Protein-protein interactions by molecular modeling and biochemical characterization of PSI-LHCI supercomplexes from Chlamydomonas reinhardtii

The physiological function of Photosystem I (PSI) is a sunlight energy converter, catalyzing one of the initial steps in driving oxygenic photosynthesis in cyanobacteria, algae and higher plants. The Chlamydomonas reinhardtii PSI structure was not known since it contains a unique structure having additional light harvesting complex I (LHCI) subunits, which play a major role in the transfer of sunlight energy to the reaction center. Here, individual subunits of LHC and core subunits are built based on the PDB taken from RCSB Protein Data Bank. The model gives information about the geometrical existence of subunits following a flanking order of Lhca5, Lhca1, Lhca6, Lhca4, Lhca2, Lhca8, Lhca9, Lhca7, and Lhca3. The new subunit PsaO is located close to the PsaH, PsaI and PsaL subunits, thus it may be involved in the state transition mechanism and stabilization of PSI-LHCI supercomplexes. The modeled PSI-LHCI structure of C. reinhardtii shows a unique arrangement of PsaN, PsaO of PSI core subunits and Lhca5 to Lhca9 of LHCI subunits. There are many non-covalent interactions among the PSI and LHCI subunits, which suggest that C. reinhardtii PSI-LHCI supercomplexes are more complex than higher plants. These results strongly support the experimental data that, even with harsh treatment of the PSI-LHCI supercomplexes with detergent, the complexes do not dissociate due to strong interactions between the PSI core and LHCI. Thus, our 3D model may give valid structural information of the PSI-LHCI arrangement and its physiological role in C. reinhardtii.     

Intracellular Localization of Three l-Glutamate Dehydrogenase Isozymes from Chlamydomonas reinhardtii

The intracellular localization of the activity and synthesis of three isozymes of NAD(P)⁺-glutamate dehydrogenase from the unicellular green alga Chlamydomonas reinhardtii cw-92 has been established. Isozyme activities have been located within mitochondria by using differential centrifugation techniques and discontinuous Percoll gradient separations. Experiments with protein synthesis inhibitors cycloheximide, rifampicin, chloramphenicol, and actinomycin D, under dark and carbon starvation conditions, revealed that synthesis of the three isozymes was likely to occur in cytosol as precursor proteins that are then transported and processed inside the mitochondria.     

Structural and functional changes of PSI-LHCI supercomplexes of Chlamydomonas reinhardtii cells grown under high salt conditions

The effect of high salt concentration (100 mM NaCl) on the organization of photosystem I-light harvesting complex I supercomplexes (PSI-LHCI) of Chlamydomonas reinhardtii was studied. The electron transfer activity was reduced by 39% in isolated PSI-LHCI supercomplexes. The visible circular dichroism (CD) spectra associated with strongly coupled chlorophyll (Chl) dimers were reduced in intensity, indicating that pigment–pigment interactions were disrupted. This data is consistent with results from fluorescence streak camera spectroscopy, which suggest that red-shifted pigments in the PSI-LHCI antenna had been lost. Denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI reaction center proteins PsaD, PsaE and PsaF were reduced due to salt stress. PsaE is almost completely absent under high salt conditions. It is known that the membrane-extrinsic subunits PsaD and E form the ferredoxin-docking site. Our results indicate that the PSI-LHCI supercomplex is damaged by reactive oxygen species at high salt concentration, with particular impact on the ferredoxin-docking site and the PSI-LHCI interface.     

Atomic resolution modeling of the ferredoxin:[FeFe] hydrogenase complex from Chlamydomonas reinhardtii

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H(2) from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.     

Three new yellow loci in Chlamydomonas reinhardtii

Summary Three phenotypically yellow, mendelian mutants of Chlamydomonas reinhardtii have been isolated and tested for allelism with the yellow mutant v-1a ¹ and with each other. The three mutants represent three new yellow loci, two of which are located on linkage group I. Like y-1a, the mutants accumulate protochlorophyllide when grown under dim light, but have a wildtype phenotype when grown in the light. We conclude that the control of light-independent protochlorophyllide reduction is more complex than has been thought previously.     

NMR structures of thioredoxin m from the green alga Chlamydomonas reinhardtii

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.     

Glutamate synthase from Chlamydomonas reinhardtii: Interaction studies with its substrate ferredoxin and molecular cloning

Glutamate synthase (GOGAT) from Chlamydomonas reinhardtii is able to form functional covalent complexes with its substrate ferredoxin (Fd), either wild-type (^WTFd) or recombinant form (^rFd). However, when Fd carboxyl groups were chemically modified (^mdFd), no complexes were detected and its ability to serve as electron donor for glutamate synthase activity was also decreased. By site-directed mutagenesis, we have demonstrated that Fd glu91 and a negative core in the helix α1 are critical for Fd interaction with this enzyme and its functionality as electron carrier for glutamate synthase. As a previous step to elucidate the specific positive charged residues involved in glutamate synthase interaction with Fd, we have isolated a cDNA, CrFG-3, encoding Fd-GOGAT from C. reinhardtii. The cDNA comprised about 60% of the protein and sequence comparison showed that CrFG-3 was structurally more similar to higher plant enzymes than to the corresponding prokaryotic GOGAT. Two conserved domains were present in this protein fragment, the FMN-binding domain and the cysteines involved in the iron–sulfur cluster binding. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.     

