RESUMO
Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling, oxidative stress resistance and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. Initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity eventually resolved, as populations gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while severity of others like membrane stress responses diverged from Pi scarcity fitness. Among preliminary candidate genes for contributors to fitness recovery, those with links to TORC1 were overrepresented. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth, stress resistance and virulence.
RESUMO
Candida albicans is a commensal fungus, opportunistic pathogen, and the most common cause of fungal infection in humans. The biosynthesis of phosphatidylcholine (PC), a major eukaryotic glycerophospholipid, occurs through two primary pathways. In Saccharomyces cerevisiae and some plants, a third PC synthesis pathway, the PC deacylation/reacylation pathway (PC-DRP), has been characterized. PC-DRP begins with the acylation of the lipid turnover product, glycerophosphocholine (GPC), by the GPC acyltransferase, Gpc1, to form Lyso-PC. Lyso-PC is then acylated by lysolipid acyltransferase, Lpt1, to produce PC. Importantly, GPC, the substrate for Gpc1, is a ubiquitous metabolite available within the host. GPC is imported by C. albicans, and deletion of the major GPC transporter, Git3, leads to decreased virulence in a murine model. Here we report that GPC can be directly acylated in C. albicans by the protein product of orf19.988, a homolog of ScGpc1. Through lipidomic studies, we show loss of Gpc1 leads to a decrease in PC levels. This decrease occurs in the absence of exogenous GPC, indicating that the impact on PC levels may be greater in the human host where GPC is available. A gpc1Δ/Δ strain exhibits several sensitivities to antifungals that target lipid metabolism. Furthermore, loss of Gpc1 results in both a hyphal growth defect in embedded conditions and a decrease in long-term cell viability. These results demonstrate for the first time the importance of Gpc1 and this alternative PC biosynthesis route (PC-DRP) to the physiology of a pathogenic fungus.
Assuntos
Aciltransferases , Animais , Humanos , Camundongos , Aciltransferases/genética , Aciltransferases/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Glicerilfosforilcolina/metabolismo , Fosfatidilcolinas/metabolismoRESUMO
The unfolded protein response (UPR) is sensitive to proteotoxic and membrane bilayer stress, both of which are sensed by the ER protein Ire1. When activated, Ire1 splices HAC1 mRNA, producing a transcription factor that targets genes involved in proteostasis and lipid metabolism, among others. The major membrane lipid phosphatidylcholine (PC) is subject to phospholipase-mediated deacylation, producing glycerophosphocholine (GPC), followed by reacylation of GPC through the PC deacylation/reacylation pathway (PC-DRP). The reacylation events occur via a two-step process catalyzed first by the GPC acyltransferase Gpc1, followed by acylation of the lyso-PC molecule by Ale1. However, whether Gpc1 is critical for ER bilayer homeostasis is unclear. Using an improved method for C14-choline-GPC radiolabeling, we first show that loss of Gpc1 results in abrogation of PC synthesis through PC-DRP and that Gpc1 colocalizes with the ER. We then probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR-inducing compounds tunicamycin, DTT, and canavanine results in a Hac1-dependent increase in GPC1 message. Further, cells lacking Gpc1 exhibit increased sensitivity to those proteotoxic stressors. Inositol limitation, known to induce the UPR via bilayer stress, also induces GPC1 expression. Finally, we show that loss of GPC1 induces the UPR. A gpc1Δ mutant displays upregulation of the UPR in strains expressing a mutant form of Ire1 that is unresponsive to unfolded proteins, indicating that bilayer stress is responsible for the observed upregulation. Collectively, our data indicate an important role for Gpc1 in yeast ER bilayer homeostasis.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aciltransferases/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não DobradasRESUMO
BACKGROUND INFORMATION: Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. RESULTS: We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. CONCLUSIONS: The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. SIGNIFICANCE: To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.
