Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts.

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light-grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.

Localization of 13 one-helix integral membrane proteins in photosystem II subcomplexes.

Photosystem II is a multimeric protein complex of the thylakoid membrane in chloroplasts. Approximately half of the at least 26 different integral membrane protein subunits have molecular masses lower than 10 kDa. After one-dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) separation, followed by enzymatic digestion of detected proteins, hardly any of these low-molecular-weight (LMW) subunits are detectable. Therefore, we developed a method for the analysis of highly hydrophobic LMW proteins. Intact proteins are extracted from acrylamide gels using a mixture of formic acid and organic solvent, precipitated with acetone, and analyzed by "top-down" mass spectrometry (MS). After offline nanoESI (electrospray ionization) MS, all LMW one-helix proteins from photosystem II were detected. In the four detected photosystem II supercomplexes of Nicotiana tabacum wild-type plants, 11 different one-helix proteins were identified as PsbE, -F, -H, -I, -K, -L, -M, -Tc, -W, and two isoforms of PsbX. The proteins PsbJ, -Y1, and -Y2 were localized in the buffer front after blue native (BN) PAGE, indicating their release during solubilization. Assembled PsbW is detected exclusively in supercomplexes, whereas it is absent in photosystem II core complexes, corroborating the protein's function for assembly of the light-harvesting complexes. This approach will substantiate gel-blot immunoanalysis for localization and identification of LMW protein subunits in any membrane protein complex.

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco.

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn(4)Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in DeltapsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in DeltapsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in DeltapsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.

Phospho-mimicry mutant of atToc33 affects early development of Arabidopsis thaliana.

The precursor protein receptor at the chloroplast outer membrane atToc33 is a GTPase, which can be inactivated by phosphorylation in vitro, being arrested in the GDP loaded state. To assess the physiological function of phosphorylation, attoc33 knock out mutants were complemented with a mutated construct mimicking the constitutively phosphorylated state. Our data suggest that the reduced functionality of the mutant protein can be compensated by its upregulation. Chloroplast biogenesis and photosynthetic activity are impaired in the mutants during the early developmental stage, which is consistent with the requirement of atToc33 in young photosynthetic tissues.

PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco.

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.