Identification of the mobile light-harvesting complex II polypeptides for state transitions in Chlamydomonas reinhardtii

Abstract

State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and photosystem II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II (LHCII) antenna proteins is thought to migrate from PSII to PSI, but biochemical evidence for a physical association between LHCII proteins and PSI in state 2 is weak. Here, using the green alga Chlamydomonas reinhardtii, which has a high capacity for state transitions, we report the isolation of PSI-light-harvesting complex I (LHCI) super-complexes from cells locked into state 1 and state 2. We solubilized the thylakoid membranes with a mild detergent, separated the proteins by sucrose density gradient centrifugation, and subjected gradient fractions to gel-filtration chromatography. Three LHCII polypeptides were associated with a PSI-LHCI supercomplex only in state 2; we identified them as two minor monomeric LHCII proteins (CP26 and CP29) and one previously unreported major LHCII protein type II, or LhcbM5. These three LHCII proteins, in addition to the major trimeric LHCII proteins, were phosphorylated upon transition to state 2. The corresponding phylogenetic tree indicates that among the LHCII proteins associated with PSII, these three LHCII proteins are the most similar to the LHC proteins for PSI (LHCI). Our results are important because CP26, CP29, and LhcbM5, which have been viewed as belonging solely to the PSII complex, are now postulated to shuttle between PSI and PSII during state transitions, thereby acting as docking sites for the trimeric LHCII proteins in both PSI and PSII.

Fig. 1.

Low temperature fluorescence spectra. Fluorescence emission spectra of C. reinhardtii thylakoid membranes locked in state 1 (thin line) and state 2 (bold line) were measured at 77 K. The excitation wavelength was 440 nm. Spectra were normalized to the emission at 688 nm.

Fig. 2.

Chlorophyll-protein complexes separated on sucrose density gradients. The thylakoids from C. reinhardtii cells placed in state 1 (A) and state 2 (B) were solubilized and subjected to sucrose density gradient centrifugation. The green bands were designated A-1, A-2, A-3, and A-3′ (Top). The gradient was divided into 20 fractions and subjected to SDS/PAGE. The gels were stained with Coomasie brilliant blue R-250 (Middle) or subjected to immunoblotting by using antibodies raised against PsaA, PsbD (D2 protein), CP26, CP29, or LHCII type II (Bottom). The positions of CP26, CP29, and LHCII type II in Middle are indicated by dots.

Fig. 3.

Gel-filtration chromatogram of A-3 and A-3′. A-3 from state 1 and A-3′ from state 2 were loaded onto a gel filtration chromatograph column. Proteins were detected at 280 nm. The positions of molecular mass markers are indicated (Upper). Fractions from 30 to 68 min were subjected to immunoblotting by using antibodies against PsaA, CP26, CP29, or LHCII type II (Lower).

Fig. 4.

Phosphorylated polypeptides in state 1 and state 2 C. reinhardtii thylakoids. Polypeptides of the A-1 and A-3′ bands from the state 1 (S-1) and state 2 (S-2) thylakoids were subjected to immunoblotting with an antibody against phosphothreonine. The sample representing the A-3′ band in S-1, which was not observed, corresponds to the fraction equivalent to the A-3′ band in S-2.

Fig. 5.

Neighbor-joining phylogenetic tree of LHC proteins in C. reinhardtii. Neighbor-joining distance tree was constructed with bootstrap values (% of 1,000 replicates) displayed at the appropriate nodes. The entire coding region, including transit peptides, was used to align amino acid sequences. LI-818 protein (GenBank accession no. X95326) was used as an outgroup. Scale bar represents the expected number of amino acid substitutions per site.

Fig. 6.

A proposed model for the migration of LHCII proteins during transition from state 1 to state 2. In state 1, two LHCII trimers and three monomers [CP29, CP26, and LHCII type II (LhcbM5), gray circles] associate with each side of the PSII dimer. Upon overexcitation of PSII, LHCII proteins are phosphorylated (asterisks) and are detached from PSII. The phospho-LHCII trimers then reassociate with PSI near the PsaH subunit by using the monomeric LHCII proteins as docking sites.