M EDIATION OF PSII ASSEMBLY BY A NETWORK OF FACILITATING FACTORS

During recent years, a number of assembly factors have been found and characterized that function in regulation of PSII biogenesis. However, relatively little is known about potential interrelations between these proteins, but it is becoming more and more apparent that the different facilitating factors do not act isolated but rather are integrated in an elaborated network of interactions. More detailed characterization of already known factors as well as identification of novel proteins participating in regulation of PSII assembly can provide new insights into the function and coordination of this complex process.

To investigate potential interconnections between different facilitating factors in Synechocystis 6803, in this work, their accumulation as well as effects on their membrane distribution in various PSII mutants was studied (see Figures 2 and 3 in section 3.2). The results confirmed that lack of one assembly factor indeed specifically affected localization

and accumulation of others demonstrating that a connected network of facilitating factors exists in cyanobacteria (Figure 9A). Whether these impairments are caused by direct interactions or represent secondary effects due to a disturbed PSII assembly process or distorted membrane structure has to be elucidated in future work. At least for YCF48 and Sll0933 as well as Pitt and POR direct interactions could be confirmed by studies in yeast (see Figure 5 in section 3.4; Schottkowski et al., 2009a). Interaction between the latter two is especially interesting with regard to the connection of protein subunit assembly with pigment synthesis, since Pitt was shown to stabilize the chlorophyll synthesis enzyme POR. The presence of both proteins in PDMs and their altered membrane sublocalization upon inactivation of pratA as well as accumulation of pD1 in PDMs of a pitt¯ mutant led to the assumption that Pitt is involved in PSII assembly, perhaps by localizing POR to distinct membrane regions thus integrating pigment synthesis and protein assembly (Schottkowski et al., 2009a).

Figure 9: Interrelationships between different PSII assembly factors in Synechocystis 6803 (A) and A. thaliana (B). Synechocystis 6803results (A) are based on protein accumulations in respective mutants (see Table 1 in section 3.2). Solid lines indicate positive, dotted lines negative regulations. Complex formation was confirmed between YCF48 and Sll0933 as well as Pitt and POR (see sections 3.2 and 3.4; Schottkowski et al., 2009a). (B) Known interactions of PSII assembly factors in

A. thaliana (see section 3.1; Ma et al., 2007; Cai et al., 2010). Homologous proteins in

Synechocystis 6803 and A. thaliana are identically coloured.

Evidence for interaction between YCF48 and Sll0933 in Synechocystis 6803 was obtained in this work (see sections 3.2 and section 3.4). This interaction seems to be evolutionary conserved up to chloroplasts of higher plants, since it was also demonstrated for the respective homologous proteins, HCF136 and PAM68, in A. thaliana (see Figure 10 in section 3.1). However, in Synechocystis 6803, lack of these factors seems to be better substituted by other

proteins, since the observed phenotypes of the respective mutants were not as severe as reported for A. thaliana (see section 3.1; Plücken et al., 2002; Komenda et al., 2008). Nevertheless, the main mode of action appears to be similar in Synechocystis 6803 and A. thaliana.

In membrane fractionation studies, Sll0933 was exclusively detected in TMs whereas YCF48 was also present in PDMs, hence, YCF48 probably acts upstream of Sll0933 during PSII assembly (see Figure 2 in section 3.2). This is in agreement with the finding that Sll0933 assists the attachment of the PSII inner antenna proteins CP47 and CP43 and, thus, is involved in later steps of PSII biogenesis compared to YCF48, which mediates RC assembly (see Figure 4 in section 3.4; Komenda et al., 2008). Interaction between YCF48 and Sll0933 therefore might take place during conversion from RC to RC47 complexes. It can be speculated that the YCF48/Sll0933 complex is involved in distribution of chlorophyll among chlorophyll-binding proteins as it was already suggested for YCF48 and its plant counterpart HCF136 (Plücken et al., 2002; Komenda et al., 2008). Reduced levels of newly synthesized CP47 and CP43 in sll0933¯ and ycf48¯sll0933¯ could hence be possibly due to a defect in pigment insertion. It furthermore can be hypothesized that YCF48 is indirectly involved in chlorophyll integration by passing on chlorophyll molecules, which are newly synthesized in PDMs, to the TM-localized Sll0933 that subsequently mediates their insertion into PSII inner antenna proteins.

The A. thaliana homolog of Sll0933, PAM68, was shown to be able to interact – besides with HCF136 – with a variety of different PSII subunits (D1, D2, CP43, CP47, PsbH, PsbI) and, interestingly, several other assembly factors (Alb3, LPA1, HCF136, LPA2) (Figure 9B; see Figure 10 in section 3.1). Whereas interaction of PAM68 with D1, D2, PsbI, Alb3, LPA1 and HCF136 underlines a function in formation of RC complexes, interaction with CP43, CP47, PsbH and LPA2 points to a role in attachment of the PSII inner antenna proteins as it was also suggested for Sll0933. Due to its interaction with Alb3, which is an integrase involved in membrane integration of light-harvesting chlorophyll binding proteins and potentially of other PSII proteins such as D1, D2 and CP43, PAM68 could indirectly even play a role in the very early steps of the assembly process, i.e. integration of D1 (Moore et al., 2000; Pasch et al., 2005). This is furthermore supported by the interrelationship with LPA1, a chloroplast- specific PSII assembly factor which directly binds to D1 and probably also assists in the correct integration of D1 into the membrane (Peng et al., 2006). Based on analyses of protein complexes in pam68 and lpa1 mutants, it was speculated, that LPA1 acts upstream of PAM68 and growing PSII complexes are passed to PAM68 thus displacing LPA1 (see Figure 9 in

section 3.1). These PSII intermediates seem to contain pD1 and D2 but lack CP47 and CP43, therefore arguing for the involvement of PAM68 in steps leading to formation of RC complexes (see Figure 9 in section 3.1). PAM68 additionally interacts with LPA2, substantiating a function of PAM68 in assembly of the inner PSII antennae (see Figure 10 in section 3.1). LPA2 is a facilitating factor only present in higher plants, which acts at the level of CP43 integration into RC47 complexes (Ma et al., 2007). This step is likely assisted by LPA3, an eukaryotic PSII assembly factor which, again, was shown to interact with LPA2 and Alb3 in A. thaliana protoplasts (Cai et al., 2010). Alb3, again, was shown to interact with LPA2 and, as mentioned above, with PAM68 (Figure 9B; Ma et al., 2007). However, no homologous proteins to LPA1 or LPA2 can be found in Synechocystis 6803, indicating that the regulatory network acting in PSII assembly has undergone several changes during evolution.

In general, the presented data illustrate that different PSII assembly factors act in a tightly regulated network rather than fulfilling their functions separately (Figure 9). Hence, it can be speculated that developing PSII complexes are passed in an assembly line built up by a consecutive machinery of facilitating factors, which is probably also linked to proteins mediating the insertion of newly synthesized pigments and the therein involved enzymes (see also section 4.1).