Effects of osmotic stress and growth stage on cellular pH and polyphosphate metabolism in Neurospora crassa as studied by 31P nuclear magnetic resonance spectroscopy

Yang, YunChung; Bastos, Margarita; Chen, Kuang Yu

doi:10.1016/0167-4889(93)90135-c

Cited by 37 publications

(26 citation statements)

References 23 publications

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“…Similar studies have been reported in eukaryotic cells, such as yeast (46), fungi (47), and algae (48). It has long been recognized that the poly P content is low during rapid growth and increases under conditions of nutritional imbalance unfavorable for growth in different organisms from bacteria to yeast (49).…”

Section: Number Of Acidocalcisomes/cellsupporting

confidence: 51%

Target of Rapamycin (TOR)-like 1 Kinase Is Involved in the Control of Polyphosphate Levels and Acidocalcisome Maintenance in Trypanosoma brucei

Jesus

Tonelli

Nardelli

et al. 2010

Journal of Biological Chemistry

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Target of rapamycin (TOR) kinases are highly conserved protein kinases that integrate signals from nutrients and growth factors to coordinate cell growth and cell cycle progression. It has been previously described that two TOR kinases control cell growth in the protozoan parasite Trypanosoma brucei, the causative agent of African trypanosomiasis. Here we studied an unusual TOR-like protein named TbTOR-like 1 containing a PDZ domain and found exclusively in kinetoplastids. TbTORlike 1 localizes to unique cytosolic granules. After hyperosmotic stress, the localization of the protein shifts to the cell periphery, different from other organelle markers. Ablation of TbTOR-like 1 causes a progressive inhibition of cell proliferation, producing parasites accumulating in the S/G 2 phase of the cell cycle. TbTOR-like 1 knocked down cells have an increased area occupied by acidic vacuoles, known as acidocalcisomes, and are enriched in polyphosphate and pyrophosphate. These results suggest that TbTOR-like 1 might be involved in the control of acidocalcisome and polyphosphate metabolism in T. brucei.African trypanosomiasis, or sleeping sickness, is a disease caused by the parasitic protist Trypanosoma brucei that affects a half-million people in sub-Saharan Africa. Transmitted by the tsetse fly vector (Glossina spp.), trypanosomiasis represents an important public health problem and has a strong impact in the economic development of that region. T. brucei has a complex life cycle involving different morphological and functional stages. These parasites alternate between insect and mammalian hosts. The adaptation to these diverse environments requires rapid changes in gene expression to fulfill metabolic or morphological changes (1). This parasite also has the ability to survive a wide range of environmental conditions as it progresses through its life cycle, including extreme fluctuations in external osmolarity (2).Target of rapamycin (TOR) 5 proteins are evolutionarily conserved protein kinases that integrate information from energy levels, mitogenic signals, and nutrient supplies regulating cell growth accordingly. Studies on mammalian cells and yeast signaling pathways have shown that nutrient starvation inhibits TOR activities, which results in G 1 cell cycle arrest, and triggers a stress response program leading to a blockade of translation initiation. Similar stress responses can be observed in cells treated with rapamycin, an immunosuppressant drug, which binds to FKBP12, a prolyl-isomerase, forming a complex with the TOR kinase (reviewed in Refs. 3 and 4).Two distinct aspects of cell growth are regulated by two functionally different TOR kinases in T. brucei, termed TbTOR1 and TbTOR2. TbTOR1 and TbTOR2 function in two distinct TOR-containing multiprotein complexes (TORC) conserved along eukaryote evolution, named TORC1 and TORC2. TbTOR1, located in the nucleus, controls the synthesis and accumulation of cell mass through TORC1 signaling, whereas TbTOR2, associated with the mitochondrion or endoplasmic reticulum, con...

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Section: Number Of Acidocalcisomes/cellsupporting

confidence: 51%

Target of Rapamycin (TOR)-like 1 Kinase Is Involved in the Control of Polyphosphate Levels and Acidocalcisome Maintenance in Trypanosoma brucei

Jesus

Tonelli

Nardelli

et al. 2010

Journal of Biological Chemistry

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“…A key role for polyphosphate in stationary phase cells is further corroborated by the fact that polyphosphate was practically absent in younger (18-24-h-old) mycelium. In N. crassa, the ratio of polyphosphate to orthophosphate in vacuoles increased from 2.4 in early log phase cells to 13.5 in stationary phase cells [15]. When early log phase cells were exposed to a hypo-osmotic shock, both pH cyt and pH vac increased and cells lost 95% of their total polyphosphate content.…”

