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J Heitman

Publications and source records attributed to J Heitman.

At least 55 records · Page 3Linked to original sources
PubMed2000

Sok2 regulates yeast pseudohyphal differentiation via a transcription factor cascade that regulates cell-cell adhesion.

In response to nitrogen limitation, Saccharomyces cerevisiae undergoes a dimorphic transition to filamentous pseudohyphal growth. In previous studies, the transcription factor Sok2 was found to negatively regulate pseudohyphal differentiation. By genome array and Northern analysis, we found that genes encoding the transcription factors Phd1, Ash1, and Swi5 were all induced in sok2/sok2 hyperfilamentous mutants. In accord with previous studies of others, Swi5 was required for ASH1 expression. Phd1 and Ash1 regulated expression of the cell surface protein Flo11, which is required for filamentous growth, and were largely required for filamentation of sok2/sok2 mutant strains. These findings reveal that a complex transcription factor cascade regulates filamentation. These findings also reveal a novel dual role for the transcription factor Swi5 in regulating filamentous growth. Finally, these studies illustrate how mother-daughter cell adhesion can be accomplished by two distinct mechanisms: one involving Flo11 and the other involving regulation of the endochitinase Cts1 and the endoglucanase Egt2 by Swi5.

Cell Adhesion↗
PubMed2000

Signal transduction cascades regulating fungal development and virulence.

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.

Calcineurin↗
PubMed1999

The TOR signaling cascade regulates gene expression in response to nutrients.

Rapamycin inhibits the TOR kinases, which regulate cell proliferation and mRNA translation and are conserved from yeast to man. The TOR kinases also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis. We have analyzed gene expression in yeast cells exposed to rapamycin using arrays representing the whole yeast genome. TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator Gln3, and globally represses ribosomal protein expression. gln3 mutations were found to confer rapamycin resistance, whereas ure2 mutations confer rapamycin hypersensitivity, even in cells expressing dominant rapamycin-resistant TOR mutants. We find that Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation. In summary, our results reveal that the TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis, and amino acid permease stability.

Culture Media↗
PubMed1999

TOR kinase homologs function in a signal transduction pathway that is conserved from yeast to mammals.

Rapamycin is a natural product with potent antifungal and immunosuppressive activities. Rapamycin binds to the FKBP12 prolyl isomerase, and the resulting protein-drug complex inhibits the TOR kinase homologs. Both the FKBP12 and the TOR proteins are highly conserved from yeast to man, and genetic and biochemical studies reveal that these proteins are the targets of rapamycin in vivo. Treatment of yeast or mammalian cells with rapamycin inhibits translational initiation of a subset of mRNAs and dramatically represses ribosomal mRNA and tRNA transcription. Furthermore, rapamycin exposure blocks cell cycle progression in the early G1 phase of the cell cycle, driving cells into a G0 state and, ultimately, triggering autophagy. Recent findings reveal that the upstream factors regulating the TOR signaling cascade are involved in detecting amino acids, nutrients, or growth factors. These findings indicate that the TOR proteins function in a signal transduction pathway that coordinates nutritional and mitogenic signals to control protein biosynthesis and degradation.

Animals↗
PubMed1999

R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays.

Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69-73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein-folding assay.

Arginine↗
PubMed1999

Molecular analysis of the Cryptococcus neoformans ADE2 gene, a selectable marker for transformation and gene disruption.

Cryptococcus neoformans is an important fungal pathogen of man. The incidence of cryptococcal disease has increased dramatically in patients immunocompromised because of HIV infection, organ transplantation, or treatment with cytotoxic chemotherapy or corticosteroids. This organism is an excellent model for molecular dissection of fungal pathogenesis and virulence factors. Here we report the nucleotide sequence of the C. neoformans serotype D genomic ADE2 gene, which encodes a phosphoribosylaminoimidazole carboxylase required for purine biosynthesis. Importantly, this version of the ADE2 gene has been used as the selectable marker for virtually all gene disruptions by transformation and homologous recombination in C. neoformans. We compare the nucleotide and amino acid sequences of the ADE2 gene and product to other highly related adenine biosynthetic genes and enzymes from other yeasts and fungi. We also describe a series of convenient ADE2 cassettes for gene disruption construct preparation. Finally, we have identified the ade2 mutations in strains M001 and M049, adenine auxotrophic mutants derived from the serotype A strain H99. These mutant strains have served as recipients for targeted gene disruptions using the ADE2 gene. These studies should facilitate transformation and gene disruption approaches using the ADE2 selectable marker in this important human fungal pathogen.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of Aspergillus nidulans cyclophilin B.

