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Reversible phosphorylation of chl a/b protein complex II (LHCII), the mobile light-harvesting antenna, regulates its association and energy transfer/dissipation to photosystem (PS) II or I (state transition). Excitation of LHCII induces conformational changes affecting the exposure of the phosphorylation site at the N-terminal domain to protein kinase(s) [Zer, H., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282; Zer, H., et al. (2003) Biochemistry 42, 728-738]. Thus, it was of interest to examine whether the pigment composition of LHCII affects the light-induced modulation of LHCII phosphorylation and state transition. To this end, we have used thylakoids of wild-type Chlamydomonas reinhardtii and xanthophyll deficient mutants npq1, lor1, npq2, npq1 lor1, and npq2 lor1. Phosphorylated protein bands P11, P13, and P17 are considered components of the mobile C. reinhardtii LHCII complex. The protein composition of these bands has been analyzed by mass spectrometry using Qtof-2 with a nanospray attachment. P11 and P13 contain C. reinhardtii light-harvesting chlorophyll a/b binding protein LhcII type I. P17 contains C. reinhardtii LhcII types III and IV. Illumination of isolated thylakoids inhibits the redox-controlled phosphorylation of polypeptide bands P13 and P17 and to a lower extent that of P11. The light-induced inhibition of LHCII phosphorylation and the state transition process are not influenced by extensive differences in the xanthophyll composition of the mutants. Thus, LHCII can be visualized as possessing two functionally distinct, independent domains: (i) the pigment binding transmembrane domain regulating the extent of energy transfer/dissipation and (ii) the surface-exposed phosphorylation site regulating the association of LHCII with PSII or PSI.

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.