Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

Gulis, Galina; Narasimhulu, K.; Fox, Lisa N.; Redding, Kevin

doi:10.1007/s11120-007-9283-9

Cited by 30 publications

(30 citation statements)

References 35 publications

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“…To provide an experimental proof for a link between LHCSR3 and the PSI-LHCI-FNR supercomplex, we took advantage of a C. reinhardtii His 6 -tagged PSAA strain (Gulis et al, 2008) and purified the supercomplex from cells grown for 24 h in high light (Fig. 6A) using detergent solubilization of isolated thylakoids and Suc density ultracentrifugation.…”

Section: Resultsmentioning

confidence: 99%

“…Wildtype strain CC124 (nit2 2 , mt 2 ) served as a control for the comparison of photoinhibition sensitivity with C. reinhardtii pgrl1 (Tolleter et al, 2011), npq4 (Peers et al, 2009), and the pgrl1 npq4 double mutant (Kukuczka et al, 2014). The C. reinhardtii PSAA-His strain (Gulis et al, 2008) was employed to purify His-tagged PSI from supercomplex fractions. …”

Section: Strainsmentioning

confidence: 99%

“…To determine whether LHCSR3 was specifically bound to the PSI-LHCI-FNR supercomplex, Ni-NTA chromatography of PSI-containing supercomplexes was carried out using the PSAA-His strain carrying a His 6 -tagged PSAA protein (Gulis et al, 2008).…”

Section: Nickel Affinity Chromatographymentioning

confidence: 99%

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STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

et al. 2015

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In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions. Oxygenic photosynthesis converts solar energy into chemical energy. This energy is utilized for carbon dioxide assimilation, allowing the formation of complex organic material. Plant photosynthesis is performed by a series of reactions in and at the thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). These light reactions are catalyzed by two photosystems (PSI and PSII). A third multiprotein complex, also embedded in the thylakoid membrane, is the cytochrome b 6 f (cyt b 6 f) complex that links photosynthetic electron transfer processes between the two photosystems and functions in proton translocation. The ATP synthase takes advantage of the proton-motive force that is generated by the light reactions (Mitchell, 1961) to produce ATP. ATP and NADPH, generated through linear electron flow from PSII to PSI, drive the CalvinBenson-Bassham cycle (Bassham et al., 1950) to fix CO 2 . Alternatively, cyclic electron flow (CEF) between PSI and the cyt b 6 f complex solely produces ATP (Arnon, 1959).Under normal growth conditions, CEF provides additionally required ATP for CO 2 fixation (Lucker and Kramer, 2013), counteracts overreduction of the PSI acceptor side under stressful environmental cues,...

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Section: Resultsmentioning

confidence: 99%

Section: Strainsmentioning

confidence: 99%

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STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

et al. 2015

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“…Strain and Growth Conditions-A C. reinhardtii strain (JVD-1B[pGG1]) in which a hexahistidine tag had been added to the N terminus of PsaA (38) was grown in liquid Tris-acetate-phosphate medium (39). Cells were cultured at room temperature (25°C) on a rotary shaker under an illumination flux of 10 mol of photons photosynthetically active radiation m Ϫ2 s Ϫ1 .…”

Section: Methodsmentioning

confidence: 99%

Photosystem I of Chlamydomonas reinhardtii Contains Nine Light-harvesting Complexes (Lhca) Located on One Side of the Core

Drop

Webber-Birungi

Fusetti

et al. 2011

Journal of Biological Chemistry

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108

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Background: Photosystem I is a multiprotein complex essential for the photosynthetic process. Results: Photosystem I of Chlamydomonas reinhardtii contains nine Lhca complexes arranged on one side of the core. Conclusion: A model of the subunits organization in the Photosystem I supercomplex is presented. Significance: The sequence of the system (dis)assembly relates to the function of the subunits.

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“…The His 6 peptides were, however, accessible only after harsh urea treatment and could not be used for purification of catalytically active PS I particles. The attachment of a His 6 tag to the N terminus of PsaA from the green alga Chlamydomonas reinhardtii yielded an active PS I version that could be purified via IMAC (25). Since the His tag was located at the cytoplasmic side of PS I, a linkage to NTA-modified surfaces would hide the acceptor site of the protein.…”

Section: Discussionmentioning

confidence: 99%

Requirements for Construction of a Functional Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase

Schwarze

Kopczak

Rögner

et al. 2010

Appl Environ Microbiol

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The development of cellular systems in which the enzyme hydrogenase is efficiently coupled to the oxygenic photosynthesis apparatus represents an attractive avenue to produce H 2 sustainably from light and water. Here we describe the molecular design of the individual components required for the direct coupling of the O 2 -tolerant membrane-bound hydrogenase (MBH) from Ralstonia eutropha H16 to the acceptor site of photosystem I (PS I) from Synechocystis sp. PCC 6803. By genetic engineering, the peripheral subunit PsaE of PS I was fused to the MBH, and the resulting hybrid protein was purified from R. eutropha to apparent homogeneity via two independent affinity chromatographical steps. The catalytically active MBH-PsaE (MBH PsaE ) hybrid protein could be isolated only from the cytoplasmic fraction. This was surprising, since the MBH is a substrate of the twin-arginine translocation system and was expected to reside in the periplasm. We conclude that the attachment of the additional PsaE domain to the small, electron-transferring subunit of the MBH completely abolished the export competence of the protein. Activity measurements revealed that the H 2 production capacity of the purified MBH PsaE fusion protein was very similar to that of wild-type MBH. In order to analyze the specific interaction of MBH PsaE with PS I, His-tagged PS I lacking the PsaE subunit was purified via Ni-nitrilotriacetic acid affinity and subsequent hydrophobic interaction chromatography. Formation of PS Ihydrogenase supercomplexes was demonstrated by blue native gel electrophoresis. The results indicate a vital prerequisite for the quantitative analysis of the MBH PsaE -PS I complex formation and its light-driven H 2 production capacity by means of spectroelectrochemistry.

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Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

Cited by 30 publications

References 35 publications

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

Photosystem I of Chlamydomonas reinhardtii Contains Nine Light-harvesting Complexes (Lhca) Located on One Side of the Core

Requirements for Construction of a Functional Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase

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