With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of Chlamydomonas reinhardtii light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific lhcbm gene products. Interestingly, lhcbm6 gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different lhca-related gene products were predicted by comparison of the mass spectrometric data against Chlamydomonas expressed sequence tag and genomic databases, demonstrating the extensive variability of the C. reinhardtii Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in C. reinhardtii and will be an important basis of future functional studies addressing this diversity.In all eukaryotic oxygenic photosynthetic organisms, lightharvesting chlorophyll a-or b-binding proteins (LHC proteins) function in the collection and transfer of light energy to the reaction centers of photosystem II (PSII) (Lhcb proteins) and photosystem I (PSI) (Lhca proteins). Additionally these proteins are also involved in light dissipation and energy quenching. Therefore, light-harvesting proteins are important components of the photosynthetic machinery that optimize photosynthetic function and minimize photooxidative damage in response to light quantity and quality. It has been known for several years that light-harvesting proteins are products of many genes. This concept is illustrated by a recent analysis of the Arabidopsis genome which revealed that the lhc gene family is composed of more than 20 genes (24). Besides the large number of lhc gene products, posttranslational modifications, such as phophorylation, contribute to even more complexity at the protein level (31, 45). Phosphorylation of the major Lhcb proteins of PSII is important in the process of state transitions. This process leads to a redistribution of excitation energy between PSII and PSI by reorganization of the antennae and thereby regulates energy flow between the photosystems. The importance of phosphorylation for state transitions is shown by the phenotype of the Chlamydomonas reinhardtii Stt7 mutant. This mutant is markedly ...

Photosynthetic organisms respond to changes in ambient light by modulating the size and composition of their lightharvesting complexes, which in the case of the green alga Chlamydomonas reinhardtii consists of >15 members of a large extended family of chlorophyll binding subunits. How their expression is coordinated is unclear. Here, we describe the analysis of an insertion mutant, state transitions mutant3 (stm3), which we show has increased levels of LHCBM subunits associated with the light-harvesting antenna of photosystem II. The mutated nuclear gene in stm3 encodes the RNA binding protein NAB1 (for putative nucleic acid binding protein). In vitro and in vivo RNA binding and protein expression studies have confirmed that NAB1 differentially binds to LHCBM mRNA in a subpolysomal high molecular weight RNA-protein complex. Binding of NAB1 stabilizes LHCBM mRNA at the preinitiation level via sequestration and thereby represses translation. The specificity and affinity of binding are determined by an RNA sequence motif similar to that used by the Xenopus laevis translation repressor FRGY2, which is conserved to varying degrees in the LHCBM gene family. We conclude from our results that NAB1 plays an important role in controlling the expression of the light-harvesting antenna of photosystem II at the posttranscriptional level. The similarity of NAB1 and FRGY2 of Xenopus implies the existence of similar RNA-masking systems in animals and plants.

SummaryFunctional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild-type and mutant strains of Chlamydomonas reinhardtii. Thylakoid membrane proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and analysed by immunoblotting and mass spectrometry for the presence of membrane-spanning proteins. Our data show that light-harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identi®ed more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2-DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2-DE revealed the presence of 18 LHCI protein spots. The use of two peptide-speci®c antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post-translational modi®cations. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2-DE. Comparison between 2-DE maps from thylakoid membrane proteins isolated from a PSI-de®cient (Dycf4) and a crd1 mutant, which is conditionally reduced in PSI and LHCI under copper-de®ciency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2-DE, and (ii) that high-resolution 2-DE facilitates the comparative analysis of membrane protein complexes in wild-type and mutants cells.

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Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta, were analysed by two‐dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20‐day‐old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa, and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta. Crosses were made between this line and the wild type Landsberg erecta. F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.