The role of the slr2034 (ycf48) gene product in the assembly and repair of photosystem II (PSII) has been studied in the cyanobacterium Synechocystis PCC 6803. YCF48 (HCF136) is involved in the assembly of Arabidopsis thaliana PSII reaction center (RC) complexes but its mode of action is unclear. We show here that YCF48 is a component of two cyanobacterial PSII RC-like complexes in vivo and is absent in larger PSII core complexes. Interruption of ycf48 slowed the formation of PSII complexes in wild type, as judged from pulse-labeling experiments, and caused a decrease in the final level of PSII core complexes in wild type and a marked reduction in the levels of PSII assembly complexes in strains lacking either CP43 or CP47. Absence of YCF48 also led to a dramatic decrease in the levels of the COOH-terminal precursor (pD1) and the partially processed form, iD1, in a variety of PSII mutants and only low levels of unassembled mature D1 were observed. Yeast two-hybrid analyses using the split ubiquitin system showed an interaction of YCF48 with unassembled pD1 and, to a lesser extent, unassembled iD1, but not with unassembled mature D1 or D2. Overall our results indicate a role for YCF48 in the stabilization of newly synthesized pD1 and in its subsequent binding to a D2-cytochrome b 559 pre-complex, also identified in this study. Besides a role in assembly, we show for the first time that YCF48 also functions in the selective replacement of photodamaged D1 during PSII repair. The photosystem II (PSII)2 complex of plants, algae, and cyanobacteria is a multisubunit pigment-protein complex responsible for water oxidation during oxygenic photosynthesis (1). The complex consists of over 20 intrinsic and peripheral membrane proteins, which must be assembled in a highly coordinated manner to ensure proper functioning of the complex (2). At the heart of the complex is a heterodimer of the D1 (the psbA gene product) and related D2 (the psbD gene product) polypeptides, which binds most of the redox-active cofactors involved in PSII electron transfer (3). The isolated PSII reaction center (RC) complex contains, in addition to D1 and D2, the intrinsic PsbI subunit and cytochrome b 559 , which bind to D1 and D2, respectively (4). Surrounding the PSII RC complex are two chlorophyll-binding proteins, CP43 (the psbC gene product) and CP47 (the psbB gene product). D1 and CP43 also provide several amino acid ligands to the Mn 4 Ca cluster involved in the oxidation of water (5, 6).In vivo, the D1 protein exhibits much faster synthesis and degradation (or protein turnover) than the other PSII subunits (7). This feature reflects the operation of a PSII repair cycle to overcome the effects of PSII photoinhibition, which primarily causes irreversible damage to D1 (8). In most PSII-containing organisms, D1 is synthesized as a precursor protein (pD1) with a carboxyl-terminal extension (9). In the cyanobacterium Synechocystis sp. PCC 6803 this extension consists of 16 amino acid residues, which is removed in two steps by the CtpA endoprotein...

Accumulation of monomer and dimer photosystem (PS) II reaction center core complexes has been analyzed by two-dimensional Blue-native/SDS-PAGE in Synechocystis PCC 6803 wild type and in mutant strains lacking genes psbA, psbB, psbC, psbDIC/DII, or the psbEFLJ operon. In vivo pulse-chase radiolabeling experiments revealed that mutant cells assembled PSII precomplexes only. In ⌬psbC and ⌬psbB, assembly of reaction center cores lacking CP43 and reaction center complexes was detected, respectively. In ⌬psbA, protein subunits CP43, CP47, D2, and cytochrome b 559 were synthesized, but proteins did not assemble. Similarly, in ⌬psbD/C lacking D2, and CP43, the de novo synthesized proteins D1, CP47, and cytochrome b 559 did not form any mutual complexes, indicating that assembly of the reaction center complex is a prerequisite for assembly with core subunits CP47 and CP43. Finally, although CP43 and CP47 accumulated in ⌬psbEFLJ, D2 was neither expressed nor accumulated. We, furthermore, show that the amount of D2 is high in the strain lacking D1, whereas the amount of D1 is low in the strain lacking D2. We conclude that expression of the psbEFLJ operon is a prerequisite for D2 accumulation that is the key regulatory step for D1 accumulation and consecutive assembly of the PSII reaction center complex. The photosystem II (PSII)1 reaction center core (RCC) complex of higher plants, algae, and cyanobacteria can be subdivided into a heterodimer containing D1 and D2, the antenna proteins CP47 and CP43, and a large number of low molecular weight integral membrane proteins including the ␣ and ␤ subunits of cytochrome b 559 (␣ and ␤ cytochrome b 559 ) (1-3). The heterodimer and antenna proteins are essential for binding the prosthetic groups needed for energy and electron transfer (4) as well as for binding the multitude of plastid-encoded small subunits, e.g. Psb-H, -J, -K, -L, and Psb-T, which affect the function of PSII (5-8). Furthermore, plastid-encoded subunit psbZ has been shown to be required for attachment of CP26 during assembly of PSII-LHC supercomplexes, whereas the nucleusencoded subunit psbW was demonstrated to be required for RCC dimer formation (9 -11). The role of plastid-encoded subunits Psb-I, -M, and -N and the nucleus-encoded small subunits Psb-R, and X remains unclear. A striking feature of PSII is the fast turnover of the D1 protein that is believed to be required for PSII repair and restoration of its photochemical activity after photoinactivation (12, 13). Maintaining PSII function may require selective replacement of this central PSII subunit including an efficient apparatus to recognize inactive complexes, and remove damaged and insert a new D1 copy (5, 14, 15). Zhang et al. (16) suggested that D1 replacement in higher plants may occur cotranslationally in a PSII subcomplex consisting of at least D2 and CP47, hence eliminating the need for complete disassembly and de novo assembly from PSII subunits.Cyanobacteria are an excellent model organism to study PSII assembly. The strain used most frequently i...