Assuntos
Arrestinas , Proteínas de Saccharomyces cerevisiae , Arrestinas/metabolismo , Endocitose , Homeostase , Fosfatos de Inositol , Fosfolipídeos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Phospholipase B-mediated hydrolysis of phosphatidylcholine (PC) results in the formation of free fatty acids and glycerophosphocholine (GPC) in the yeast Saccharomyces cerevisiae GPC can be reacylated by the glycerophosphocholine acyltransferase Gpc1, which produces lysophosphatidylcholine (LPC), and LPC can be converted to PC by the lysophospholipid acyltransferase Ale1. Here, we further characterized the regulation and function of this distinct PC deacylation/reacylation pathway in yeast. Through in vitro and in vivo experiments, we show that Gpc1 and Ale1 are the major cellular GPC and LPC acyltransferases, respectively. Importantly, we report that Gpc1 activity affects the PC species profile. Loss of Gpc1 decreased the levels of monounsaturated PC species and increased those of diunsaturated PC species, whereas Gpc1 overexpression had the opposite effects. Of note, Gpc1 loss did not significantly affect phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine profiles. Our results indicate that Gpc1 is involved in postsynthetic PC remodeling that produces more saturated PC species. qRT-PCR analyses revealed that GPC1 mRNA abundance is regulated coordinately with PC biosynthetic pathways. Inositol availability, which regulates several phospholipid biosynthetic genes, down-regulated GPC1 expression at the mRNA and protein levels and, as expected, decreased levels of monounsaturated PC species. Finally, loss of GPC1 decreased stationary phase viability in inositol-free medium. These results indicate that Gpc1 is part of a postsynthetic PC deacylation/reacylation remodeling pathway (PC-DRP) that alters the PC species profile, is regulated in coordination with other major lipid biosynthetic pathways, and affects yeast growth.
Assuntos
Aciltransferases/metabolismo , Glicerilfosforilcolina/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Acilação , Aciltransferases/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).
Assuntos
Aciltransferases/metabolismo , Fosfatidilcolinas/biossíntese , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Acilação , Aciltransferases/genética , Fosfatidilcolinas/genética , Proteínas de Plantas/genética , Plantas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Ypk1, the yeast homolog of the human serum- and glucocorticoid-induced kinase (Sgk1), affects diverse cellular activities, including sphingolipid homeostasis. We now report that Ypk1 also impacts the turnover of the major phospholipid, phosphatidylcholine (PC). Pulse-chase radiolabeling reveals that a ypk1Δ mutant exhibits increased PC deacylation and glycerophosphocholine production compared with wild type yeast. Deletion of PLB1, a gene encoding a B-type phospholipase that hydrolyzes PC, in a ypk1Δ mutant curtails the increased PC deacylation. In contrast to previous data, we find that Plb1 resides in the ER and in the medium. Consistent with a link between Ypk1 and Plb1, the levels of both Plb1 protein and PLB1 message are elevated in a ypk1Δ strain compared with wild type yeast. Furthermore, deletion of PLB1 in a ypk1Δ mutant exacerbates phenotypes associated with loss of YPK1, including slowed growth and sensitivity to cell wall perturbation, suggesting that increased Plb1 activity buffers against the loss of Ypk1. Because Plb1 lacks a consensus phosphorylation site for Ypk1, we probed other processes under the control of Ypk1 that might be linked to PC turnover. Inhibition of sphingolipid biosynthesis by the drug myriocin or through utilization of a lcb1-100 mutant results in increased PLB1 expression. Furthermore, we discovered that the increase in PLB1 expression observed upon inhibition of sphingolipid synthesis or loss of Ypk1 is under the control of the Crz1 transcription factor. Taken together, these results suggest a functional interaction between Ypk1 and Plb1 in which altered sphingolipid metabolism up-regulates PLB1 expression via Crz1.
Assuntos
Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Lisofosfolipase/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação , Alelos , Colina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos Monoinsaturados/química , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glicerilfosforilcolina/metabolismo , Homeostase , Hidrólise , Lipídeos/química , Proteínas de Membrana/metabolismo , Mutação , Fenótipo , Fosforilação , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity.
