ECS-based investigation of chloroplast ATP synthase regulation

Mapping Intimacies ◽

10.1101/2020.04.28.066100 ◽

2020 ◽

Author(s):

Felix Buchert ◽

Benjamin Bailleul ◽

Pierre Joliot

Keyword(s):

Atp Synthase ◽

Redox Regulation ◽

Spectroscopic Method ◽

Atp Synthesis ◽

Redox States ◽

Chloroplast Atp Synthase ◽

Γ Subunit ◽

Synthase Regulation

AbstractThe chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain which contains a cysteine couple and interacts with the torque-generating βDELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock. In vitro, γ-disulfide formation slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient . Here, we utilize in vivo absorption spectroscopy measurements for functional CF1Fo activity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure the present in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extra generated by an illumination, including its osmotic and electric components, and from there we estimate the lifetime of the light-generated ATP. In contrast with a previous report [Joliot and Joliot, Biochim. Biophys. Acta, 1777 (2008) 676-683], the CF1Fo γ-subunit exists mostly in an oxidized form in the dark-adapted state. To study the redox regulation of the CF1Fo, we used thiol agent infiltration in WT and a mutant that does not form the γ-disulfide. The obtained -dependent CF1Fo activity profile in the two γ-redox states in vivo reconciles with previous biochemical in vitro findings [Junesch and Gräber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two γ-redox state were similar at high . In the presence of the γ-dithiol, similar rates were obtained at a ~45 mV lower value compared to the oxidized state, which closely resembled the energetic gap of 0.7 ΔpH units reported in vitro.

Download Full-text

Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

Journal of Biological Chemistry ◽

10.1074/jbc.m115.665059 ◽

2015 ◽

Vol 290 (34) ◽

pp. 21032-21041 ◽

Cited By ~ 20

Author(s):

Naman B. Shah ◽

Thomas M. Duncan

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Intrinsic Rate ◽

Synthesis Rate ◽

Final Segment ◽

Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

Download Full-text

Abstract MP247: Mitochondrial Complex V Is Regulated By GRK2 In The Heart

Circulation Research ◽

10.1161/res.129.suppl_1.mp247 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Kimberly Ferrero ◽

Jessica M Pfleger ◽

Kurt Chuprun ◽

Eric Barr ◽

Erhe Gao ◽

...

Keyword(s):

Atp Synthase ◽

Cardiac Injury ◽

Atp Synthesis ◽

Cardiac Metabolism ◽

Neonatal Rat ◽

Interacting Proteins ◽

Atp Production

The GPCR kinase GRK2 is highly expressed the heart; importantly, during cardiac injury or heart failure (HF) both levels and activity of GRK2 increase. The role of GRK2 during HF is canonically studied upstream of β-adrenergic desensitization. However, GRK2 has a large interactome and noncanonical functions for this kinase are being uncovered. We have discovered that in the heart, GRK2 translocates to mitochondria ( mtGRK2 ) following injury and is associated with negative effects on cardiac metabolism. Thus, we have sought to identify the mechanism(s) by which GRK2 can regulate mitochondrial function. We hypothesize that mtGRK2 interacts with proteins which regulate bioenergetics and substrate utilization, and this never-before-described role may partially explain the altered mitochondrial phenotype seen following cardiac injury or HF. Stress-induced mitochondrial translocation of GRK2 was validated in neonatal rat ventricular myocytes, murine heart tissue and a cardiac-derived cell line. Consequently, the GRK2 interactome was mapped basally and under stress conditions in vitro, in vivo , and with tagged recombinant peptides. GRK2-interacting proteins were isolated via immunoprecipitation and analyzed via liquid chromatography-mass spectroscopy (LCMS). Proteomics analysis (IPA; Qiagen) identified mtGRK2 interacting proteins which were also involved in mitochondrial dysfunction. Excitingly, Complexes I, II, IV and V (ATP synthase) of the electron transport chain (ETC) were identified in the subset of mtGRK2-dysfunction partners. Several mtGRK2-ETC interactions were increased following stress, particularly those in Complex V. We further established that mtGRK2 phosphorylates some of the subunits of Complex V, particularly the ATP synthase barrel which is critical for ATP production in the heart. Specific amino acid residues on these subunits have been identified using PTM-LCMS and are currently being validated in a murine model of myocardial infarction. To support these data, we have also determined that alterations in either the levels or kinase activity of GRK2 appear to alter the enzymatic activity of Complex V in vitro , thus altering ATP production. In summary, the phosphorylation of the ATP synthesis machinery by mtGRK2 may be regulating some of the phenotypic effects of injured or failing hearts such as increased ROS production and reduced fatty acid metabolism. Research is ongoing in our lab to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death, thus uncovering an exciting, druggable novel target for rescuing cardiac function in patients with injured and/or failing hearts.

