Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de Novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation

Abstract Cellular homeostasis is maintained by the proteasomal degradation of regulatory and misfolded proteins, which sustains the amino acid pool. Although proteasomes alleviate stress by removing damaged proteins, mounting evidence indicates that severe stress caused by salt, metal(oids), and some pathogens can impair the proteasome. However, the consequences of proteasome inhibition in plants are not well understood and even less is known about how its malfunctioning alters metabolic activities. Lethality causes by proteasome inhibition in non-photosynthetic organisms stem from amino acid depletion, and we hypothesized that plants respond to proteasome inhibition by increasing amino acid biosynthesis. To address these questions, the short-term effects of proteasome inhibition were monitored for 3, 8 and 48 h in the roots of Brassica napus treated with the proteasome inhibitor MG132. Proteasome inhibition did not affect the pool of free amino acids after 48 h, which was attributed to elevated de novo amino acid synthesis; these observations coincided with increased levels of sulfite reductase and nitrate reductase activities at earlier time points. However, elevated amino acid synthesis failed to fully restore protein synthesis. In addition, transcriptome analysis points to perturbed abscisic acid signaling and decreased sugar metabolism after 8 h of proteasome inhibition. Proteasome inhibition increased the levels of alternative oxidase but decreased aconitase activity, most sugars and tricarboxylic acid metabolites in root tissue after 48 h. These metabolic responses occurred before we observed an accumulation of reactive oxygen species. We discuss how the metabolic response to proteasome inhibition and abiotic stress partially overlap in plants.

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The inorganic carbon requirements for nitrogen assimilation

Canadian Journal of Botany ◽

10.1139/b91-146 ◽

1991 ◽

Vol 69 (5) ◽

pp. 1139-1145 ◽

Cited By ~ 26

Author(s):

David H. Turpin ◽

Greg C. Vanlerberghe ◽

Alan M. Amory ◽

Robert D. Guy

Keyword(s):

Amino Acid ◽

Phosphoenolpyruvate Carboxylase ◽

Nitrogen Assimilation ◽

Inorganic Carbon ◽

Dissolved Inorganic Carbon ◽

Sufficient Conditions ◽

Acid Synthesis ◽

Amino Acid Synthesis ◽

Bisphosphate Carboxylase ◽

N Assimilation

In the green alga Selenastrum minutum (Naeg.) Collins the assimilation of NH4+ into the full suite of protein amino acids requires at least three separate and distinct inorganic carbon fixing reactions, catalyzed by the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), and carbamoyl phosphate synthetase. In this paper we examine the requirements for CO2 fixation of NH4+ assimilation in this organism. When grown under N-sufficient conditions, NH4+ assimilation is directly dependent upon photosynthetic CO2 fixation to provide carbon skeletons for amino acid synthesis. When cultured under N-limited conditions, the cells accumulate starch, which is then available for amino acid synthesis. This alleviates the requirement of photosynthetic CO2 fixation for NH4+ assimilation. N-limited cells, however, still exhibit a nonphotosynthetic CO2 requirement for N assimilation that is mediated through PEPC. This activity of PEPC increases during N assimilation to replenish TCA cycle intermediates consumed during amino acid synthesis. The in vivo activity of this enzyme is tightly regulated so that there are ~0.3 moles C fixed per mole N assimilated. In S. minutum PEPC is regulated primarily by the ratio of glutamine/glutamate, thus providing a mechanism by which primary NH4+ assimilation modulates the supply of carbon for amino acid biosynthesis. Activation of PEPC during NH4+ assimilation occurs in both the light and the dark. Key words: dissolved inorganic carbon, nitrogen assimilation, phosphoenolpyruvate carboxylase, photosynthesis, amino acid synthesis, respiration.

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Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

Applied and Environmental Microbiology ◽

10.1128/aem.02135-09 ◽

2010 ◽

Vol 76 (5) ◽

pp. 1507-1515 ◽

Cited By ~ 36

Author(s):

Motoyuki Shimizu ◽

Tatsuya Fujii ◽

Shunsuke Masuo ◽

Naoki Takaya

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aspergillus Nidulans ◽

De Novo ◽

Acid Synthesis ◽

Branched Chain Amino Acids ◽

Amino Acid Synthesis ◽

Lactate Dehydrogenase Activity ◽

Branched Chain Amino Acid ◽

Branched Chain

ABSTRACT Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD+ and NADPH/NADP+ under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD+ and NADP+ regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

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Glucose and insulin effects on de novo amino acid synthesis in young men: Studies with stable isotope labeled alanine, glycine, leucine, and lysine

Metabolism ◽

10.1016/0026-0495(82)90006-3 ◽

1982 ◽

Vol 31 (12) ◽

pp. 1210-1218 ◽

Cited By ~ 68

Author(s):

Jean-Jacques Robert ◽

Dennis M. Bier ◽

X.H. Zhao ◽

Dwight E. Matthews ◽

Vernon R. Young

Keyword(s):

Amino Acid ◽

Stable Isotope ◽

De Novo ◽

Young Men ◽

Acid Synthesis ◽

Amino Acid Synthesis

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Transient increase of de novo amino acid synthesis and its physiological significance in water-stressed white clover

Functional Plant Biology ◽

10.1071/fp05022 ◽

2005 ◽

Vol 32 (9) ◽

pp. 831 ◽

Cited By ~ 12

Author(s):

Bok-Rye Lee ◽

Woo-Jin Jung ◽

Kil-Yong Kim ◽

Jean-Christophe Avice ◽

Alain Ourry ◽

...