Assuntos
Ácido Acético/toxicidade , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Expressão Gênica , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Estresse Fisiológico , Proteínas Adaptadoras de Transporte Vesicular/genética , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismoRESUMO
Candida albicans contains four ORFs (GIT1,2,3,4) predicted to encode proteins involved in the transport of glycerophosphodiester metabolites. Previously, we reported that Git1, encoded by ORF 19.34, is responsible for the transport of intact glycerophosphoinositol but not glycerophosphocholine (GroPCho). Here, we report that a strain lacking both GIT3 (ORF 19.1979) and GIT4 (ORF 19.1980) is unable to transport [(3)H]GroPCho into the cell. In the absence of a GroPCho transporter, C. albicans can utilize GroPCho via a mechanism involving extracellular hydrolysis. Upon reintegration of either GIT3 or GIT4 into the genome, measurable uptake of [(3)H]GroPCho is observed. Transport assays and kinetic analyses indicate that Git3 has the greater transport velocity. We present evidence that GDE1 (ORF 19.3936) codes for an enzyme with glycerophosphodiesterase activity against GroPCho. Homozygous deletion of GDE1 results in a buildup of internal GroPCho that is restored to wild type levels by reintegration of GDE1 into the genome. The transcriptional regulator, Pho4, is shown to regulate the expression of GIT3, GIT4, and GDE1. Finally, Git3 is shown to be required for full virulence in a mouse model of disseminated candidiasis, and Git3 sequence orthologs are present in other pathogenic Candida species. In summary, we have characterized multiple aspects of GroPCho utilization by C. albicans and have demonstrated that GroPCho transport plays a key role in the growth of the organism in the host.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Candida albicans/metabolismo , Candida albicans/patogenicidade , Candidíase/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Candida albicans/genética , Candidíase/genética , Feminino , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Transporte de Íons/genética , Camundongos , Camundongos Endogâmicos BALB C , Fases de Leitura Aberta , Éteres Fosfolipídicos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genéticaRESUMO
Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. In this process, initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity were eventually lost as populations presumably gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while that of others like membrane stress responses diverged from these fitness phenotypes. C. albicans therefore has diverse options to reconfigure Pi management during prolonged scarcity. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth and virulence.
RESUMO
Acetic acid inhibition of yeast fermentation has a negative impact in several industrial processes. As an initial step in the construction of a Saccharomyces cerevisiae strain with increased tolerance for acetic acid, mutations conferring resistance were identified by screening a library of deletion mutants in a multiply auxotrophic genetic background. Of the 23 identified mutations, 11 were then introduced into a prototrophic laboratory strain for further evaluation. Because none of the 11 mutations was found to increase resistance in the prototrophic strain, potential interference by the auxotrophic mutations themselves was investigated. Mutants carrying single auxotrophic mutations were constructed and found to be more sensitive to growth inhibition by acetic acid than an otherwise isogenic prototrophic strain. At a concentration of 80 mM acetic acid at pH 4.8, the initial uptake of uracil, leucine, lysine, histidine, tryptophan, phosphate, and glucose was lower in the prototrophic strain than in a non-acetic acid-treated control. These findings are consistent with two mechanisms by which nutrient uptake may be inhibited. Intracellular adenosine triphosphate (ATP) levels were severely decreased upon acetic acid treatment, which likely slowed ATP-dependent proton symport, the major form of transport in yeast for nutrients other than glucose. In addition, the expression of genes encoding some nutrient transporters was repressed by acetic acid, including HXT1 and HXT3 that encode glucose transporters that operate by facilitated diffusion. These results illustrate how commonly used genetic markers in yeast deletion libraries complicate the effort to isolate strains with increased acetic acid resistance.
Assuntos
Ácido Acético/toxicidade , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Aminoácidos/metabolismo , Fermentação , Deleção de Genes , Glucose/metabolismo , Fosfatos/metabolismo , Uracila/metabolismoRESUMO
IMPORTANCE: Candida albicans is the most commonly isolated species from patients suffering from invasive fungal disease. C. albicans is most commonly a commensal organism colonizing a variety of niches in the human host. The fungus must compete for resources with the host flora to acquire essential nutrients such as phosphate. Phosphate acquisition and homeostasis have been shown to play a key role in C. albicans virulence, with several genes involved in these processes being required for normal virulence and several being upregulated during infection. In addition to inorganic phosphate (Pi), C. albicans can utilize the lipid-derived metabolite glycerophosphocholine (GPC) as a phosphate source. As GPC is available within the human host, we examined the role of GPC in phosphate homeostasis in C. albicans. We find that GPC can substitute for Pi by many though not all criteria and is likely a relevant physiological phosphate source for C. albicans.
