (MCL), which catalyzes the last reaction in the methylcitrate cycle (i.e., the cleavage of methylisocitrate into pyruvate and succinate) in fungal virulence, has not been determined, although deletion of the MCL gene inhibits hyphal growth and conidiation in Aspergillus nidulans (4). The protein sequences of several fungal MCLs show high similarity to fungal ICLs of the glyoxylate cycle (4, 30). In the pathogenic bacterium Mycobacterium tuberculosis, the methylcitrate cycle, only when working together with the glyoxylate cycle, is involved in virulence as well as fatty acid metabolism and intracellular growth (34, 35).Here, we focused on the roles of these two cycles during disease development caused by the devastating cereal pathogen Gibberella zeae (anamorph: Fusarium graminearum). G. zeae is a ubiquitously distributed ascomycete fungus that causes major disease in cereal crops such as corn, wheat, barley, and rice (33). Severe epidemics of these diseases result in serious economic consequences due to yield losses and contamination by fungal mycotoxins (32, 33). Wind-disseminated sexual spores (ascospores), which are produced in perithecia formed on plant debris, can infect plant spikes during anthesis (13, 39, 45). Detailed studies of the G. zeae infection process on wheat and barley heads have shown that fungal hyphae on the inner surfaces of the spike penetrate epicarp cells through pits or pores and grow into the caryopses through the pericarp (21). Thus, the glyoxylate cycle, either alone or in conjunction with the methylcitrate cycle, is likely employed by G. zeae during the infection process, as in other fungus-plant interactions (20, 46). G. zeae genome searches have identified orthologs of fungal ICL and MCL genes, designated GzICL1 and GzMCL1, respectively. Here, we performed functional analyses of these genes to provide new insight into their importance in lipid metabolism during the G. zeae infection process in host plants.     

African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali,Nigeria, Cameroon,and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.Mycobacterium bovis causes bovine tuberculosis (TB), an important disease of domesticated cattle that has a major economic and health impact throughout the world (61, 64, 65). The pathogen is a member of the Mycobacterium tuberculosis complex, which includes many species and subspecies that cause similar pathologies in a variety of mammalian hosts. The most notable member of the complex is M. tuberculosis, the most important bacterial pathogen of humans. In contrast to M. tuberculosis, which is largely host restricted to humans, M. bovis is primarily maintained in bovids, in particular, domesticated cattle, although the pathogen can frequently be recovered from other mammals, including humans (61). Bovine TB is found in cattle throughout the world and has been reported on every continent where cattle are farmed (3).Bovine TB has been reduced or eliminated from domestic cattle in many developed countries by the application of a test-and-cull policy that removes infected cattle (3, 8, 16, 17, 61, 64, 65). However, in Africa, although bovine TB is known to be common in both cattle and wildlife, control policies have not been enforced in many countries due to cost implications, lack of capacity, and infrastructure limitations (8, 16, 17, 57). In 1998, Cosivi et al. reported of bovine TB, “Of all nations in Africa, only seven apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease; the remaining 48 control the disease inadequately or not at all” (16). In the intervening years, the situation is not thought to have improved (8); however, preliminary surveys of bovine TB have been carried out in some African countries (4, 7, 12, 37, 44, 49, 53, 54, 56).The most common epidemiological molecular-typing method applied to strains of M. bovis is spoligotyping. This method identifies polymorphism in the presence of spacer units in the direct-repeat (DR) region in strains of the M. tuberculosis complex (36, 67). The DR is composed of multiple, virtually identical 36-bp regions interspersed with unique DNA spacer sequences of similar size (direct variant repeat [DVR] units). Spacer sequences are unique to the DR region, and copies are not located elsewhere in the chromosome (68). The DR region may contain over 60 DVR units; however, 43 of the spacer units were selected from the spacer sequences of the M. tuberculosis reference strain H37Rv and M. bovis BCG strain P3 and are used in the standard application of spoligotyping to strains of the M. tuberculosis complex (29, 36). The DR region is polymorphic because of the loss (deletion) of single or multiple spacers, and each