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Directed mutagenesis of the transmembrane domain of the PsbL subunit of photosystem II in Synechocystis sp. PCC 6803

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Abstract

The PsbL protein is one of three low-molecular-weight subunits identified at the monomer–monomer interface of photosystem II (PSII) [Ferreira et al. (2004) Science 303:1831–1838; Loll et al. (2005) Nature 438:1040–1044]. We have employed site-directed mutagenesis to investigate the role of PsbL in Synechocystis sp. PCC 6803 cells. Truncation of the C-terminus by deleting the last four residues (Tyr-Phe-Phe-Asn) prevented association of PsbL with the CP43-less monomeric sub-complex and therefore blocked PSII assembly resulting in an obligate photoheterotrophic strain. Replacement of these residues with Ala created four photoautotrophic mutants. Compared to wild type, the F37A, F38A, and N39A strains had reduced levels of assembled PSII centers and F37A and F38A cells were readily photodamaged. In contrast, Y36A and Y36F mutants were similar to wild type. However, each of these strains had elevated levels of the CP43-less inactive monomeric complex. Mutations targeting a putative hydrogen bond between Arg-16 and sulfoquinovosyldiacylglycerol resulted in mutants that were also highly susceptible to photodamage. Similarly mutations targeting a conserved Tyr residue (Tyr-20) also destabilized PSII under high light and suggest that Tyr-20–lipid interactions or interactions of Tyr-20 with PsbT influence the ability of PSII to recover from photodamage.

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Abbreviations

BN-PAGE:

Blue-native polyacrylamide gel electrophoresis

DCMU:

3-(3,4-dichlorophenyl)-1,1-dimethylurea

DM:

n-dodecyl β-d-maltoside

DMBQ:

2,5-dimethyl-p-benzoquinone

EDTA:

Ethylenediamine tetra-acetic acid (di-sodium salt)

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Kb:

Kilobases

kDa:

Kilodaltons

OD:

Optical density

PCC:

Pasteur Culture Collection

PCR:

Polymerase chain reaction

PQ:

Plastoquinone

PSII:

Photosystem II

QA :

Primary quinone electron acceptor of photosystem II

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

SDS polyacrylamide gel electrophoresis

TES:

2-[tris(hydroxymethyl)methyl]amino-1-ethanesulfonic acid

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgment

This work was supported by a grant (UOO309) from the New Zealand Marsden Fund to J.J.E.-R.

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  1. Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, New Zealand

    Hao Luo & Julian J. Eaton-Rye

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  1. Hao Luo
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  2. Julian J. Eaton-Rye
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Correspondence to Julian J. Eaton-Rye.

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Luo, H., Eaton-Rye, J.J. Directed mutagenesis of the transmembrane domain of the PsbL subunit of photosystem II in Synechocystis sp. PCC 6803. Photosynth Res 98, 337–347 (2008). https://doi.org/10.1007/s11120-008-9375-1

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