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1.  Synthesis and antifungal activities of miltefosine analogs 
Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a–3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5–5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5–3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.
doi:10.1016/j.bmcl.2013.06.096
PMCID: PMC3760157  PMID: 23891181
alkylphosphocholine; miltefosine; antifungal; cryptococcosis; candidiasis
2.  A functional variomics tool for discovering drug resistance genes and drug targets 
Cell reports  2013;3(2):577-585.
Comprehensive discovery of genetic mechanisms of drug resistance and identification of in vivo drug targets represent significant challenges. Here we present a functional variomics technology in the model organism Saccharomyces cerevisiae. This tool analyzes numerous genetic variants and effectively tackles both problems simultaneously. Using this tool, we discovered almost all genes that, due to mutations or modest overexpression, confer resistance to rapamycin, cycloheximide, and amphotericin B. Most significant among the resistance genes were drug targets, including multiple targets of a given drug. With amphotericin B, we discovered the highly conserved membrane protein Pmp3 as a potent resistance factor and a possible novel target. Widespread application of this tool should allow rapid identification of conserved resistance mechanisms and targets of many more compounds. New genes and alleles that confer resistance to other stresses can also be discovered. Similar tools in other systems such as human cell lines will also be useful.
doi:10.1016/j.celrep.2013.01.019
PMCID: PMC3594652  PMID: 23416056
3.  Discovering Thiamine Transporters as Targets of Chloroquine Using a Novel Functional Genomics Strategy 
PLoS Genetics  2012;8(11):e1003083.
Chloroquine (CQ) and other quinoline-containing antimalarials are important drugs with many therapeutic benefits as well as adverse effects. However, the molecular targets underlying most such effects are largely unknown. By taking a novel functional genomics strategy, which employs a unique combination of genome-wide drug-gene synthetic lethality (DGSL), gene-gene synthetic lethality (GGSL), and dosage suppression (DS) screens in the model organism Saccharomyces cerevisiae and is thus termed SL/DS for simplicity, we found that CQ inhibits the thiamine transporters Thi7, Nrt1, and Thi72 in yeast. We first discovered a thi3Δ mutant as hypersensitive to CQ using a genome-wide DGSL analysis. Using genome-wide GGSL and DS screens, we then found that a thi7Δ mutation confers severe growth defect in the thi3Δ mutant and that THI7 overexpression suppresses CQ-hypersensitivity of this mutant. We subsequently showed that CQ inhibits the functions of Thi7 and its homologues Nrt1 and Thi72. In particular, the transporter activity of wild-type Thi7 but not a CQ-resistant mutant (Thi7T287N) was completely inhibited by the drug. Similar effects were also observed with other quinoline-containing antimalarials. In addition, CQ completely inhibited a human thiamine transporter (SLC19A3) expressed in yeast and significantly inhibited thiamine uptake in cultured human cell lines. Therefore, inhibition of thiamine uptake is a conserved mechanism of action of CQ. This study also demonstrated SL/DS as a uniquely effective methodology for discovering drug targets.
Author Summary
By using a novel SL/DS methodology in the model organism yeast, we discovered that the antimalarial drug CQ inhibits thiamine transporters and consequently causes thiamine (vitamin B1) deficiency and growth defects. This mechanism of action (MOA) is conserved in human cells and possibly also in other organisms. Given that both thiamine deficiency and treatment with CQ cause retinal, neurological, and cardiovascular disorders in humans, our results suggest that thiamine deficiency might be a root cause of some of CQ's adverse effects, which might be preventable with concomitant dietary thiamine supplementation. Such a MOA by CQ could also be responsible for its therapeutic effects against malarial parasites, which need exogenous thiamine for survival. Such a possibility needs to be investigated before dietary thiamine supplementation can be used to prevent CQ's adverse effects.
doi:10.1371/journal.pgen.1003083
PMCID: PMC3510038  PMID: 23209439
4.  The Saccharomyces cerevisiae Nrd1-Nab3 Transcription Termination Pathway Acts in Opposition to Ras Signaling and Mediates Response to Nutrient Depletion 
Molecular and Cellular Biology  2012;32(10):1762-1775.
The Saccharomyces cerevisiae Nrd1-Nab3 pathway directs the termination and processing of short RNA polymerase II transcripts. Despite the potential for Nrd1-Nab3 to affect the transcription of both coding and noncoding RNAs, little is known about how the Nrd1-Nab3 pathway interacts with other pathways in the cell. Here we present the results of a high-throughput synthetic lethality screen for genes that interact with NRD1 and show roles for Nrd1 in the regulation of mitochondrial abundance and cell size. We also provide genetic evidence of interactions between the Nrd1-Nab3 and Ras/protein kinase A (PKA) pathways. Whereas the Ras pathway promotes the transcription of genes involved in growth and glycolysis, the Nrd1-Nab3 pathway appears to have a novel role in the rapid suppression of some genes when cells are shifted to poor growth conditions. We report the identification of new mRNA targets of the Nrd1-Nab3 pathway that are rapidly repressed in response to glucose depletion. Glucose depletion also leads to the dephosphorylation of Nrd1 and the formation of novel nuclear speckles that contain Nrd1 and Nab3. Taken together, these results indicate a role for Nrd1-Nab3 in regulating the cellular response to nutrient availability.
doi:10.1128/MCB.00050-12
PMCID: PMC3347421  PMID: 22431520
5.  Sampangine Inhibits Heme Biosynthesis in both Yeast and Human ▿ † 
Eukaryotic Cell  2011;10(11):1536-1544.
