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1.
Marco Ivn Valle‐Maldonado Jos Alberto Patio‐Medina Carlos Prez‐Arques Nancy Yadira Reyes‐Mares Irvin Eduardo Jcome‐Galarza Rafael Ortíz‐Alvarado Sandeep Vellanki Martha Isela Ramírez‐Díaz Soo Chan Lee Victoriano Garre Víctor Meza‐Carmen 《Cellular microbiology》2020,22(10)
Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G‐protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1‐encoding gene is up‐regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild‐type strain. Mutation in a regulatory subunit of cAMP‐dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild‐type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels. 相似文献
2.
Yarrowia lipolytica is an ascomycete with biotechnological potential. In common media, the fungus grows as a mixture of yeast-like and short mycelial cells. The environmental factors that affect dimorphism in the wild-type strain, W29, and its auxotrophic derivative, PO1a, were analyzed. In both strains, pH was the most important factor regulating the dimorphic transition. Mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to become almost null at pH 3. Carbon and nitrogen sources, namely glucose and ammonium, were also important for mycelium formation; and their effect was antagonized by some alternative carbon and nitrogen sources. Citrate was an important positive effector of mycelium growth. Anaerobic stress induced formation of mycelial cells. The importance of the protein kinase A pathway was suggested by the inhibition of mycelium growth by cAMP. We propose that the interplay of these factors regulates the adaptation of the fungus, to better exploit its natural ecological niches. 相似文献
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As a result of mating of cells carrying plasmid F' lac with cells carrying plasmid F'his merodiploids carrying plasmid complex F'lacF'his were isolated. The plasmid genes of the isolated merodiploids are donated together into the recipient cells and are eliminated jointly from host-cells both spontaneously and by acridine orange. As the formation of the plasmid complex took place in E. coli cells carrying mutation in rec A gene the recombination of plasmids F' is supposed to take place in the absence of the product of gene rec. A. 相似文献
4.
Yarrowia lipolytica is a dimorphic fungus whose morphology is controlled by several factors such as pH and different compounds. To determine if the STE11-mitogen-activated protein kinase (MAPK) pathway plays a role in dimorphism of Y. lipolytica, we isolated the gene encoding a Mapkkk. The isolated gene (STE11) has an ORF of 2832 bp without introns, encoding a protein of 944 amino acids, with a theoretical Mr of 100.9 kDa, that exhibits high homology to fungal Mapkkks. Disruption of the STE11 gene was achieved by the pop-in/pop-out procedure. Growth rate and response to osmotic stress or agents affecting wall integrity were unaffected in the deleted mutants, but they lost the capacity to mate and to grow in the mycelial form. Both alterations were reverted by transformation with the wild-type STE11 gene. The Y. lipolytica STE11 gene driven by two different promoters was unable to complement Saccharomyces cerevisiae ste11Delta mutants, although the gene was transcribed. Also, a wild-type MAPKKK gene from Ustilago maydis failed to complement Y. lipolyticaDeltaste11 mutants. Both negative results were attributed to a failure of the transgenic gene products to interact with the corresponding regulatory and scaffold proteins. This hypothesis was supported by the observation that a truncated version of the U. maydis MAPKKK gene reversed mating and dimorphic defects in the mutants. All these results demonstrate that the MAPK pathway is essential for both morphogenesis and mating in Y. lipolytica. 相似文献
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A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form 总被引:4,自引:0,他引:4
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《The Journal of cell biology》1994,125(1):113-127
The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino- terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms. 相似文献
7.
Banno S Ochiai N Noguchi R Kimura M Yamaguchi I Kanzaki S Murayama T Fujimura M 《Genes & genetic systems》2005,80(1):25-34
A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity. 相似文献
8.