Download Full-text

Feedback limitation of photosynthesis at high CO2 acts by modulating the activity of the chloroplast ATP synthase

Functional Plant Biology ◽

10.1071/fp09129 ◽

2009 ◽

Vol 36 (11) ◽

pp. 893 ◽

Cited By ~ 30

Author(s):

Olavi Kiirats ◽

Jeffrey A. Cruz ◽

Gerald E. Edwards ◽

David M. Kramer

Keyword(s):

Electron Transfer ◽

Atp Synthase ◽

Atp Synthesis ◽

Nicotiana Sylvestris ◽

O2 Evolution ◽

High Co2 ◽

Chloroplast Atp Synthase ◽

Low Co2 ◽

Photosynthetic Electron Transfer

It was previously shown that photosynthetic electron transfer is controlled under low CO2 via regulation of the chloroplast ATP synthase. In the current work, we studied the regulation of photosynthesis under feedback limiting conditions, where photosynthesis is limited by the capacity to utilise triose-phosphate for synthesis of end products (starch or sucrose), in a starch-deficient mutant of Nicotiana sylvestris Speg. & Comes. At high CO2, we observed feedback control that was progressively reversed by increasing O2 levels from 2 to 40%. The activity of the ATP synthase, probed in vivo by the dark-interval relaxation kinetics of the electrochromic shift, was proportional to the O2-induced increases in O2 evolution from PSII (JO2), as well as the sum of Rubisco oxygenation (vo) and carboxylation (vc) rates. The altered ATP synthase activity led to changes in the light-driven proton motive force, resulting in regulation of the rate of plastoquinol oxidation at the cytochrome b6f complex, quantitatively accounting for the observed control of photosynthetic electron transfer. The ATP content of the cell decreases under feedback limitation, suggesting that the ATP synthesis was downregulated to a larger extent than ATP consumption. This likely resulted in slowing of ribulose bisphosphate regeneration and JO2). Overall, our results indicate that, just as at low CO2, feedback limitations control the light reactions of photosynthesis via regulation of the ATP synthase, and can be reconciled with regulation via stromal Pi, or an unknown allosteric affector.

Download Full-text

C-Terminal Mutations in the Chloroplast ATP Synthase γ Subunit Impair ATP Synthesis and Stimulate ATP Hydrolysis†

Biochemistry ◽

10.1021/bi701581y ◽

2008 ◽

Vol 47 (2) ◽

pp. 836-844 ◽

Cited By ~ 7

Author(s):

Feng He ◽

Hardeep S. Samra ◽

Eric A. Johnson ◽

Nicholas R. Degner ◽

Richard E. McCarty ◽

...

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Chloroplast Atp Synthase ◽

Γ Subunit

Download Full-text

Functional analysis of membranous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis

Biochemical Journal ◽

10.1042/bj20111284 ◽

2012 ◽

Vol 442 (3) ◽

pp. 631-638 ◽

Cited By ~ 38

Author(s):

Yutetsu Kuruma ◽

Toshiharu Suzuki ◽

Sakurako Ono ◽

Masasuke Yoshida ◽

Takuya Ueda

Keyword(s):

Protein Synthesis ◽

Atp Synthase ◽

Transmembrane Helix ◽

Atp Synthesis ◽

Membrane Vesicles ◽

F1fo Atp Synthase ◽

Pure System ◽

A Subunit

The a subunit of F1Fo (F1Fo-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H+-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed Foa-less F1Fo complex in Escherichia coli membrane vesicles and liposomes. The resulting F1Fo has a H+-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F1Fo. By using this procedure, we analysed five mutations of F1Fo, where the conserved residues in the a subunit (Asn90, Asp112, Arg169, Asn173 and Gln217) were individually replaced with alanine. All of the mutant Foa subunits were successfully incorporated into F1Fo, showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F1Fo. The N173A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn90 is located in the middle of putative second transmembrane helix and likely to play an important role in H+-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.