spoligotype pattern from strains of M. bovis is given an identifier (http://www.Mbovis.org).Several studies of the DR regions in closely related strains of M. tuberculosis have concluded that the evolutionary trend for this region is primarily loss of single DVRs or multiple contiguous DVRs (22, 29, 68); duplication of DVR units or point mutations in spacer sequences were found to be rare. The loss of discrete units observed by Groenen et al. (29) led them to suggest that the mechanism for spacer loss was homologous recombination between repeat units. However, a study by Warren et al. (69) suggested that for strains of M. tuberculosis, insertion of IS6110 sequences into the DR region and recombination between adjacent IS6110 elements were more important mechanisms for the loss of spacer units.The population structure of the M. tuberculosis group of organisms is apparently highly clonal, without any transfer and recombination of chromosomal sequences between strains (15, 30, 60, 61). In a strictly clonal population, the loss by deletion of unique chromosomal DNA cannot be replaced by recombination from another strain, and the deleted region will act as a molecular marker for the strain and all its descendants. Deletions of specific chromosomal regions (regions of difference [RDs] or large sequence polymorphisms) have been very successful at identifying phylogenetic relationships in the M. tuberculosis complex (11, 25, 26, 35, 48, 50, 61, 62, 66). However, because the loss of spoligotype spacer sequences is so frequent, identical spoligotype patterns can occur independently in unrelated lineages (homoplasy), and therefore, the deletion of spoligotype spacers may be an unreliable indicator of phylogenetic relationship (61, 69).In samples of M. bovis strains from Cameroon, Nigeria, Chad, and Mali, spoligotyping was used to show that many of the strains had similar spoligotype patterns that lacked spacer 30, and it has been suggested that strains from these four countries are phylogenetically related (12, 18, 49, 53). We have extended the previous observations of spoligotype similarities between strains from these countries and confirmed the existence of a unique clonal complex of M. bovis, all descended from a single strain in which a specific deletion of chromosomal DNA occurred. We have named this clonal complex of M. bovis strains African 1 (Af1), and we show that this clonal complex is dominant in these four west-central African countries but rare in eastern and southern Africa. Extended genotyping, using variable-number tandem repeats (VNTR), of strains with the most common spoligotype patterns suggests that each of these four west-central African countries has a unique population structure. Evolutionary scenarios that may have led to the present day distribution of the Af1 clonal complex are discussed.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

TOR Complex 2 Controls Gene Silencing,Telomere Length Maintenance,and Survival under DNA-Damaging Conditions

The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of Δtor1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.The TOR protein kinase is a major cell growth regulator that links cellular growth with cell divisions (18, 42, 64, 65). TOR is an atypical protein kinase conserved from yeast to humans that was isolated as the target of the immunosuppressive and anticancer drug rapamycin (28). TOR proteins can be found in two distinct complexes, known as TORC1 and TORC2 (27, 64). These complexes mediate their distinct cellular functions via phosphorylation and activation of different sets of AGC-like kinases, including mammalian p70S6K, downstream of TORC1, and AKT/protein kinase B (PKB) downstream of TORC2 (18). TORC1 in mammals contains mTOR (Tor1 or Tor2 in Saccharomyces cerevisiae; Tor2 in Schizosaccharomyces pombe) and the Raptor protein (Kog1 in S. cerevisiae; Mip1 in S. pombe). TORC1 in many different eukaryotes plays a central role in the control of growth (mass accumulation) in response to external stimuli, particularly nutrient availability. Disruption of TORC1, either by mutating its components or by rapamycin treatment, can lead to a starvation-like phenotype (64). The cellular roles of TORC2, on the other hand, are less well defined. TORC2 in mammals contains mTOR (Tor2 in S. cerevisiae; Tor1 in S. pombe) together with Rictor (Avo3 in S. cerevisiae; Ste20 in S. pombe) and mSin1 (Avo1 in S. cerevisiae; Sin1 in S. pombe). TORC2 plays a role in regulating the actin cytoskeleton and cell wall integrity pathway in S. cerevisiae (3, 15, 27), a function that is at least partially conserved in human cells (17, 47).Fission yeast contains two TOR homologues, Tor1 and Tor2 (59), which form the TORC2 and TORC1 complexes, respectively (14, 32). Disruption tor2⁺ (TORC1) mimics nitrogen starvation responses (1, 14, 32, 56, 57, 62), while disruption of tor1⁺ (TORC2) results in pleiotropic defects, including elongated cells, sensitivity to osmotic and oxidative stress, inability to execute developmental processes in response to nutrient depletion, and a decrease in amino acid uptake (16, 22, 59). Tor1 regulates cell survival under stress conditions and starvation responses via the AGC protein kinase Gad8, a putative homologue of mammalian AKT/PKB (16).In budding yeast and mammalian cells, TORC1 mediates the rapamycin-sensitive signaling branch while TORC2 is far less sensitive to inhibition by this drug (27, 48). Curiously, rapamycin does not inhibit growth of S. pombe cells but partially inhibits sexual development and amino acid uptake (60-62). Inhibition of amino acid uptake is likely a result of inhibiting Tor1 (61, 62). Accordingly, a tor1 rapamycin-defective allele (tor1^S1834E) confers rapamycin resistance to strains that are dependent on amino acid uptake for their growth (61). Yet rapamycin also induces a response similar to that for a shift from rich to poor nitrogen conditions, an effect that may involve inhibition of both Tor1 and Tor2 (41).While other members of the phosphatidylinositol-3-kinase-related kinase (PIKK) family of proteins, such as ATM and ATR, have been shown to play central roles in the DNA damage response, little is known about roles that TOR proteins might play in such processes. Recently it was shown that the rapamycin-sensitive TORC1 complex participates in regulating cell survival under DNA-damaging conditions (24, 42, 49). Currently, no such role has been attributed to TORC2.Here we show that Tor1 (TORC2) is critical for cell survival under DNA-damaging conditions, gene silencing at heterochromatic regions, and telomere length maintenance and for regulation of cell cycle progression. Since the TOR complexes are highly conserved in evolution, this novel TORC2 function may also be conserved in other organisms.     

Spitzenkorper localization and intracellular traffic of green fluorescent protein-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of Neurospora crassa

The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.Fungal hyphae elongate and branch by a complex process based on polarized secretion. Many studies have investigated the cellular and molecular components involved in shaping fungal cells, but no detailed understanding of the mechanisms that govern and regulate polarized fungal growth has been achieved (4, 25). In the yeast Saccharomyces cerevisiae, many of the main components of the secretory pathway, including some of the enzymes involved in cell wall formation, have been extensively characterized (32). Filamentous fungi encode homologues of some key components known from the yeast secretory pathway, but despite their apparent orthology, relatively little is known about how this pathway is organized to accomplish the highly polarized growth typical of hyphae. There are some differences in cell wall synthesis between filamentous fungi and S. cerevisiae. In hyphae of septate fungi, vesicles and other components accumulate at the apex, as part of the Spitzenkörper (Spk) (14, 22-24, 28). The composition and mode of action of this pleomorphic and dynamic structure have intrigued fungal biologists for many decades.Fungal cells have at least two types of well-defined secretory vesicles (5). It has been suggested that macrovesicles, or conventional secretory vesicles, carry the components of the amorphous phase of the cell wall, in addition to the load of extracellular enzymes (5, 27). There is a large body of evidence characterizing the chitin synthase (CHS)-carrying microvesicles as chitosomes (3, 8, 13, 30). CHS are β-glycosyltransferases that catalyze the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin (47), a major structural polymer of the fungal cell wall (2). Chitin synthesis occurs in highly localized fashion both at the hyphal apices (7) and at nascent septa (29). Chitosomes are the smallest vesicles with the ability to form chitin microfibrils in vitro and have been suggested to carry and transport CHS to the cell surface at the apex of hyphae for cell wall synthesis (13, 37, 48, 55, 56). In recent years, studies on fungal CHS have concentrated mainly on gene identification. Given this wealth of information, we chose CHS as candidate markers to investigate vesicle traffic in fungal hyphae.Fungi have multiple chs genes grouped into two divisions, with seven classes, primarily on the basis of similarities in the primary sequence of the predicted