I Abraham K V Chin M M Gottesman J K Mayo K E Sampson 《Experimental cell research》1990,189(1):133-141
Wild-type Chinese hamster ovary (CHO) cells were transfected with a DNA clone (MT-REV, site A) carrying a mouse gene for a dominant mutant regulatory subunit (RI) gene of cAMP-dependent protein kinase (PKA) from S49 cells along with a marker for G418 resistance. G418-resistant transfectant clone R-2D1 was resistant to 8-Br-cAMP-induced growth inhibition and morphological changes. The cells also did not phosphorylate a 50-kDa protein after cAMP stimulation and had decreased PKA activity, both characteristics of PKA mutants. Northern blot analysis indicated that clone R-2D1 was actively transcribing the MT-REV (site A)-specific RNA. We also tested clone R-2D1 for sensitivity to certain natural product hydrophobic drugs and found increased sensitivity to several drugs including adriamycin. Hypersensitivity to these drugs has previously been shown by us to be a characteristic of a CHO PKA mutant cell line. Expression of the mutant RI gene is also associated with a decrease in expression of the multidrug resistance associated P-glycoprotein (gp170) mRNA and protein. These results show that the PKA mutant RI gene from S49 cells acts as a dominant mutation to reduce the total PKA activity in the CHO transfectants as it does in mouse S49 cells. This study also confirms that reduced PKA activity modulates the basal multidrug resistance of these cells, apparently by causing decreased expression of the mdr gene at the protein and mRNA level. 相似文献
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Ramesh MA Laidlaw RD Dürrenberger F Orth AB Kronstad JW 《Fungal genetics and biology : FG & B》2001,32(3):183-193
The cAMP signal transduction pathway mediates the switch between yeast-like and filamentous growth and influences both sexual development and pathogenicity in the smut fungus Ustilago maydis. Signaling via cAMP may also play a role in fungicide resistance in U. maydis. In particular, the adr1 gene, which encodes the catalytic subunit of the U. maydis cAMP-dependent protein kinase (PKA), is implicated in resistance to the dicarboximide and aromatic hydrocarbon fungicides. In this study, we examined the sensitivity of PKA to vinclozolin and could not demonstrate direct inhibition of protein kinase activity. However, we did find that mutants with disruptions in the ubc1 gene, which encodes the regulatory subunit of PKA, were resistant to both vinclozolin and chloroneb. We also found that these fungicides altered the morphology of both wild-type and ubc1 mutant cells. In addition, strains that are defective in ubc1 display osmotic sensitivity, a property often associated with vinclozolin and chloroneb resistance in other fungi. 相似文献
10.
Efficient transition to growth on fermentable carbon sources in Saccharomyces cerevisiae requires signaling through the Ras pathway. 总被引:1,自引:0,他引:1
Strains carrying ras2(318S) as their sole RAS gene fail to elicit a transient increase in cAMP levels following addition of glucose to starved cells but maintain normal steady-state levels of cAMP under a variety of growth conditions. Such strains show extended delays in resuming growth following transition from a quiescent state to glucose-containing growth media, either in emerging from stationary phase or following inoculation as spores onto fresh media. Otherwise, growth of such strains is indistinguishable from that of RAS2(+) strains. ras2(318S) strains also exhibit a delay in glucose-stimulated phosphorylation and turnover of fructose-1,6-bisphosphatase, a substrate of the cAMP-dependent protein kinase A (PKA) and a key component of the gluconeogenic branch of the glycolytic pathway. Finally Tpk(w) strains, which fail to modulate PKA in response to fluctuations in cAMP levels, show the same growth delay phenotypes, as do ras2(318S) strains. These observations indicate that the glucose-induced cAMP spike results in a transient activation of PKA, which is required for efficient transition of yeast cells from a quiescent state to resumption of rapid growth. This represents the first demonstration that yeast cells use the Ras pathway to transmit a signal to effect a biological change in response to an upstream stimulus. 相似文献
11.
Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea. 总被引:8,自引:2,他引:8
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cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis. 相似文献
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The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize. 总被引:4,自引:0,他引:4
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In the plant, filamentous growth is required for pathogenicity of the corn smut pathogen Ustilago maydis. Earlier, we identified a role for the cAMP signal transduction pathway in the switch between budding and filamentous growth for this fungus. A gene designated ubc1 (for Ustilago bypass of cyclase) was found to be required for filamentous growth and to encode the regulatory subunit of a cAMP-dependent protein kinase (PKA). Here, we show that ubc1 is important for the virulence of the pathogen. Specifically, ubc1 mutants are able to colonize maize plants and, like the wild-type pathogen, cause localized symptoms in association with the presence of hyphae. However, in contrast to plants infected with wild-type cells that often developed galls from initially chlorotic tissue, plants infected with the ubc1 mutant did not produce galls. These data suggest that PKA regulation is critical for the transition from saprophytic to pathogenic growth and from vegetative to reproductive development. Plate mating assays in which exogenous cAMP was applied suggested that the cAMP and b mating-type morphogenetic pathways may be coordinated. 相似文献
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Peroxisomal peripheral membrane protein YlInp1p is required for peroxisome inheritance and influences the dimorphic transition in the yeast Yarrowia lipolytica
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Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast Yarrowia lipolytica. Most peroxisomes are anchored at the periphery of cells of Y. lipolytica. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in Y. lipolytica. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in Y. lipolytica, as cells lacking the YlINP1 gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in Y. lipolytica. 相似文献
17.