The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism—by hyperactivating uroporhyrinogen III synthase—to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.
doi:10.1128/EC.05170-11
PMCID: PMC3209050  PMID: 21908598
6.  Making temperature-sensitive mutants 
Methods in enzymology  2010;470:181-204.
The study of temperature sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter we describe two “shuffling” methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the “plasmid” version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the “chromosomal” version, The Ts allele is integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have the ir strengths and weaknesses, which are discussed here.
doi:10.1016/S0076-6879(10)70008-2
PMCID: PMC2957654  PMID: 20946811
7.  A Microarray-Based Genetic Screen for Yeast Chronological Aging Factors 
PLoS Genetics  2010;6(4):e1000921.
Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA “bar-code” sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Δ mutant and exacerbated by a short-lived atg16Δ autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process.
Author Summary
The aging process is associated with the onset of several age-associated diseases including diabetes and cancer. In rodent model systems, the dietary regimen known as caloric restriction (CR) is known to delay or prevent these diseases and to extend lifespan. As a result, there is a great deal of interest in understanding the mechanisms by which CR functions. The budding yeast, Saccharomyces cerevisiae, has proven to be an effective model for the analysis of genes and cellular pathways that contribute to the regulation of aging. In this study we have performed a microarray-based genetic screen in yeast that identified short- and long-lived mutants from a population that contained each of the viable haploid gene deletion mutants from the yeast gene knockout collection that were pooled together. Using such an approach, we were able to identify genes from several pathways that had not been previously implicated in aging, including some that appear to contribute to the CR effect induced by restriction of either amino acids or sugar. These results are expected to provide new groundwork for future mechanistic aging studies in more complex organisms.
doi:10.1371/journal.pgen.1000921
PMCID: PMC2858703  PMID: 20421943
8.  Microarray-based genetic screen defines SAW1, a new gene required for Rad1/Rad10-dependent processing of recombination intermediates 
Molecular cell  2008;30(3):325-335.
Elimination of a DSB flanked by direct repeat sequences is mediated by single strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA using a plasmid based SSA assay coupled to a bar-code microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3’-flap cleavage step. Slx4 and Saw1 also contribute to integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2 are defective in 3’-flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.
doi:10.1016/j.molcel.2008.02.028
PMCID: PMC2398651  PMID: 18471978
9.  A comprehensive synthetic genetic interaction network governing yeast histone acetylation and deacetylation 
Genes & development  2008;22(15):2062-2074.
Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at lysine 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes.
doi:10.1101/gad.1679508
PMCID: PMC2492751  PMID: 18676811
systems biology; histone; NuA4; acetylation; DNA repair
10.  dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae 
Methods (San Diego, Calif.)  2007;41(2):206-221.
Analysis of genetic interactions has been extensively exploited to study gene functions and to dissect pathway structures. One such genetic interaction is synthetic lethality, in which the combination of two non-lethal mutations leads to loss of organism viability. We have developed a dSLAM (heterozygote diploid-based synthetic lethality analysis with microarrays) technology that effectively studies synthetic lethality interactions on a genome-wide scale in the budding yeast Saccharomyces cerevisiae. Typically, a query mutation is introduced en masse into a population of ~6,000 haploid-convertible heterozygote diploid Yeast Knockout (YKO) mutants via integrative transformation. Haploid pools of single and double mutants are freshly generated from the resultant heterozygote diploid double mutant pool after meiosis and haploid selection and studied for potential growth defects of each double mutant combination by microarray analysis of the “molecular barcodes” representing each YKO. This technology has been effectively adapted to study other types of genome-wide genetic interactions including gene-compound synthetic lethality, secondary mutation suppression, dosage-dependent synthetic lethality and suppression.
doi:10.1016/j.ymeth.2006.07.033
PMCID: PMC2491713  PMID: 17189863
11.  Gene function prediction from congruent synthetic lethal interactions in yeast 
Molecular Systems Biology  2005;1:2005.0026.