proteins (12, 16, 37, 50). Division I includes classes I, II, and III, which share a catalytic domain surrounded by a hydrophilic N-terminal region and a hydrophobic C-terminal region (12). Division II includes classes IV, V, and VII, all with a catalytic domain preceded by a cytochrome b₅-like domain. In addition, classes V and VII contain an N-terminal myosin motor-like domain, suggesting a direct interaction with the actin cytoskeleton (15, 20, 58). Class VI has not been assigned to either division and includes recently identified CHS of unknown function (16). Earlier studies suggest that the various CHS have specific roles in chitin cell wall synthesis that are time or space dependent (60). In contrast to most filamentous fungi, S. cerevisiae (46) and Candida albicans (40) have only three or four CHS isozymes, respectively. S. cerevisiae Chs1p, C. albicans Chs2p, and C. albicans Chs8p belong to class I; S. cerevisiae Chs2p and C. albicans Chs1p belong to class II; and S. cerevisiae Chs3p and C. albicans Chs3p belong to class IV (46). While potential roles in hyphal growth have been suggested for some of the seven CHS classes described in filamentous fungi (9, 64, 65), we lack specific information on the cellular localization and trafficking to their sites of action in regions of active cell wall growth for most of these proteins.The goal of this study was to elucidate the traffic of CHS-containing vesicles en route from their site of genesis to their site of exocytosis in living hyphae of Neurospora crassa. The availability of an almost-complete genome sequence for this fungus allowed the identification of seven open reading frames with high homology to previously described chs genes (10). We chose to trace the intracellular location and secretory paths of CHS-3 and CHS-6. Neurospora CHS-3 belongs to the previously reported class I CHS with known homologues in all fungi tested, including S. cerevisiae Chs1p. In contrast, CHS-6 is a newly identified CHS assigned to class VI, homologous to Aspergillus fumigatus ChsD (39) and Coccidioides posadasii CHS-6 (34) but with no apparent homologues in S. cerevisiae or C. albicans. To trace both proteins, we fused green fluorescent protein (GFP) to the carboxyl terminus of the CHS coding regions and analyzed the fate of the resulting CHS-3-GFP and CHS-6-GFP fusion proteins by high-resolution confocal laser scanning microscopy (CLSM) in living hyphae of N. crassa.     

The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.DNA replication must be highly coordinated with chromatin assembly and cell division for correct propagation of genetic information and cell survival. Errors arising during DNA replication are corrected through the functions of numerous pathways including checkpoints and a diversity of DNA repair mechanisms (32, 33, 35). However, in the absence of these critical cellular responses, replication errors can lead to the accumulation of mutations and gross chromosomal rearrangements (GCRs) as well as chromosome loss, a condition generally termed genomic instability (33). Genome instability is a hallmark of many cancers as well as other human diseases (24). There are many mechanisms by which GCRs can arise, and over the last few years numerous genes and pathways have been implicated in playing a role in the suppression of GCRs in Saccharomyces cerevisiae and in some cases in the etiology of cancer (27, 28, 33, 39-47, 51, 53, 56, 58, 60), including S. cerevisiae ASF1, which encodes the main subunit of the replication coupling assembly factor (37, 62).Asf1 is involved in the deposition of histones H3 and H4 onto newly synthesized DNA during DNA replication and repair (62), and correspondingly, asf1 mutants are sensitive to chronic treatment with DNA-damaging agents (2, 30, 62). However, asf1 mutants do not appear to be repair defective and can recover from acute treatment with at least some DNA-damaging agents (2, 8, 30, 31, 54), properties similar to those described for rad9 mutants (68). In the absence of Asf1, both the DNA damage and replication checkpoints become activated during normal cell growth, and in the absence of checkpoint execution, there is a further increase in checkpoint activation in asf1 mutants (30, 46, 54). It has been suggested that asf1 mutants are defective for checkpoint shutoff and that this might account for the increased steady-state levels of checkpoint activation seen in asf1 mutants (8); however, another study has shown that asf1 mutants are not defective for checkpoint shutoff and that in fact Asf1 and the chromatin assembly factor I (CAF-I) complex act redundantly or cooperate in checkpoint shutoff (31). Furthermore, Asf1 might be involved in proper