Role for YakA,cAMP, and protein kinase A in regulation of stress responses of Dictyostelium discoideum cells
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Taminato A Bagattini R Gorjão R Chen G Kuspa A Souza GM 《Molecular biology of the cell》2002,13(7):2266-2275
The Dictyostelium protein kinase YakA is required for the growth-to-development transition. During growth YakA controls the cell cycle, regulating the intervals between cell divisions. When starved for nutrients Dictyostelium cells arrest growth and undergo changes in gene expression, decreasing vegetative mRNAs and inducing the expression of pkaC. YakA is an effector of these changes, being necessary for the decrease of vegetative mRNA expression and the increase of protein kinase A (PKA) activity that will ultimately regulate expression of adenylyl cyclase, cAMP synthesis, and the induction of development. We report a role for this kinase in the response to nitrosoative or oxidative stress of Dictyostelium cells. Hydrogen peroxide and sodium nitroprusside arrest the growth of cells and trigger cAMP synthesis and activation of PKA in a manner similar to the well-established response to nutrient starvation. We have found that yakA null cells are hypersensitive to nitrosoative/oxidative stress and that a second-site mutation in pkaC suppresses this sensitivity. The response to different stresses has been investigated and YakA, cAMP, and PKA have been identified as components of the pathway that regulate the growth arrest that follows treatment with compounds that generate reactive oxygen species. The effect of different types of stress was evaluated in Dictyostelium and the YakA/PKA pathway was also implicated in the response to heat stress. 相似文献
18.
A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A
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Zhang L Duan CJ Binkley C Li G Uhler MD Logsdon CD Simeone DM 《Molecular and cellular biology》2004,24(5):2169-2180
Transforming growth factor beta (TGFbeta) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFbeta signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFbeta activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFbeta activation of CREB, induction of p21(Cip1), and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFbeta and PKA signaling pathways. 相似文献
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We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition. 相似文献
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The Ras-cyclic AMP pathway is connected to other nutrient-regulated signaling pathways and mediates the global stress responses of Saccharomyces cerevisiae. Here, we show that Rom2p, the Rho1 GTP/GDP exchange factor, can mediate stress responses and cell growth via the Ras-cAMP pathways. ROM2 was isolated as a suppresser of heat and NaCl sensitivity caused by the lack of the Ras-GTPase activator Ira2p or of cAMP phosphodiesterases. Subsequent analysis of strains with a rom2 deletion showed that Rom2p is essential for resistance to a variety of stresses caused by freeze-thawing, oxidants, cycloheximide, NaCl, or cobalt ions. Stress sensitivity and the growth defect caused by the rom2 deletion could be suppressed by depleting Ras or protein kinase A (PKA) activity or by overexpressing the high affinity cAMP phosphodiesterase Pde2p. In addition, overexpression of ROM2 could not rescue cells lacking the regulatory subunit of PKA, indicating that the Ras-adenylate, cyclase-PKA cascade is essential for Rom2p-mediated stress responses and cell growth. Deletion of IRA2 exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant, indicating that Rom2p signaling may control Ras independently of IRA2. Increases in cAMP levels were detected in the rom2 deletion mutants, and these were comparable with the effects of an ira2 mutation. The effects of the deletion of ROM2 on sensitivity to hydrogen peroxide, paraquat, and cobalt ions, but not to caffeine, were reduced when a constitutive allele of RHO1 was introduced on a single copy plasmid. However, the effects of the deletion of ROM2 on sensitivity to diamide and NaCl were exacerbated. Taken together, our data indicate that Rom2p can regulate PKA activity by controlling cAMP levels via the Ras-cAMP pathway and that for those stresses related to oxidative stress, this cross-talk is probably mediated via the Rho1p-activated MAPK pathway. 相似文献