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the ␤-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the ␣-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.biliprotein biosynthesis ͉ light-harvesting ͉ photosynthesis ͉ phycobilisome P hycobilisomes, the extramembraneous light-harvesting antennas in cyanobacteria and red algae, use four different types of linear tetrapyrrole chromophores to harvest light in the green gap of chlorophyll absorption (1-6). These phycobilins are covalently bound to seven or more proteins, each carrying one to four binding sites. The chromophores are biosynthesized from the cyclic irontetrapyrrole, heme, by ring opening at C-5, followed by reduction and, sometimes, also by isomerization (7-9). In the last step, these phycobilins are covalently attached to cysteines of the apoprotein via a thioether bond to C-3 1 on ring A ( Fig. 1) and in some cases by an additional thioether bond to C-18 1 on ring D (6, 10-12). This step, the binding to the apoprotein, is presently only poorly understood; it involves a considerable number of binding sites and chromophores, as well as the proper regulation and coordination of events. ¶ An increasing number of lyases has recently been identified that catalyze the chromophore addition and are specific not only for the chromophore but also for the apoprotein and the binding site (12-16). Based on the capacity of several of the respective apoproteins to also bind the chromophores autocatalytically (17-21), a chaperone-like function has been suggested (12). It enhances and guides the autocatalytic binding, which is generally of low fidelity, possibly by conformational control of the chromophore (18). At the same time, this autocatalytic binding interferes with the lyase analyses (22). The situation is some...

In the postgenomic era, the transformation of genetic information into biochemical meaning is required. We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach. Based on its evolutionary relation to the outer membrane of Gram-negative bacteria, the outer envelope membrane should contain a large number of ␤-barrel proteins. We therefore calculated the probability for the existence of ␤-sheet, ␤-barrel, and hairpin structures among all proteins of the Arabidopsis thaliana genome. According to the existence of these structures, a number of candidates were selected. This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts. In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope. The approach developed here can be used to predict the topology of ␤-barrel proteins. For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts from Pisum sativum were analyzed by ESI-MS/MS mass spectrometry. In addition to the known components, four new proteins of the outer envelope membranes were identified in this study.

Hcf136 encodes a hydrophilic protein localized in the lumen of stroma thylakoids. Its mutational inactivation in Arabidopsis thaliana results in a photosystem II (PHII)-less phenotype. Under standard illumination, PSII is not detectable and the amount of photosystem I (PSI) is reduced, which implies that HCF136p may be required for photosystem biogenesis in general. However, at low light, a comparison of mutants with defects in PSII, PSI, and the cytochrome b 6 f complex reveals that HCF136p regulates selectively biogenesis of PSII. We demonstrate by in vivo radiolabeling of hcf136 that biogenesis of the reaction center (RC) of PSII is blocked. Gel blot analysis and a⁄nity chromatography of solubilized thylakoid membranes suggest that HCF136p associates with a PSII precomplex containing at least D2 and cytochrome b 559 . We conclude that HCF136p is essential for assembly of the RC of PSII and discuss its function as a chaperone-like assembly factor.