scholarly journals Independent evolution of polymerization in the Actin ATPase clan regulates hexokinase activity

2019 ◽  
Author(s):  
Patrick R Stoddard ◽  
Eric M. Lynch ◽  
Daniel P. Farrell ◽  
Quincey A. Justman ◽  
Annie M. Dosey ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Maximum Rate ◽  
Protein Fold ◽  
Hexokinase Activity ◽  
Sugar Addition ◽  
Glucose Phosphorylation ◽  
Ascomycete Fungi ◽  
Actin Protein

AbstractThe actin protein fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, like the carbohydrate kinases, do not polymerize. We find that Glk1, aSaccharomyces cerevisiaeglucokinase, forms two-stranded filaments with unique ultrastructure, distinct from that of cytoskeletal polymers. In cells, Glk1 polymerizes upon sugar addition and depolymerizes upon sugar withdrawal. Glk1 polymerization inhibits its enzymatic activity, thus the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation eliminating Glk1 polymerization alleviates concentration-dependent enzyme inhibition, causing glucokinase activity to become unconstrained. Polymerization-based regulation of Glk1 activity serves an important functionin vivo: yeast containing non-polymerizing Glk1 are less fit when growing on sugars and more likely to die when refed glucose. Glucokinase polymerization arose within the ascomycete fungi and is conserved across a group of divergent (150-200 mya) yeast. We show that Glk1 polymerization arose independently from other actin-related filaments and allows yeast to rapidly modulate glucokinase activity as nutrient availability changes.One-sentence summaryYeast glucokinase activity is limited by its polymerization, which is critical for cell viability during glucose refeeding.

scholarly journals Polymerization in the actin ATPase clan regulates hexokinase activity in yeast

Science ◽  
2020 ◽  
Vol 367 (6481) ◽  
pp. 1039-1042 ◽  
Author(s):  
Patrick R. Stoddard ◽  
Eric M. Lynch ◽  
Daniel P. Farrell ◽  
Annie M. Dosey ◽  
Frank DiMaio ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Enzyme Inhibition ◽  
Nutrient Availability ◽  
Maximum Rate ◽  
Metabolic Enzymes ◽  
Hexokinase Activity ◽  
Sugar Addition ◽  
Glucose Phosphorylation ◽  
In Cells

The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.

scholarly journals Trm11p and Trm112p Are both Required for the Formation of 2-Methylguanosine at Position 10 in Yeast tRNA

2005 ◽  
Vol 25 (11) ◽  
pp. 4359-4370 ◽  
Author(s):  
Suresh K. Purushothaman ◽  
Janusz M. Bujnicki ◽  
Henri Grosjean ◽  
Bruno Lapeyre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Zinc Binding ◽  
Growth Defect ◽  
Putative Protein ◽  
Yeast Trna ◽  
Protein Methyltransferase ◽  
Severe Growth Defect ◽  
Severe Growth

ABSTRACT N 2 -Monomethylguanosine-10 (m2G10) and N 2 ,N 2 -dimethylguanosine-26 (m2 2G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m2 2G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m2G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m2G10 and m2 2G26 affects tRNA metabolism or functioning.

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

2011 ◽  
Vol 392 (6) ◽  
Author(s):  
Patrycja Zembek ◽  
Urszula Perlińska-Lenart ◽  
Katarzyna Rawa ◽  
Wioletta Górka-Nieć ◽  
Grażyna Palamarczyk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Analysis ◽  
Cell Wall ◽  
Enzymatic Activity ◽  
Trichoderma Reesei ◽  
Cell Wall Composition ◽  
Regulatory Subunit ◽  
Dolichyl Phosphate ◽  
Key Enzyme

AbstractInTrichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, thedpm2anddpm3genes coding for the DPMII and DPMIII subunits ofT. reeseiDPM synthase were cloned and functionally analyzed after expression in theSaccharomyces cerevisiae dpm1Δ[genotype (BY4743;his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterizedS. cerevisiaestrains expressing thedpm1,2,3ordpm1, 3genes and analyzed the consequences ofdpmexpression on protein O-glycosylationin vivoand on the cell wall composition.

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (12) ◽  
pp. 5643-5649
Author(s):  
H Ma ◽  
L M Bloom ◽  
C T Walsh ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Point Mutations ◽  
Inverse Correlation ◽  
Invertase Activity ◽  
Yeast Cells ◽  
Hexokinase Ii ◽  
Mutant Proteins ◽  
Hexokinase Activity

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.

scholarly journals The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (12) ◽  
pp. 5643-5649 ◽  
Author(s):  
H Ma ◽  
L M Bloom ◽  
C T Walsh ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Point Mutations ◽  
Inverse Correlation ◽  
Invertase Activity ◽  
Yeast Cells ◽  
Hexokinase Ii ◽  
Mutant Proteins ◽  
Hexokinase Activity

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.

scholarly journals Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions

2004 ◽  
Vol 24 (16) ◽  
pp. 6931-6946 ◽  
Author(s):  
Jason C. Tanny ◽  
Donald S. Kirkpatrick ◽  
Scott A. Gerber ◽  
Steven P. Gygi ◽  
Danesh Moazed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Protein Interactions ◽  
Budding Yeast ◽  
Protein A ◽  
Physical Interaction ◽  
Dna Repeats ◽  
Protein Protein Interactions

ABSTRACT Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.

scholarly journals PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (10) ◽  
pp. 5744-5753 ◽  
Author(s):  
C E Brown ◽  
S Z Tarun ◽  
R Boeck ◽  
A B Sachs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
A Subunit ◽  
Two Hybrid

The Pab1p-dependent poly(A) nuclease (PAN) from Saccharomyces cerevisiae copurifies with polypeptides of approximately 127 and 76 kDa. Previously, it was demonstrated that the 127-kDa Pan2 protein is required for PAN activity (R. Boeck, S. Tarun, M. Reiger, J. Deardorff, S. Müller-Auer, and A.B. Sachs, J. Biol. Chem. 271:432-438, 1996). Here we demonstrate that the 76-kDa protein, encoded by the nonessential PAN3 gene, is also required for enzymatic activity. Deletion of PAN3 resulted in the loss of PAN activity in yeast extracts, and immunodepletion of Pan3p from purified PAN fractions abolished enzymatic activity. We show by coimmunoprecipitation and directed two-hybrid studies that the Pan2 and Pan3 proteins physically interact. In addition, we demonstrate that a deletion of PAN2, PAN3, or both resulted in similar increases in mRNA poly(A) tail lengths in vivo. These data strongly suggest that both Pan2p and Pan3p are required subunits of the PAN enzyme and that PAN functions in vivo to shorten mRNA poly(A) tails.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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