Assuntos
Candida albicans , Proteínas Fúngicas , Humanos , Proteínas Fúngicas/genética , Fosfatos/metabolismo , Fenótipo , VirulênciaRESUMO
White lupin (Lupinus albus) is a legume that is very efficient in accessing unavailable phosphorus (Pi). It develops short, densely clustered tertiary lateral roots (cluster/proteoid roots) in response to Pi limitation. In this report, we characterize two glycerophosphodiester phosphodiesterase (GPX-PDE) genes (GPX-PDE1 and GPX-PDE2) from white lupin and propose a role for these two GPX-PDEs in root hair growth and development and in a Pi stress-induced phospholipid degradation pathway in cluster roots. Both GPX-PDE1 and GPX-PDE2 are highly expressed in Pi-deficient cluster roots, particularly in root hairs, epidermal cells, and vascular bundles. Expression of both genes is a function of both Pi availability and photosynthate. GPX-PDE1 Pi deficiency-induced expression is attenuated as photosynthate is deprived, while that of GPX-PDE2 is strikingly enhanced. Yeast complementation assays and in vitro enzyme assays revealed that GPX-PDE1 shows catalytic activity with glycerophosphocholine while GPX-PDE2 shows highest activity with glycerophosphoinositol. Cell-free protein extracts from Pi-deficient cluster roots display GPX-PDE enzyme activity for both glycerophosphocholine and glycerophosphoinositol. Knockdown of expression of GPX-PDE through RNA interference resulted in impaired root hair development and density. We propose that white lupin GPX-PDE1 and GPX-PDE2 are involved in the acclimation to Pi limitation by enhancing glycerophosphodiester degradation and mediating root hair development.
Assuntos
Aclimatação/fisiologia , Lupinus/enzimologia , Lupinus/crescimento & desenvolvimento , Diester Fosfórico Hidrolases/metabolismo , Fósforo/deficiência , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Aclimatação/efeitos dos fármacos , Escuridão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Ensaios Enzimáticos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes de Plantas/genética , Genes Reporter , Teste de Complementação Genética , Lupinus/efeitos dos fármacos , Lupinus/genética , Dados de Sequência Molecular , Mutação/genética , Fosfitos/farmacologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Fósforo/farmacologia , Fotossíntese/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Glycerophosphodiesters are the products of phospholipase-mediated deacylation of phospholipids. In Saccharomyces cerevisiae, a single gene, GIT1, encodes a permease responsible for importing glycerophosphodiesters, such as glycerophosphoinositol and glycerophosphocholine, into the cell. In contrast, the Candida albicans genome contains four open reading frames (ORFs) with a high degree of similarity to S. cerevisiae GIT1 (ScGIT1) Here, we report that C. albicans utilizes glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) as sources of phosphate at both mildly acidic and physiological pHs. Insertional mutagenesis of C. albicans GIT1 (CaGIT1) (orf19.34), the ORF most similar to ScGit1, abolished the ability of cells to use GroPIns as a phosphate source at acidic pH and to transport [(3)H]GroPIns at acidic and physiological pHs, while reintegration of a GIT1 allele into the genome restored those functions. Several lines of evidence, including the detection of internal [(3)H]GroPIns, indicated that GroPIns is transported intact through CaGit1. GroPIns transport was shown to conform to Michaelis-Menten kinetics, with an apparent K(m) of 28 ± 6 µM. Notably, uptake of label from [(3)H]GroPCho was found to be roughly 50-fold greater than uptake of label from [(3)H]GroPIns and roughly 500-fold greater than the equivalent activity in S. cerevisiae. Insertional mutagenesis of CaGIT1 had no effect on the utilization of GroPCho as a phosphate source or on the uptake of label from [(3)H]GroPCho. Growth under low-phosphate conditions was shown to increase label uptake from both [(3)H]GroPIns and [(3)H]GroPCho. Screening of a transcription factor deletion set identified CaPHO4 as required for the utilization of GroPIns, but not GroPCho, as a phosphate source.
Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Glicerilfosforilcolina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfolipases/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Genoma Fúngico , Concentração de Íons de Hidrogênio , Fosfatos de Inositol/metabolismo , Cinética , Proteínas de Membrana Transportadoras/genética , Mutagênese , Mutagênese Insercional , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Biofilms of Candida albicans include both yeast cells and hyphae. Prior studies indicated that a zap1Δ/Δ mutant, defective in zinc regulator Zap1, has increased accumulation of yeast cells in biofilms. This altered yeast-hypha balance may arise from internal regulatory alterations or from an effect on the production of diffusible quorum-sensing (QS) molecules. Here, we develop biosensor reporter strains that express yeast-specific YWP1-RFP or hypha-specific HWP1-RFP, along with a constitutive TDH3-GFP normalization standard. Seeding these biosensor strains into biofilms allows a biological activity assay of the surrounding biofilm milieu. A zap1Δ/Δ biofilm induces the yeast-specific YWP1-RFP reporter in a wild-type biosensor strain, as determined by both quantitative reverse transcription-PCR (qRT-PCR) gene expression measurements and confocal microscopy. Remediation of the zap1Δ/Δ zinc uptake defect through zinc transporter gene ZRT2 overexpression reverses induction of the yeast-specific YWP1-RFP reporter. Gas chromatography-mass spectrometry (GC-MS) measurements of known organic QS molecules show that the zap1Δ/Δ mutant accumulates significantly less farnesol than wild-type or complemented strains and that ZRT2 overexpression does not affect farnesol accumulation. Farnesol is a well-characterized inhibitor of hypha formation; hence, a reduction in farnesol levels in zap1Δ/Δ biofilms is unexpected. Our findings argue that a Zap1- and zinc-dependent signal affects the yeast-hypha balance and that it is operative in the low-farnesol environment of the zap1Δ/Δ biofilm. In addition, our results indicate that Zap1 is a positive regulator of farnesol accumulation.
Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Proteínas Fúngicas/metabolismo , Transdução de Sinais , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Transporte de Cátions/biossíntese , Farneseno Álcool/análise , Farneseno Álcool/metabolismo , Farneseno Álcool/farmacologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Cromatografia Gasosa-Espectrometria de Massas , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hifas/genética , Hifas/metabolismo , Glicoproteínas de Membrana/biossíntese , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/genéticaRESUMO
To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Delta mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Delta cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Delta cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Delta cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.
Assuntos
Carboxiliases/metabolismo , Citocinese , Fosfatidiletanolaminas/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Carboxiliases/química , Carboxiliases/genética , Meios de Cultura/metabolismo , Dados de Sequência Molecular , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de SequênciaRESUMO
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.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fosfatos de Inositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas Ativadoras de GTPase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The turnover of phospholipids plays an essential role in membrane lipid homeostasis by impacting both lipid head group and acyl chain composition. This review focusses on the degradation and acyl chain remodeling of the major phospholipid classes present in the ER membrane of the reference eukaryote Saccharomyces cerevisiae, i.e. phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Phospholipid turnover reactions are introduced, and the occurrence and important functions of phospholipid remodeling in higher eukaryotes are briefly summarized. After presenting an inventory of established mechanisms of phospholipid acyl chain exchange, current knowledge of phospholipid degradation and remodeling by phospholipases and acyltransferases localized to the yeast ER is summarized. PC is subject to the PC deacylation-reacylation remodeling pathway (PC-DRP) involving a phospholipase B, the recently identified glycerophosphocholine acyltransferase Gpc1p, and the broad specificity acyltransferase Ale1p. PI is post-synthetically enriched in C18:0 acyl chains by remodeling reactions involving Cst26p. PE may undergo turnover by the phospholipid: diacylglycerol acyltransferase Lro1p as first step in acyl chain remodeling. Clues as to the functions of phospholipid acyl chain remodeling are discussed.
Assuntos
Retículo Endoplasmático/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acilação , Animais , Retículo Endoplasmático/química , Humanos , Fosfatidilcolinas/análise , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/análise , Fosfatidilinositóis/metabolismo , Fosfolipídeos/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.
Assuntos
Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Colina/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Deleção de Genes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Espectrometria de Massas , Fenótipo , Fosfatidilcolinas/química , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/metabolismo , Fosfolipídeos/química , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estearoil-CoA Dessaturase , TemperaturaRESUMO
Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.