activation of the Rad53 checkpoint protein, as Asf1 physically interacts with Rad53 and this interaction is abrogated in response to exogenous DNA damage (15, 26); however, the physiological relevance of this interaction is unclear. Asf1 is also required for K56 acetylation of histone H3 by Rtt109, and both rtt109 mutants and histone H3 variants that cannot be acetylated (38) share many of the properties of asf1 mutants, suggesting that at least some of the requirement for Asf1 in response to DNA damage is mediated through Rtt109 (11, 14, 22, 61). Subsequent studies of checkpoint activation in asf1 mutants have led to the hypothesis that replication coupling assembly factor defects result in destabilization of replication forks which are then recognized by the replication checkpoint and stabilized, suggesting that the destabilized replication forks account for both the increased GCRs and increased checkpoint activation seen in asf1 mutants (30). This hypothesis is supported by other recent studies implicating Asf1 in the processing of stalled replication forks (16, 57). This role appears to be independent of CAF-I, which can cooperate with Asf1 in chromatin assembly (63). Asf1 has also been shown to function in disassembly of chromatin, suggesting other possibilities for the mechanism of action of Asf1 at the replication fork (1, 2, 34). Thus, while Asf1 is thought to be involved in progression of the replication fork, both the mechanism of action and the factors that cooperate with Asf1 in this process remain obscure.Stalled replication forks, particularly those that stall at sites of DNA damage, can be processed by homologous recombination (HR) (6) or by a mechanism known as postreplication repair (PRR) (reviewed in reference 67). There are two PRR pathways, an error-prone pathway involving translesion synthesis (TLS) by lower-fidelity polymerases and an error-free pathway thought to involve template switching (TS) (67). In S. cerevisiae, the PRR pathways are under the control of the RAD6 epistasis group (64). The error-prone pathway depends on monoubiquitination of proliferating cell nuclear antigen (PCNA) on K164 by Rad6 (an E2 ubiquitin-conjugating enzyme) by Rad18 (E3 ubiquitin ligase) (23). This results in replacement of the replicative DNA polymerase with nonessential TLS DNA polymerases, such as REV3/REV7-encoded DNA polymerase ζ (polζ) and RAD30-encoded DNA polη, which can bypass different types of replication-blocking damage (67). The error-free pathway is controlled by Rad5 (E3) and a complex consisting of Ubc13 and Mms2 (E2 and E2 variant, respectively), which add a K63-linked polyubiquitin chain to monoubiquitinated PCNA, leading to TS to the undamaged nascent sister chromatid (4, 25, 65). Furthermore, in addition to modification with ubiquitin, K164 of PCNA can also be sumoylated by Siz1, resulting in subsequent recruitment of the Srs2 helicase and inhibition of deleterious Rad51-dependent recombination events (50, 52, 55), although it is currently unclear if these are competing PCNA modifications or if both can exist on different subunits in the same PCNA trimer. A separate branch of the Rad6 pathway involving the E3 ligase Bre1 monoubiquitinates the histone H2B (29, 69) as well as Swd2 (66), which stimulates Set1-dependent methylation of K4 and Dot1-dependent methylation of K79 of histone H3 (48, 49, 66). Subsequently, K79-methylated H3 recruits Rad9 and activates the Rad53 checkpoint (19, 70). Activation of Rad53 is also bolstered by Rad6-Rad18-dependent ubiquitination of Rad17, which is part of the 9-1-1 complex that functions upstream in the checkpoint pathway (17). Finally, Rad6 complexes with the E3 Ubr1, which mediates protein degradation by the N-end rule pathway (13).Due to the role of the PRR pathways at stalled replication forks and a recent study implicating the Rad6 pathway in the suppression of GCRs (39), we examined the relationship between these ubiquitination and sumoylation pathways and the Asf1 pathway in order to gain additional insights into the function of Asf1 during DNA replication and repair. Our findings suggest that Asf1 has multiple functions that prevent replication damage or act in the cellular responses to replication damage and that these functions are modified by and interact with the PRR pathways. The TLS PRR pathway does not appear to be involved, and both a Dun1-dependent replication checkpoint and HR are important for preventing the deleterious effects of PRR and Asf1 pathway defects. We hypothesize that this newly observed cooperation between Asf1 and the PRR pathways may be required for resolving stalled replication forks, leading to suppression of GCRs and successful DNA replication.