scholarly journals Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program

2020 ◽  
Author(s):  
Adhish S. Walvekar ◽  
Ganesh Kadamur ◽  
Sreesa Sreedharan ◽  
Ritu Gupta ◽  
Rajalakshmi Srinivasan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Carbon Metabolism ◽  
Protein Phosphatase ◽  
Ribosome Biogenesis ◽  
Catalytic Subunit ◽  
Master Regulator ◽  
Nucleotide Biosynthesis ◽  
Induction Mechanism ◽  
Precursor Supply

AbstractMethionine, through S-adenosylmethionine, activates multifaceted growth programs where ribosome biogenesis, carbon metabolism, amino acid and nucleotide biosynthesis are induced. This growth program requires activity of the Gcn4 transcription factor (called ATF4 in mammals), which enables metabolic precursor supply essential for anabolism. Here, we discover how the Gcn4 protein is induced by methionine, despite conditions of high translation and anabolism. This induction mechanism is independent of transcription, as well as the conventional Gcn2/eIF2α mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 ubiqitination and therefore degradation is reduced, due to the decreased phosphorylation of this protein. This Gcn4 stabilization is mediated by the activity of the conserved methyltransferase, Ppm1, which specifically methylates the catalytic subunit of protein phosphatase PP2A when methionine is abundant. This methylation of PP2A shifts the balance of Gcn4 to a dephosphorylated state, which stabilizes the protein. The loss of Ppm1, or PP2A-methylation destabilizes Gcn4 when methionine is abundant, and the Gcn4-dependent anabolic program collapses. These findings reveal a novel signaling and regulatory axis, where methionine directs a conserved methyltransferase Ppm1, via its target phosphatase PP2A, to selectively stabilize Gcn4. Thereby, when methionine is abundant, cells conditionally modify a major phosphatase in order to stabilize a metabolic master-regulator and drive anabolism.

scholarly journals Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program

2020 ◽  
Vol 295 (52) ◽  
pp. 18390-18405
Author(s):  
Adhish S. Walvekar ◽  
Ganesh Kadamur ◽  
Sreesa Sreedharan ◽  
Ritu Gupta ◽  
Rajalakshmi Srinivasan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Carbon Metabolism ◽  
Protein Phosphatase ◽  
Ribosome Biogenesis ◽  
Stable State ◽  
High Cell ◽  
Nucleotide Biosynthesis ◽  
Protein Levels ◽  
Phosphatase 2A ◽  
Metabolic Precursors

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, and amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (in which the roles of Gcn4 are not well-studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase 2A (PP2A); our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 toward a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs the conserved methyltransferase Ppm1 via its target phosphatase PP2A to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master regulator and drive anabolism.

scholarly journals The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo.

1994 ◽  
Vol 269 (23) ◽  
pp. 16311-16317 ◽  
Author(s):  
B. Favre ◽  
S. Zolnierowicz ◽  
P. Turowski ◽  
B.A. Hemmings
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Phosphatase 2A

scholarly journals N-Terminal Acetyltransferase Naa40p Whereabouts Put into N-Terminal Proteoform Perspective

2021 ◽  
Vol 22 (7) ◽  
pp. 3690
Author(s):  
Veronique Jonckheere ◽  
Petra Van Damme
Keyword(s):  
Amino Acid ◽  
Subcellular Localization ◽  
Nuclear Import ◽  
Ribosome Biogenesis ◽  
Regulatory Protein ◽  
Ectopic Expression ◽  
Cytoplasmic Localization ◽  
Histone H2a ◽  
Histone H4 ◽  
Nucleolar Proteins

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.

scholarly journals The Role of Arg13 in Protein Phosphatase M tPphA from Thermosynechococcus elongatus

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Jiyong Su ◽  
Karl Forchhammer
Keyword(s):  
Amino Acid ◽  
Protein Phosphatase ◽  
Arginine Residue ◽  
Side Chain ◽  
Wild Type ◽  
Catalytic Center ◽  
Nitrophenyl Phosphate ◽  
Reduced Activity ◽  
Amino Acid Variants

A highly conserved arginine residue is close to the catalytic center of PPM/PP2C-type protein phosphatases. Different crystal structures of PPM/PP2C homologues revealed that the guanidinium side chain of this arginine residue can adopt variable conformations and may bind ligands, suggesting an important role of this residue during catalysis. In this paper, we randomly mutated Arginine 13 of tPphA, a PPM/PP2C-type phosphatase from Thermosynechococcus elongatus, and obtained 18 different amino acid variants. The generated variants were tested towards p-nitrophenyl phosphate and various phosphopeptides. Towards p-nitrophenyl phosphate as substrate, twelve variants showed 3–7 times higher Km values than wild-type tPphA and four variants (R13D, R13F, R13L, and R13W) completely lost activity. Strikingly, these variants were still able to dephosphorylate phosphopeptides, although with strongly reduced activity. The specific inability of some Arg-13 variants to hydrolyze p-nitrophenyl phosphate highlights the importance of additional substrate interactions apart from the substrate phosphate for catalysis. The properties of the R13 variants indicate that this residue assists in substrate binding.

scholarly journals The Growth-Promoting and Stress Response Activities of the Bacillus subtilis GTP Binding Protein Obg Are Separable by Mutation

2008 ◽  
Vol 190 (20) ◽  
pp. 6625-6635 ◽  
Author(s):  
Shrin Kuo ◽  
Borries Demeler ◽  
W. G. Haldenwang
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Binding Protein ◽  
Normal Growth ◽  
Gtp Binding Protein ◽  
Missense Change ◽  
Gtp Binding ◽  
Growth Promoting ◽  
Carboxy Terminal

ABSTRACT Bacillus subtilis Obg is a ribosome-associating GTP binding protein that is needed for growth, sporulation, and induction of the bacterium's general stress regulon (GSR). It is unclear whether the roles of Obg in sporulation and stress responsiveness are direct or a secondary effect of its growth-promoting functions. The present work addresses this question by an analysis of two obg alleles whose phenotypes argue for direct roles for Obg in each process. The first allele [obg(G92D)] encodes a missense change in the protein's highly conserved “obg fold” region. This mutation impairs cell growth and the ability of Obg to associate with ribosomes but fails to block sporulation or the induction of the GSR. The second obg mutation [obg(Δ22)] replaces the 22-amino-acid carboxy-terminal sequence of Obg with an alternative 26-amino-acid sequence. This Obg variant cofractionates with ribosomes and allows normal growth but blocks sporulation and impairs the induction of the GSR. Additional experiments revealed that the block on sporulation occurs early, preventing the activation of the essential sporulation transcription factor Spo0A, while inhibition of the GSR appears to involve a failure of the protein cascade that normally activates the GSR to effectively catalyze the reactions needed to activate the GSR transcription factor (σB).

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