Section: Discussionmentioning

confidence: 99%

“…A few reports have dealt with cytoplasmic pH of citric acidproducing A. niger mycelium [13,14]. More detailed knowledge about intracellular pH homeostasis in filamentous fungi is to date only available for N. crassa [1,15,16]. To investigate the ability of A. niger to maintain cellular energy levels, cells were subjected to several stresses like low pH ex , citric acid producing conditions, increased proton permeability by an uncoupler and inhibition of ATP synthesis by sodium azide.…”

mentioning

confidence: 99%

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger

Hesse

Ruijter

Dijkema

et al. 2002

European Journal of Biochemistry

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Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31 P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca 2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pH cyt ) and vacuolar pH (pH vac ) values of 7.6 and 6.2, respectively, when the extracellular pH (pH ex ) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a DpH over the cytoplasmic membrane of 6.6-6.7 was reached (pH ex 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH ex 5.8. Perfusion in the presence of various hexoses and pentoses (pH ex 5.8) revealed that the magnitude of DpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger DpH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 lM CCCP), transient (2 lM CCCP) or permanent (10 lM CCCP) collapse of the vacuolar membrane DpH. Nonlethal levels of the metabolic inhibitor azide (N 3 -, 0.1 mM) caused a transient decrease in pH cyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H + influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31 P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH ex (1.8) and a high acidsecreting capacity, pH cyt and pH vac values were still well maintained (pH 7.5 and 6.4, respectively).

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“…The involvement of poly P in the adaptation of eukaryotic cells to osmotic stress has been investigated before in yeast (3), fungi (5), and algae (6 -8) following changes in the 31 P NMR spectra of cells exposed to hyposmotic or hyperosmotic stresses. For example when the algae Dunaliella salina was submitted to hyposmotic conditions there was a rapid hydrolysis of long-chain poly P with generation of shorter poly P chains, whereas hyperosmotic stress resulted in the elongation of poly P chains (58).…”

Section: Discussionmentioning

confidence: 99%

“…Poly P is essential for bacterial responses to stresses and starvation, as well as for survival and virulence (1,2). Similar functions in adaptation to stress have been attributed to poly P in eukaryotic cells such as yeast (3,4), fungi (5), and algae (6 -8). Poly P is also involved in blood clotting (9), eukaryotic cell proliferation (10,11), and induction of apoptosis in plasma and myeloma cells (12).…”

mentioning

confidence: 99%

Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

Fang

Ruiz²,

Docampo

et al. 2007

Journal of Biological Chemistry

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We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn 2؉ , high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in longchain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation. Polyphosphate (poly P)8 is a ubiquitous polymer of a few to several hundred orthophosphate residues linked by high-energy phosphoanhydride bonds. Poly P is found in the environment and in all life forms, where it is involved in a number of functions (1, 2). Poly P is essential for bacterial responses to stresses and starvation, as well as for survival and virulence (1, 2). Similar functions in adaptation to stress have been attributed to poly P in eukaryotic cells such as yeast (3, 4), fungi (5), and algae (6 -8). Poly P is also involved in blood clotting (9), eukaryotic cell proliferation (10, 11), and induction of apoptosis in plasma and myeloma cells (12).In Trypanosoma cruzi, the etiologic agent of Chagas disease, most poly P is accumulated in acidocalcisomes. Acidocalcisomes are acidic, calcium-containing organelles that have been demonstrated to be involved in osmoregulation (13-16). Rapid hydrolysis of acidocalcisome poly P occurs when epimastigotes of T. cruzi are exposed to hyposmotic stress (16) resulting in increased osmolarity and swelling of the organelle (15). In addition, a microtubule and cAMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of an aquaporin results in water movement and a regulatory volume decrease (15), indicating a link between acidocalcisomes and osmotic homeostasis.In eukaryotic cells the hydrolysis of poly P is performed by the action of endopolyphosphatases and exopolyphosphatases (1, 2). Genes encoding for exopolyphosphatases from Saccharomyces cerevisiae (17), Trypanosoma brucei (18), and Leishmania major (19) have been cloned and expressed in Escherichia coli, whereas a gene encoding an endopolyphosphatase from S. cerevisiae (20) is the only one that has been cloned and expressed. The L. major exopolyphosphatase (LmPPx) has been characterized recently and demonstrated to be located in acidocalcisomes and in the cytosol (19). An exopolyphosphatase activity has also been detected in acidocalcisomes of T. cruzi (16). The crystal structures...

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Effects of osmotic stress and growth stage on cellular pH and polyphosphate metabolism in Neurospora crassa as studied by 31P nuclear magnetic resonance spectroscopy

Cited by 37 publications

References 23 publications

Target of Rapamycin (TOR)-like 1 Kinase Is Involved in the Control of Polyphosphate Levels and Acidocalcisome Maintenance in Trypanosoma brucei

Target of Rapamycin (TOR)-like 1 Kinase Is Involved in the Control of Polyphosphate Levels and Acidocalcisome Maintenance in Trypanosoma brucei

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger

Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

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