We predicted gene function using synthetic lethal genetic interactions between null alleles in Saccharomyces cerevisiae. Phenotypic and protein interaction data indicate that synthetic lethal gene pairs function in parallel or compensating pathways. Congruent gene pairs, defined as sharing synthetic lethal partners, are in single pathway branches. We predicted benomyl sensitivity and nuclear migration defects using congruence; these phenotypes were uncorrelated with direct synthetic lethality. We also predicted YLL049W as a new member of the dynein–dynactin pathway and provided new supporting experimental evidence. We performed synthetic lethal screens of the parallel mitotic exit network (MEN) and Cdc14 early anaphase release pathways required for late cell cycle. Synthetic lethal interactions bridged genes in these pathways, and high congruence linked genes within each pathway. Synthetic lethal interactions between MEN and all components of the Sin3/Rpd3 histone deacetylase revealed a novel function for Sin3/Rpd3 in promoting mitotic exit in parallel to MEN. These in silico methods can predict phenotypes and gene functions and are applicable to genomic synthetic lethality screens in yeast and analogous RNA interference screens in metazoans.
doi:10.1038/msb4100034
PMCID: PMC1681444  PMID: 16729061
congruence score; function prediction; quantitative phenotype; synthetic lethality; yeast
12.  Improved microarray methods for profiling the yeast knockout strain collection 
Nucleic Acids Research  2005;33(12):e103.
A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens.
doi:10.1093/nar/gni105
PMCID: PMC1169235  PMID: 15994458
13.  FKBP12 Controls Aspartate Pathway Flux in Saccharomyces cerevisiae To Prevent Toxic Intermediate Accumulation 
Eukaryotic Cell  2004;3(5):1287-1296.
FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate β-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate β-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.
doi:10.1128/EC.3.5.1287-1296.2004
PMCID: PMC522611  PMID: 15470257
14.  Protein Kinase A Operates a Molecular Switch That Governs Yeast Pseudohyphal Differentiation 
Molecular and Cellular Biology  2002;22(12):3981-3993.
The yeast Saccharomyces cerevisiae undergoes a dimorphic filamentous transition in response to nutrient cues that is affected by both mitogen-activated protein kinase and cyclic AMP-protein kinase A signaling cascades. Here two transcriptional regulators, Flo8 and Sfl1, are shown to be the direct molecular targets of protein kinase A. Flo8 and Sfl1 antagonistically control expression of the cell adhesin Flo11 via a common promoter element. Phosphorylation by the protein kinase A catalytic subunit Tpk2 promotes Flo8 binding and activation of the Flo11 promoter and relieves repression by prohibiting dimerization and DNA binding by Sfl1. Our studies illustrate in molecular detail how protein kinase A combinatorially effects a key developmental switch. Similar mechanisms may operate in pathogenic fungi and more complex multicellular eukaryotic organisms.
doi:10.1128/MCB.22.12.3981-3993.2002
PMCID: PMC133872  PMID: 12024012
15.  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.
PMCID: PMC99013  PMID: 11104818
16.  Sok2 Regulates Yeast Pseudohyphal Differentiation via a Transcription Factor Cascade That Regulates Cell-Cell Adhesion 
Molecular and Cellular Biology  2000;20(22):8364-8372.
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.
PMCID: PMC102143  PMID: 11046133
17.  Cyclic AMP-Dependent Protein Kinase Regulates Pseudohyphal Differentiation in Saccharomyces cerevisiae 
Molecular and Cellular Biology  1999;19(7):4874-4887.
In response to nitrogen starvation, diploid cells of the yeast Saccharomyces cerevisiae differentiate to a filamentous growth form known as pseudohyphal differentiation. Filamentous growth is regulated by elements of the pheromone mitogen-activated protein (MAP) kinase cascade and a second signaling cascade involving the receptor Gpr1, the Gα protein Gpa2, Ras2, and cyclic AMP (cAMP). We show here that the Gpr1-Gpa2-cAMP pathway signals via the cAMP-dependent protein kinase, protein kinase A (PKA), to regulate pseudohyphal differentiation. Activation of PKA by mutation of the regulatory subunit Bcy1 enhances filamentous growth. Mutation and overexpression of the PKA catalytic subunits reveal that the Tpk2 catalytic subunit activates filamentous growth, whereas the Tpk1 and Tpk3 catalytic subunits inhibit filamentous growth. The PKA pathway regulates unipolar budding and agar invasion, whereas the MAP kinase cascade regulates cell elongation and invasion. Epistasis analysis supports a model in which PKA functions downstream of the Gpr1 receptor and the Gpa2 and Ras2 G proteins. Activation of filamentous growth by PKA does not require the transcription factors Ste12 and Tec1 of the MAP kinase cascade, Phd1, or the PKA targets Msn2 and Msn4. PKA signals pseudohyphal growth, in part, by regulating Flo8-dependent expression of the cell surface flocculin Flo11. In summary, the cAMP-dependent protein kinase plays an intimate positive and negative role in regulating filamentous growth, and these findings may provide insight into the roles of PKA in mating, morphogenesis, and virulence in other yeasts and pathogenic fungi.
PMCID: PMC84286  PMID: 10373537
18.  The TOR Signal Transduction Cascade Controls Cellular Differentiation in Response to Nutrients 
Molecular Biology of the Cell  2001;12(12):4103-4113.
Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiae to humans. In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis. We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation. Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42–Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.
PMCID: PMC60779  PMID: 11739804

Results 1-18 (18)