The FtsH Protease slr0228 Is Important for Quality Control of Photosystem II in the Thylakoid Membrane of Synechocystis sp. PCC 6803

Myles Barker,Remco de Vries,Josef Komenda,Conrad W. Mullineaux,Stanislava Kuviková,Martin Tichý,Peter J. Nixon

doi:10.1074/jbc.m503852200

Abstract

The cyanobacterium Synechocystis sp. PCC 6803 contains four members of the FtsH protease family. One of these, FtsH (slr0228), has been implicated recently in the repair of photodamaged photosystem II (PSII) complexes. We have demonstrated here, using a combination of blue native PAGE, radiolabeling, and immunoblotting, that FtsH (slr0228) is required for selective replacement of the D1 reaction center subunit in both wild type PSII complexes and in PSII subcomplexes lacking the PSII chlorophyll a-binding subunit CP43. To test whether FtsH (slr0228) has a more general role in protein quality control in vivo, we have studied the synthesis and degradation of PSII subunits in wild type and in defined insertion and missense mutants incapable of proper assembly of the PSII holoenzyme. We discovered that, when the gene encoding FtsH (slr0228) was disrupted in these strains, the overall level of assembly intermediates and unassembled PSII proteins markedly increased. Pulse-chase experiments showed that this was due to reduced rates of degradation in vivo. Importantly, analysis of epitope-tagged and green fluorescent protein-tagged strains revealed that slr0228 was present in the thylakoid and not the cytoplasmic membrane. Overall, our results show that FtsH (slr0228) plays an important role in controlling the removal of PSII subunits from the thylakoid membrane and is not restricted to selective D1 turnover.

Highlights

Our results show that FtsH plays an important role in controlling the removal of photosystem II (PSII) subunits from the thylakoid membrane and is not restricted to selective D1 turnover
PCC 6803 [22], referred to as wild type (WT), and its following previously constructed mutants were used in the study: (i) the CP43-less strain ⌬CP43 with the psbC gene inactivated by a kanamycin resistance cassette [21]; (ii) the D1-less strain ⌬D1 with the psbA1, psbA2, and psbA3 genes inactivated by chloramphenicol, kanR, and spectinomycin resistance cassettes, respectively [23]; and (iii) the site-directed mutants D1-H198L and D1-H198V having the psbA1 and psbA2 genes inactivated by cmR and kanR, respectively, and the codon for residue His198 in the psbA3 gene replaced by codons for Leu or Val [24]
In accordance with earlier work, we found that WT accumulated the monomeric PSII reaction center core (RCC) together with smaller amounts of RC47 and unassembled CP47 and CP43 (Fig. 1A) [28]

Summary

Introduction

The precise mechanism of PSII repair is unknown, recent work has emphasized the importance of FtsH proteases for D1 turnover in vivo [7] This class of proteases is found throughout nature and is involved in the degradation of both soluble and membrane proteins [8]. Deletion of sll1463 does not result in any apparent phenotypic change [12], whereas ftsH (slr0228) insertion mutants showing lightsensitive growth are impaired in PSII repair and display slower rates of D1 degradation in vivo [13]. FtsH2 (VAR2) has been shown to be involved in D1 degradation [17], but other FtsH members

Methods

Results

Discussion

Conclusion