Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii

Abstract

Mapping of in vivoThe thylakoid membranes in chloroplasts of plants and green algae carry out oxygenic photosynthesis. Two multisubunit pigment-containing protein complexes, photosystem II (PSII) 1 and photosystem I (PSI), located in this membrane system work in series to generate an electrochemical potential gradient of protons across the membrane following vectorial electron flow from PSII to PSI via the cytochrome b 6 f complex. PSII uses light to oxidize water, whereas PSI, via a second photoact, uses reducing equivalents derived from PSII to reduce NADP ϩ to NADPH. The electrochemical potential gradient of protons is used to power conversion of ADP to ATP (1). Several thylakoid membrane proteins that make up the PSII complex and its LHCII (light-harvesting chlorophyll a/b-binding proteins of PSII) antennae undergo light-and redox-dependent phosphorylation (2, 3) as discovered more than 2 decades ago (4 -6). Phosphorylation of LHCII in plants and algae controls photosynthetic state transitions, which optimize efficient use of the absorbed light energy by both photosystems. Thus, in State 1 more energy is transferred to PSII, whereas in State 2 a proportion of the excitation energy is redistributed to PSI (7-9). The essential role of protein phosphorylation in state transitions has recently been proven in the studies using mutants of Arabidopsis thaliana plants and the green alga Chlamydomonas reinhardtii deficient in protein kinases STN7 (9) and Stt7 (8), respectively. However, despite the generally assumed similarity in thylakoid protein phosphorylation between plants and algae and a high homology between the plant STN7 and the algal Stt7 protein kinases (8), the extent of photosynthetic state transitions differs between these species. In plant thylakoids, only 15-20% of LHCII participates in the lateral migration between the photosystems, whereas up to 80% of the excitation energy absorbed by the LHCII antenna can be redistributed from PSII to PSI in green alga (8 -10).

PHOTOSYSTEM II SUBUNIT R Is Required for Efficient Binding of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 to Photosystem II-Light-Harvesting Supercomplexes in Chlamydomonas reinhardtii

“…The ClustalW multiple sequence alignment of PSBR revealed that the HL/state 2-induced (Turkina et al, 2006) phosphorylation site Ser-40 of C. reinhardtii PSBR is located in a protein segment that appears to be absent in land plants (as seen for P. patens and Arabidopsis PSBR), while C-terminal sequences are more conserved (Fig. 10).…”

“…To validate PSBR phosphorylation in C. reinhardtii, we performed mass spectrometric analyses of thylakoid membranes using sequential elution from immobilized metal ion affinity chromatography (SIMAC) for phosphopeptide enrichment (Thingholm et al, 2008). Using this approach, three distinct phosphorylation sites could be detected on PSBR (Table I): two T phosphorylation sites were unknown, while the phosphorylation of S40 has already been published (Turkina et al, 2006), referred to as Ser-43. All three phosphorylation sites differ from those known for Arabidopsis as visualized in a ClustalW (Larkin et al, 2007) multiple sequence alignment (Fig.…”

“…PSBR phosphorylation is known for Arabidopsis (Reiland et al, 2009(Reiland et al, , 2011Nakagami et al, 2010) and C. reinhardtii (Turkina et al, 2006). To validate PSBR phosphorylation in C. reinhardtii, we performed mass spectrometric analyses of thylakoid membranes using sequential elution from immobilized metal ion affinity chromatography (SIMAC) for phosphopeptide enrichment (Thingholm et al, 2008).…”

“…PSBR phosphopeptides were identified by shaving thylakoid membranes, which were isolated in the presence of 10 mM sodium fluoride, with trypsin as described (Turkina et al, 2006). Phosphopeptides were enriched using the SIMAC protocol (Thingholm et al, 2008).…”

“…Functionally, the depletion of PSBR protein expression decreased rates of oxygen evolution (Allahverdiyeva et al, 2007(Allahverdiyeva et al, , 2013 and quinone reoxidation (Allahverdiyeva et al, 2007). PSBR phosphorylation is known for Arabidopsis (Arabidopsis thaliana; Reiland et al, 2009Reiland et al, , 2011Nakagami et al, 2010) and in the green alga C. reinhardtii (Turkina et al, 2006), although phosphorylation sites are not conserved between the alga and the vascular plant.…”