scholarly journals Synechocystis KaiC3 displays temperature and KaiB dependent ATPase activity and is important for growth in darkness

2019 ◽  
Author(s):  
Anika Wiegard ◽  
Christin Kobler ◽  
Katsuaki Oyama ◽  
Anja Katharina Dorrich ◽  
Chihiro Azai ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Synechococcus Elongatus ◽  
Constant Darkness ◽  
Temperature Dependent ◽  
Clock Proteins ◽  
Circadian Clock Proteins ◽  
Pcc 7942 ◽  
Fine Tune

Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of non-standard KaiC homologs in vitro. KaiC3 displayed ATPase activity, which were lower compared to the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature-dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1 and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a crosstalk between the KaiC1 and KaiC3 based systems.

scholarly journals Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness

2019 ◽  
Vol 202 (4) ◽  
Author(s):  
Anika Wiegard ◽  
Christin Köbler ◽  
Katsuaki Oyama ◽  
Anja K. Dörrich ◽  
Chihiro Azai ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Circadian Clock ◽  
Atp Hydrolysis ◽  
Synechococcus Elongatus ◽  
Constant Darkness ◽  
Content Type ◽  
Clock Proteins ◽  
Pcc 7942 ◽  
Pcc 6803

ABSTRACT Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro. KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

scholarly journals Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

2010 ◽  
Vol 21 (6) ◽  
pp. 871-884 ◽  
Author(s):  
Atanas V. Koulov ◽  
Paul LaPointe ◽  
Bingwen Lu ◽  
Abbas Razvi ◽  
Judith Coppinger ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Protein Misfolding ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Inherited Disease ◽  
Hsp90 Chaperone ◽  
Terminal Domains

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

scholarly journals A New Circadian Class 2 Gene, opcA, Whose Product Is Important for Reductant Production at Night in Synechococcus elongatus PCC 7942

2000 ◽  
Vol 182 (21) ◽  
pp. 6214-6221 ◽  
Author(s):  
Hongtao Min ◽  
Susan S. Golden
Keyword(s):  
Synechococcus Elongatus ◽  
Flavin Mononucleotide ◽  
Circadian Oscillation ◽  
Environmental Cues ◽  
Light Production ◽  
Class 1 ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942 ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT Gene expression in the cyanobacterium Synechococcus elongatus PCC 7942 is under the control of a circadian oscillator, such that peaks and troughs of expression recur with a periodicity of about 24 h in the absence of environmental cues. This can be monitored easily as light production from luciferase gene fusions to S. elongatus promoters. All promoters seem to exhibit circadian oscillation of expression, but the phasing of peak and trough times differs among different genes. The majority of genes are designated class 1, with expression peaks near dusk or subjective dusk (the time corresponding to dusk in the absence of a diurnal cycle). A minority, of which purF is an example, have expression peaks approximately 12 h out of phase with class 1 genes. A screen of Tn5 mutants for those in whichpurF phasing is altered revealed a mutant that carries an insertion in the opcA gene, previously identified as essential for glucose-6-phosphate dehydrogenase function. However, a different enzymatic reporter and in vitro luciferase assays revealed that the expression pattern of the purF promoter is not altered by opcA inactivation, but rather the reduced flavin mononucleotide substrate of luciferase is limiting at the time of the natural circadian peak. The results suggest that OpcA is involved in temporally separated reductant-generating pathways in S. elongatus and that it has a role outside of its function in activating glucose-6-phosphate dehydrogenase. The opcAgene, expected to be cotranscribed with fbp andzwf, was shown to have its own class 2 promoter, whereas the fbp promoter was determined to be in class 1. Thus, opcA expression is likely to be constitutive by virtue of the activity of two promoters in nearly opposite circadian phases.

scholarly journals The Sso7d protein ofSulfolobus solfataricus: in vitro relationship among different activities

Archaea ◽  
2002 ◽  
Vol 1 (2) ◽  
pp. 87-93 ◽  
Author(s):  
Annamaria Guagliardi ◽  
Laura Cerchia ◽  
Mosè Rossi
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Physiological Role ◽  
Protein Aggregates ◽  
Dependent Manner ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Negative Supercoiling

The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeonSulfolobus solfataricusis unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997), induces negative supercoiling (Lopez-Garcia et al. 1998), and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000). In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates.

scholarly journals Mutation of the ATP-Binding Pocket of SSA1 Indicates That a Functional Interaction Between Ssa1p and Ydj1p Is Required for Post-translational Translocation Into the Yeast Endoplasmic Reticulum

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 501-512 ◽  
Author(s):  
Amie J McClellan ◽  
Jeffrey L Brodsky
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Binding Pocket ◽  
Atp Binding ◽  
Dominant Effect ◽  
Functional Interaction ◽  
Mutant Proteins

Abstract The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

scholarly journals Single-molecular and Ensemble-level Oscillations of Cyanobacterial Circadian Clock

2018 ◽  
Author(s):  
Sumita Das ◽  
Tomoki P. Terada ◽  
Masaki Sasai
Keyword(s):  
Atpase Activity ◽  
Circadian Clock ◽  
Single Molecule ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Theoretical Models ◽  
Oscillation Period ◽  
Coarse Grained ◽  
Simulation Results