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Water Deficit ◽

White Clover ◽

De Novo ◽

Acid Synthesis ◽

Ammonia Concentration ◽

De Novo Synthesis ◽

Amino Acid Synthesis ◽

De Novo Protein Synthesis

In white clover (Trifolium repens L. cv. Regal) the kinetics of de novo synthesis of amino acid and protein were compared by tracing 15N under well-watered (control) or water-deficit conditions. The physiological relationship between ammonia concentration, in response to the change in leaf water parameters, and de novo synthesis of amino acid and protein was also assessed. Leaf and root dry mass were not significantly affected for the first 3 d, whereas metabolic parameters such as total N and ammonia were significantly affected within the first day of water-deficit treatment. Inhibitory effect of water deficit on N acquisition from the soil was significant throughout the experimental period. Water deficit induced a significant increase in ammonia concentration in leaves during the first 3 d, and in roots for only the first day. In both leaves and roots, an increase in de novo amino acid synthesis, which peaked in leaves within the first 3 d of water-deficit treatment (Ψw ≥ –1.18 MPa), was observed. The rate of decrease in de novo protein synthesis gradually accelerated as the duration of the water-deficit treatment increased. There was a significant positive relationship between ammonia production and the increase in de novo amino acid synthesis during the first 3-d period, but not during the later period (day 3–day 7). This experiment clearly indicates that the increase in de novo amino acid synthesis caused by water deficit is a transient adaptive response occurring during the first few days and that it is associated with the increased ammonia concentrations, which in turn arise in response to a decrease in de novo protein synthesis.

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De novo amino acid synthesis and turnover during N2 fixation

Limnology and Oceanography ◽

10.1002/lno.10755 ◽

2017 ◽

Vol 63 (3) ◽

pp. 1076-1092 ◽

Cited By ~ 4

Author(s):

Natalie Loick-Wilde ◽

Sarah C. Weber ◽

Elvita Eglite ◽

Iris Liskow ◽

Detlef Schulz-Bull ◽

...

Keyword(s):

Amino Acid ◽

N2 Fixation ◽

De Novo ◽

Acid Synthesis ◽

Amino Acid Synthesis

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Whole Body De Novo Amino Acid Synthesis in Type I (Insulin-dependent) Diabetes Studied With Stable Isotope-labeled Leucine, Alanine, and Glycine

Diabetes ◽

10.2337/diab.34.1.67 ◽

1985 ◽

Vol 34 (1) ◽

pp. 67-73 ◽

Cited By ~ 29

Author(s):

J. J. Robert ◽

B. Beaufrere ◽

J. Koziet ◽

J. F. Desjeux ◽

D. M. Bier ◽

...

Keyword(s):

Amino Acid ◽

Stable Isotope ◽

De Novo ◽

Acid Synthesis ◽

Insulin Dependent Diabetes ◽

Whole Body ◽

Type I ◽

Amino Acid Synthesis ◽

Insulin Dependent

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Pyruvate metabolism by Anaplasma marginale in cell-free culture

Canadian Journal of Microbiology ◽

10.1139/w98-215 ◽

1999 ◽

Vol 45 (2) ◽

pp. 185-189 ◽

Cited By ~ 2

Author(s):

Linda E McHolland ◽

Daniel R Caldwell

Keyword(s):

Amino Acid ◽

De Novo ◽

Anaplasma Marginale ◽

Acid Synthesis ◽

Pyruvate Metabolism ◽

Amino Acid Synthesis ◽

Free System ◽

Label Incorporation ◽

A Cell ◽

Body Components

Partially purified Anaplasma marginale initial bodies were cultivated in a cell-free system in the presence of [3-14C]pyruvate for 24 or 48 h. Experiments showed that a significant portion of the pyruvate supplied to the cultures was incorporated into initial body components. Label incorporation was reduced by 72% in the presence of oxytetracycline. Fractionation and chromatography of the organisms revealed radioactive incorporation as alanine. This is the first report of de novo amino acid synthesis by A. marginale demonstrating that the rickettsia is capable of using pyruvate, an erythrocyte glycolytic product, in its metabolism.Key words: Anaplasma marginale, pyruvate metabolism, amino acid synthesis.

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