S PATIAL ORGANIZATION OF INITIAL STEPS OF PSII ASSEMBLY

PDMs have been proposed in earlier studies to represent the site of early steps of PSII biogenesis. In the present work, PDMs were further characterized by biochemical means and it was speculated that this membrane subfraction is identical to the earlier described TCs and in contact to both, the PM and TMs, hence potentially representing the controversially discussed connections between these two membrane types (see section 4.1). Electron microscopic analyses revealed that both, PratA and pD1, were found to localize to distinct clusters at the cell periphery (see Figures 8 and 9 in section 3.3). In pratA¯, pD1 was still detected in such clusters, although their number was twofold diminished, indicating that their formation is at least partially dependent on the presence of PratA. This structure was assigned to so-called biogenesis centers, consisting of a granular matrix assumed to correspond to TCs, which is surrounded by a semicircular structure (Figure 10). These centers are located at sites where TMs converge to the PM and, therefore, seem to link these two membrane systems. However, only a small percentage of examined wild-type cells exhibited these structures;

although it has to be noted that merely a few slices have been analyzed per cell and thus no three-dimensional investigation of the cells has been performed. Nevertheless, the results indicate that either only one or a few biogenesis centers are present per cell, or that they are only formed transiently and dynamically. However, the membrane organization and/or biogenesis was found to be altered in pratA¯, as only the TC structures, but not the semicircle- shaped systems were detected in > 1000 analyzed pratA¯ cells, underlining that their formation is PratA-dependent. Determination of the three-dimensional organization of biogenesis centers will be subject of future work, which could also clarify whether they form cylindrical structures as it was previously described for TCs (van de Meene et al., 2006). It can be concluded that these biogenesis centers correspond to PDMs and represent the region where early steps of PSII biogenesis occur. The presented data allow the development of a detailed model of events taking place during these initial assembly steps: Mediated by PratA, D1 becomes preloaded with Mn2+ from the PP, thus bypassing an active transport of Mn2+ across the PM and TMs (Figure 10). In addition, pD1 is processed by CtpA, which has been exclusively detected in PM preparations – probably including PDMs – while PSII assembly proceeds with integration of D2/PsbEF, resulting in formation of RC complexes (Figures 4, 10; Zak et al., 2001). Probably, these RC complexes are located at the transition point from PDMs to TMs and are transferred to the latter for subsequent biogenesis steps, since the next protein assembled, CP47, is found solely in the TM system (Zak et al., 2001; Bergantino et al., 2003). Moreover, the function of the YCF48/Sll0933 complex seems to be connected to this step (see section 4.2). Additionally, chlorophyllide a accumulating in PDMs substantiates their function in pigment synthesis and insertion (Figure 10). The whole assembly process is finally completed in the TMs.

It can be speculated that the PM-near localization of initial steps of PSII assembly is due to the necessity of incorporation of metal ions at the lumenal side of PSII, as it avoids energy consuming, active metal transport processes across the PM and TM system. This might also explain that PSI-related proteins did not depict changes in their amount or localization upon inactivation of pratA (see Figure 2A, B in section 3.2): In contrast to PSII, PSI monomers do not contain inorganic cofactors at their lumenal side, therefore, their assembly might be restricted to subcellular regions independent of the cell envelope (Jordan et al., 2001).

A similar organization of PSII assembly has also been suggested for the green alga Chlamydomonas reinhardtii. Under conditions triggering de novo PSII assembly, ribosomes and mRNAs encoding PSII subunits were shown to localize at distinct regions in the periphery of the pyrenoid, which is formed by semicrystalline concentrations of the CO2-

fixing enzyme ribulose-bisphosphate carboxylase/oxygenase (McKay and Gibbs, 1991; Borkhsenious et al., 1998; Uniacke and Zerges, 2007). Furthermore, mutants defective in PSII assembly accumulate early PSII intermediates around the pyrenoid, which further substantiates the importance of this region in PSII biogenesis. Therefore, these regions were named T-zones (translation zones) and suggested to represent the place of early PSII biogenesis with a subsequent transport of newly assembled complexes to the TMs (Uniacke and Zerges, 2007). Taken together, the principle of a centered localization of PSII biogenesis might be evolutionary conserved – at least from cyanobacteria to chloroplasts of unicellular green algae.

Figure 10: Model for spatial organization of PSII biogenesis in Synechocystis 6803. PSII assembly starts in biogenesis centers consisting of thylakoid centers (TC) surrounded by semicircular structures (SS), which connect PM and TMs. Mediated by PratA the D1 precursor is preloaded with manganese ions (Mn) that have been stored in the periplasmic space. PSII complexes are further assembled while they migrate from PDMs to TMs. Chlorophyllide a (green pentagon) accumulates in PDM fractions merging with TMs. Pitt and POR as well as YCF48 and Sll0933 seem to be involved in pigment synthesis/integration. Assembly of RC47, PSII monomers and dimers (not shown), occurs in TMs. OM, outer membrane; PM, plasma membrane; TM, thylakoid membrane; RC, PSII reaction center complex lacking CP47 and CP43; RC47, PSII reaction center complex lacking CP43; PSII[1], PSII monomer. For further details, see text. According to Nickelsen and Rengstl, submitted (see appendix).