AbstractWhen three cyanobacterial proteins, KaiA, KaiB, and KaiC, are incubated with ATP in vitro, the phosphorylation level of KaiC hexamers shows stable oscillation with approximately 24 h period. In order to understand this KaiABC clockwork, we need to analyze both the macroscopic synchronization of a large number of KaiC hexamers and the microscopic reactions and structural changes in individual KaiC molecules. In the present paper, we explain two coarse-grained theoretical models, the many-molecule (MM) model and the single-molecule (SM) model, to bridge the gap between macroscopic and microscopic understandings. In the simulation results with these models, ATP hydrolysis drives oscillation of individual KaiC hexamers and ATP hydrolysis is necessary for synchronizing oscillations of a large number of KaiC hexamers. Sensitive temperature dependence of the lifetime of the ADP bound state in the CI domain of KaiC hexamers makes the oscillation period temperature insensitive. ATPase activity is correlated to the frequency of phosphorylation oscillation in the single molecule of KaiC hexamer, which should be the origin of the observed ensemble-level correlation between the ATPase activity and the frequency of phosphorylation oscillation. Thus, the simulation results with the MM and SM models suggest that ATP hydrolysis randomly occurring in each CI domain of individual KaiC hexamers is a key process for oscillatory behaviors of the ensemble of many KaiC hexamers.Significance StatementCyanobacterial proteins, KaiA, KaiB, and KaiC, can reconstitute a circadian clock when they are incubated with ATP in vitro. In order to understand this prototypical oscillator, we need to analyze both synchronization of a macroscopically large number of oscillating molecules and microscopic reactions in individual molecules. We introduced two theoretical models to unify macroscopic and microscopic viewpoints. Simulation results suggest that ATP hydrolysis is necessary for synchronization and temperature compensation and that ATPase activity is correlated to the oscillation frequency in individual molecules. Thus, ATP hydrolysis randomly occurring in individual molecules should determine important features of the ensemble-level oscillation.

scholarly journals ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Christopher Frederick Mugler ◽  
Maria Hondele ◽  
Stephanie Heinrich ◽  
Ruchika Sachdev ◽  
Pascal Vallotton ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
Posttranscriptional Gene Regulation ◽  
The Dead ◽  
Processing Body

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

scholarly journals Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

2020 ◽  
Author(s):  
Andrés López-Perrote ◽  
Nele Hug ◽  
Ana González-Corpas ◽  
Carlos F. Rodríguez ◽  
Marina Serna ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Mrna Decay ◽  
Atp Hydrolysis ◽  
Unknown Mechanism ◽  
Macromolecular Complexes ◽  
Wide Range ◽  
Nonsense Mediated Mrna Decay ◽  
Aaa Atpases ◽  
Hydrolysis Of

AbstractNonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

scholarly journals Role for intestinal CYP2E1 in alcohol-induced circadian gene-mediated intestinal hyperpermeability

2013 ◽  
Vol 305 (2) ◽  
pp. G185-G195 ◽  
Author(s):  
Christopher B. Forsyth ◽  
Robin M. Voigt ◽  
Maliha Shaikh ◽  
Yueming Tang ◽  
Arthur I. Cederbaum ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Cell Monolayer ◽  
Intestinal Cell ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Circadian Gene ◽  
Colon Tissue ◽  
Clock Proteins ◽  
Circadian Clock Proteins

We have shown that alcohol increases Caco-2 intestinal epithelial cell monolayer permeability in vitro by inducing the expression of redox-sensitive circadian clock proteins CLOCK and PER2 and that these proteins are necessary for alcohol-induced hyperpermeability. We hypothesized that alcohol metabolism by intestinal Cytochrome P450 isoform 2E1 (CYP2E1) could alter circadian gene expression ( Clock and Per2), resulting in alcohol-induced hyperpermeability. In vitro Caco-2 intestinal epithelial cells were exposed to alcohol, and CYP2E1 protein, activity, and mRNA were measured. CYP2E1 expression was knocked down via siRNA and alcohol-induced hyperpermeability, and CLOCK and PER2 protein expression were measured. Caco-2 cells were also treated with alcohol or H2O2 with or without N-acetylcysteine (NAC) anti-oxidant, and CLOCK and PER2 proteins were measured at 4 or 2 h. In vivo Cyp2e1 protein and mRNA were also measured in colon tissue from alcohol-fed mice. Alcohol increased CYP2E1 protein by 93% and enzyme activity by 69% in intestinal cells in vitro. Alcohol feeding also increased mouse colonic Cyp2e1 protein by 73%. mRNA levels of Cyp2e1 were not changed by alcohol in vitro or in mouse intestine. siRNA knockdown of CYP2E1 in Caco-2 cells prevented alcohol-induced hyperpermeability and induction of CLOCK and PER2 proteins. Alcohol-induced and H2O2-induced increases in intestinal cell CLOCK and PER2 were significantly inhibited by treatment with NAC. We concluded that our data support a novel role for intestinal CYP2E1 in alcohol-induced intestinal hyperpermeability via a mechanism involving CYP2E1-dependent induction of oxidative stress and upregulation of circadian clock proteins CLOCK and PER2.

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