The construction and analysis of a disruption mutant of psbH in Chlamydomonas reinhardtii

Ruffle, SV; O’Connor, Helen E.; Cheater, AJ; Purton, Saul; Nugent, Jha

doi:10.1007/978-94-009-0173-5_613

Cited by 2 publications

(3 citation statements)

References 6 publications

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“…These results complement and extend another report of a Chlamydomonas psbli deletion mutant in which the absence of PSII activity was assessed by oxygen evolution, electron paramagnetic resonance, and fluorescence spectroscopy (Ruffle et al, 1995). The disparity between the requirement of PSII-H in cyanobacteria and C. reinhardtii could be due to the extensive N-terminal sequence differences of the protein of these species, resulting in evolutionary divergence in its physiological role(s).…”

Section: Discussionsupporting

confidence: 83%

“…Replacement of psbH with a gene mutated at phosphorylation site(s) should aid in resolution of this problem. In fact, such a mutant has been constructed with an N-terminal substitution of Ala for Thr: it was reported to exhibit no phosphorylation of PSII-H or other PSII proteins in an in vitro assay with purified thylakoids (Cheater et al, 1995). Because phosphorylation of light-harvesting proteins was also severely affected, in vivo phosphorylation studies are needed to confirm a central role of PSII-H in regulating protein kinase activities.…”

Section: Discussionmentioning

confidence: 99%

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Requirement for the H Phosphoprotein in Photosystem II of Chlamydomonas reinhardtii

et al. 1997

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To dissect the expression of the psbB gene cluster of the Chlamydomonas reinhardtii chloroplast genome and to assess the role of the photosystem II H-phosphoprotein (PSII-H) in the biogenesis and/or stabilization of PSII, an aadA gene cassette conferring spectinomycin resistance was employed for mutagenesis. Disruption of the gene cluster has no effect on the abundance of transcripts of the upstream psbB/T locus. Likewise, interruption of psbB/T and psbH with a strong transcriptional terminator from the rbcL gene does not influence transcript accumulation. Thus, psbB/T and psbH may be independently transcribed, and the latter gene seems to have its own promoter in C. reinhardtii. In the absence of PSII-H, translation and thylakoid insertion of chloroplast PSII core proteins is unaffected, but PSII proteins do not accumulate. Because the deletion mutant also exhibits PSII deficiency when dark-grown, the effect is unrelated to photoinhibition. Turnover of proteins B and C of PSII and the polypeptides PSII protein A and PSII protein D is faster than in wild-type cells but is much slower than that observed in other PSII-deficient mutants of C. reinhardtii, suggesting a peripheral location of PSII-H in PSII. The role of PSII-H on PSII assembly was examined by sucrose gradient fractionation of pulse-labeled thylakoids; the accumulation of high-molecular-weight forms of PSII is severely impaired in the psbH deletion mutant. Thus, a primary role of PSII-H may be to facilitate PSII assembly/stability through dimerization. PSII-H phosphorylation, which possibly occurs at two sites, may also be germane to its role in regulating PSII structure, stabilization, or activity.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Requirement for the H Phosphoprotein in Photosystem II of Chlamydomonas reinhardtii

et al. 1997

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“…These findings led to the conclusion that PsbH is not an essential requirement for PSII activity but simply contributes to its optimization, by regulating the threshold of photoinduced damage at the Q B site and controlling the rate of the compensatory repair process. However, it has been reported that disruption of this gene has serious consequences in the alga Chlamydomonas reinhardtii , including incapacity for photoautotrophic growth on minimal medium, absence of measurable oxygen evolution, and alteration of the normal pattern of in vitro phosphorylation of PSII subunits [11,12]. The latter effect is brought about even by less dramatic alteration of the gene, a mutation of threonine to alanine at position 3 which abolishes PsbH phosphorylation [12,13].…”

mentioning

confidence: 99%

Construction and characterization of a functional mutant of Synechocystis 6803 harbouring a eukaryotic PSII‐H subunit

Chiaramonte

Giacometti

Bergantino

1999

European Journal of Biochemistry

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A Synechocystis 6803 mutant carrying a chimaeric photosystem II (PSII), in which the Zea mays PsbH subunit (7.7 kDa calculated molecular mass) replaces the cyanobacterial copy (7.0 kDa), was constructed. With the exception of the N-terminal 12 amino acid extension, which has a phosphorylatable threonine, the eukaryotic polypeptide is 78% homologous to its bacterial counterpart. Biochemical characterization of this mutant shows that it expresses the engineered gene correctly and is competent for photoautotrophic growth. Fluorescence analysis and oxygen evolution measurements in the presence of exogenous acceptors indicate that the observed phenotype results from a chimaeric PSII rather than from the absence of function associated with PsbH, suggesting that the heterologous protein is assembled into a functional PSII. Inhibition of oxygen evolution by herbicides belonging to different classes shows that the sensitivity of the mutant PSII is changed only towards phenolic compounds. This result indicates slight conformational modification of the Q B /herbicide binding pocket of the D1 polypeptide caused by the bulky PsbH protein in the mutant, and also suggests close structural interaction of the D1 and PsbH subunits in the topological arrangement of PSII.

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The construction and analysis of a disruption mutant of psbH in Chlamydomonas reinhardtii

Cited by 2 publications

References 6 publications

Requirement for the H Phosphoprotein in Photosystem II of Chlamydomonas reinhardtii

Requirement for the H Phosphoprotein in Photosystem II of Chlamydomonas reinhardtii

Construction and characterization of a functional mutant of Synechocystis 6803 harbouring a eukaryotic PSII‐H subunit

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