Download Full-text

Structural basis of redox modulation on chloroplast ATP synthase

Communications Biology ◽

10.1038/s42003-020-01221-8 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Jay-How Yang ◽

Dewight Williams ◽

Eaazhisai Kandiah ◽

Petra Fromme ◽

Po-Lin Chiu

Keyword(s):

Atp Synthase ◽

Higher Plants ◽

Atp Synthesis ◽

Smooth Transition ◽

Structural Basis ◽

Energy Regulation ◽

Redox Modulation ◽

Disulfide Linkage ◽

Chloroplast Atp Synthase ◽

Γ Subunit

AbstractIn higher plants, chloroplast ATP synthase has a unique redox switch on its γ subunit that modulates enzyme activity to limit ATP hydrolysis at night. To understand the molecular details of the redox modulation, we used single-particle cryo-EM to determine the structures of spinach chloroplast ATP synthase in both reduced and oxidized states. The disulfide linkage of the oxidized γ subunit introduces a torsional constraint to stabilize the two β hairpin structures. Once reduced, free cysteines alleviate this constraint, resulting in a concerted motion of the enzyme complex and a smooth transition between rotary states to facilitate the ATP synthesis. We added an uncompetitive inhibitor, tentoxin, in the reduced sample to limit the flexibility of the enzyme and obtained high-resolution details. Our cryo-EM structures provide mechanistic insight into the redox modulation of the energy regulation activity of chloroplast ATP synthase.

Download Full-text

Two genes, atpC1 and atpC2, for the γ subunit of Arabidopsis thaliana chloroplast ATP synthase

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)89450-2 ◽

1991 ◽

Vol 266 (12) ◽

pp. 7333-7338

Author(s):

N Inohara ◽

A Iwamoto ◽

Y Moriyama ◽

S Shimomura ◽

M Maeda ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Atp Synthase ◽

Chloroplast Atp Synthase ◽

Γ Subunit

Download Full-text

Sulfasalazine modifies metabolic profiles and enhances cisplatin chemosensitivity on cholangiocarcinoma cells in in vitro and in vivo models

Cancer & Metabolism ◽

10.1186/s40170-021-00249-6 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Malinee Thanee ◽

Sureerat Padthaisong ◽

Manida Suksawat ◽

Hasaya Dokduang ◽

Jutarop Phetcharaburanin ◽

...

Keyword(s):

Redox Regulation ◽

Treated Group ◽

Control Group ◽

High Recurrence Rate ◽

Nucleic Acid Metabolism ◽

In Vivo Models ◽

Tryptophan Degradation ◽

Xenograft Mice

Abstract Background Sulfasalazine (SSZ) is widely known as an xCT inhibitor suppressing CD44v9-expressed cancer stem-like cells (CSCs) being related to redox regulation. Cholangiocarcinoma (CCA) has a high recurrence rate and no effective chemotherapy. A recent report revealed high levels of CD44v9-positive cells in CCA patients. Therefore, a combination of drugs could prove a suitable strategy for CCA treatment via individual metabolic profiling. Methods We examined the effect of xCT-targeted CD44v9-CSCs using sulfasalazine combined with cisplatin (CIS) or gemcitabine in CCA in vitro and in vivo models and did NMR-based metabolomics analysis of xenograft mice tumor tissues. Results Our findings suggest that combined SSZ and CIS leads to a higher inhibition of cell proliferation and induction of cell death than CIS alone in both in vitro and in vivo models. Xenograft mice showed that the CD44v9-CSC marker and CK-19-CCA proliferative marker were reduced in the combination treatment. Interestingly, different metabolic signatures and significant metabolites were observed in the drug-treated group compared with the control group that revealed the cancer suppression mechanisms. Conclusions SSZ could improve CCA therapy by sensitization to CIS through killing CD44v9-positive cells and modifying the metabolic pathways, in particular tryptophan degradation (i.e., kynurenine pathway, serotonin pathway) and nucleic acid metabolism.

Download Full-text

Determining the Rate-Limiting Step for Light-Responsive Redox Regulation in Chloroplasts

Antioxidants ◽

10.3390/antiox7110153 ◽

2018 ◽

Vol 7 (11) ◽

pp. 153 ◽

Cited By ~ 6

Author(s):

Keisuke Yoshida ◽

Toru Hisabori

Keyword(s):

Redox Regulation ◽

Reducing Power ◽

Rubisco Activase ◽

Power Transfer ◽

Target Proteins ◽

Rate Limiting Step ◽

Limiting Step ◽

Rate Limiting

Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.

Download Full-text