Cyclophilins are an evolutionarily conserved family of proteins which serve as the intracellular receptors for the immunosuppressive drug cyclosporin A. Here we report the characterization of the first cyclophilin cloned from the filamentous fungus Aspergillus nidulans (CYPB). Sequence analysis of the cypB gene predicts an encoded protein with highest homology to the murine cyclophilin B protein. The sequence similarity includes an N-terminal sequence predicted to target the protein to the endoplasmic reticulum (ER) as well as a C-terminal sequence predicted to retain the mature protein in the ER. The bacterially expressed hexa-histidine tagged protein displays peptidyl-prolyl isomerase activity which is inhibited by cyclosporin A. In the presence of cyclosporin A, the expressed protein also inhibits purified calcineurin. When the endogenous cypB gene was disrupted and placed under the control of the regulatable alcohol dehydrogenase promoter, the strain demonstrated no detectable growth phenotype under conditions which induce or repress cypB transcription. Induction or repression of the cypB gene also did not effect sensitivity of A. nidulans to cyclosporin A. cypB mRNA levels were significantly elevated under severe heat shock conditions, indicating a possible role for the A. nidulans cyclophilin B protein during growth in high stress environments.

Amino Acid Sequence↗
PubMed1999

On the origins of congenic MATalpha and MATa strains of the pathogenic yeast Cryptococcus neoformans.

The basidiomycetous yeast Cryptococcus neoformans infects humans and causes a meningoencephalitis that is uniformly fatal if untreated. The organism has a defined sexual cycle involving mating of haploid MATa and MATalpha strains, gene disruption by transformation and homologous recombination is now readily accomplished, and robust animal models for infection have been well established. In addition, a pair of congenic MATalpha and MATa haploid strains have been constructed that permit detailed studies on physiology and virulence by classical genetic approaches. These strains represent a valuable resource for further studies in this organism, and the genomic sequence of one of these strains, JEC21 (=B-4500), was recently chosen to be sequenced by an international consortium. Because of the importance of these strains for genetic studies in C. neoformans and the fact that the genomic sequence of one of these strains is in progress, we review here how these congenic strains were originally constructed.

Animals↗
PubMed1999

Calcineurin. Structure, function, and inhibition.

Calcineurin is a serine-threonine specific Ca(2+)-calmodulin-activated protein phosphatase that is conserved from yeast to humans. Remarkably, this enzyme is the common target for two novel and structurally unrelated immunosuppressive antifungal drugs, cyclosporin A and FK506. Both drugs form complexes with abundant intracellular binding proteins, cyclosporin A with cyclophilin A and FK506 with FKBP 12, which bind to and inhibit calcineurin. The X-ray structure of an FKPB12-FK506-calcineurin AB ternary complex reveals that FKBP12-FK506 binds in a hydophobic groove between the calcineurin A catalytic and the regulatory B subunit, in accord with biochemical and genetic studies on inhibitor action. Calcineurin plays a key role in regulating the transcription factor NF-AT during T-cell activation, and in mediating responses of microorganisms to cation stress. These findings highlight the potential of yeast genetic studies to define novel drug targets and elucidate conserved elements of signal transduction cascades.

Calcineurin↗
PubMed1999

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae.

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na+/Li+-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li+-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li+ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li+-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast.

Calcineurin↗
PubMed1999

Signal transduction cascades regulating mating, filamentation, and virulence in Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycetous fungal pathogen that infects the central nervous system. The organism has a defined sexual cycle involving mating between haploid MATalpha and MATa cells. Recent studies have revealed signaling cascades that coordinately regulate differentiation and virulence of C. neoformans. One signaling cascade involves a conserved G-protein alpha subunit and cAMP, and senses nutrients during mating and virulence. The second is a conserved mitogen activated protein (MAP) kinase cascade that senses pheromone during mating, and also regulates haploid fruiting and virulence. Interestingly, some of the MAP kinase components are encoded by the MAT locus itself, which may explain the unique association of the MATalpha locus with physiology and virulence.

Cryptococcus neoformans↗
PubMed1999

The Cryptococcus neoformans genome sequencing project.

Cryptococcus neoformans is a basidiomycete that can cause life-threatening meningoencephalitis in patients with and without impaired immune function. Cryptococcosis is usually an opportunistic infection in patients with compromised immunity as a consequence of HIV-1 infection, steroid administration, cancer chemotherapy, sarcoidosis, diabetes, or inherited immune system defects. This pathogenic yeast has a defined sexual cycle, which allows classical genetic analysis. Molecular biology approaches, including transformation and gene disruption by homologous recombination, and animal models for studies of virulence are both well developed. Recently an international consortium convened to begin the C. neoformans genome sequencing project, and we review here background and arguments for this project. We also discuss the importance of this project to the biology and virulence of this organism in particular, and to virulence in general.

AIDS-Related Opportunistic Infections↗
PubMed1999

DNA nicks inflicted by restriction endonucleases are repaired by a RecA- and RecB-dependent pathway in Escherichia coli.

Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non-cognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.

DNA Damage↗
PubMed1999

Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast.

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Amino Acid Sequence↗
PubMed1999

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

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

Alleles↗
PubMed1999

Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target.

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.

Amino Acid Sequence↗