Brownian dynamics and molecular dynamics study of the association between hydrogenase and ferredoxin from Chlamydomonas reinhardtii

The [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii can catalyze the reduction of protons to hydrogen gas using electrons supplied from photosystem I and transferred via ferredoxin. To better understand the association of the hydrogenase and the ferredoxin, we have simulated the process over multiple timescales. A Brownian dynamics simulation method gave an initial thorough sampling of the rigid-body translational and rotational phase spaces, and the resulting trajectories were used to compute the occupancy and free-energy landscapes. Several important hydrogenase-ferredoxin encounter complexes were identified from this analysis, which were then individually simulated using atomistic molecular dynamics to provide more details of the hydrogenase and ferredoxin interaction. The ferredoxin appeared to form reasonable complexes with the hydrogenase in multiple orientations, some of which were good candidates for inclusion in a transition state ensemble of configurations for electron transfer.     

Comparison of the subunit compositions of the PSI-LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii

Although the light-harvesting chlorophyll protein complex I (LHCI) of photosystem I (PSI) is intimately associated with the PSI core complex and forms the PSI-LHCI supercomplex, the LHCI is normally synthesized in PSI-deficient mutants. In this paper, we compared the subunit compositions of the PSI-LHCI supercomplex and the LHCI by immunoblot analysis and two-dimensional gel electrophoresis combined with mass spectrometry. The PSI-LHCI supercomplex and the LHCI were purified by sucrose density gradient centrifugation and (diethylamino)ethyl column chromatography from n-dodecyl-beta-D-maltoside-solubilized thylakoids of the wild-type and DeltapsaB mutant of the green alga Chlamydomonas reinhardtii. The PSI-LHCI supercomplex contained all of the nine Lhca polypeptides (Lhca1-9) that are detected in wild-type thylakoids. In contrast, the LHCI retained only six Lhca polypeptides, whereas Lhca3 and two minor polypeptides, Lhca2 and Lhca9, were lost during the purification procedure. Sucrose density gradient centrifugation showed that the purified LHCI retains an oligomeric structure with an apparent molecular mass of 300-400 kDa. We therefore concluded that Lhca2, Lhca3, and Lhca9 are not required for the stable oligomeric structure of the LHCI and that the association of these polypeptides in the LHCI is stabilized by the presence of the PSI core complex. Finally, we discuss the possible localization and function of Lhca polypeptides in the LHCI.     

Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging

Oxygenic photosynthetic organisms adapt to varying light conditions by changing the distribution of light energy between Photosystem II (PS II) and photosystem I (PS I) during so-called state transitions. To identify the genes involved in this process, we have exploited a simple chlorophyll fluorescence video-imaging technique to screen a library of nuclear mutants of Chlamydomonas reinhardtii for colonies grown on agar plates that are disturbed in their ability to regulate light energy distribution between PS I and PS II. Subsequent modulated fluorescence measurements at room temperature and 77 K fluorescence emission spectra confirmed that 5 mutants (0.025% of total number screened) were defective in state transitions. [³²P]orthophosphate phosphorylation experiments in vivo revealed that in one of these mutants, designated stm1, the level of LHC II polypeptide phosphorylation was drastically reduced compared with wild type. Despite WT levels of PS I and PS II, stm1 grew photoautotrophically at reduced rates, compared with WT especially under low light conditions, which is consistent with an important physiological role for state transitions. Our results highlight the feasibility of video imaging in tandem with mutagenesis as a means of identifying the genes involved in controlling state transitions in eukaryotic photosynthetic organisms.     

Purification of Hydrogenase from Chlamydomonas reinhardtii

A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H₂ produced per milligram protein per minute. The molecular weight was determined to be 4.5 × 10⁴ by gel filtration and 4.75 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H₂ production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55°C. The K_m of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag.     

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter- or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys¹⁷⁸, whereas a second residue, Cys²⁴⁷, becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.     

Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.Cilia and flagella, which are essentially identical, are among the most ancient cellular organelles, providing motility for primitive eukaryotic cells living in aqueous environments. The assembly and motility of flagella have been studied extensively with the unicellular biflagellate green alga Chlamydomonas reinhardtii. This alga uses flagella for motility and for cell-cell recognition during mating. In basal land plants, such as bryophytes and pteridophytes, the only flagellated cells are motile sperm cells, which require water to swim to the egg. With the evolution of pollen tubes in higher gymnosperms and angiosperms, these plant species lost the ability to assemble flagella (24, 42). Flagella of animals have acquired new functions in multicellular organizations during evolution (6). In mammals, cilia and flagella can be motile or immotile. Motile cilia can be found, for example, in airways (respiratory cilia), in the brain (ependymal cilia), or in the male reproductive system (sperm flagella). Defects in cilia in humans can cause severe diseases, such as polycystic kidney disease, retinal degeneration, hydrocephalus, or changes in the left-right symmetry of organs, collectively known as ciliopathies (20, 32).Although C. reinhardtii and mammals are separated by more than 10⁹ years of evolution, C. reinhardtii flagella are amazingly similar in structure and function to the 9+2-type axonemes of most motile mammalian flagella and cilia (42). They are composed of nine microtubular doublets surrounding two central microtubular singlets. The axoneme of motile flagella includes substructures such as dynein arms and radial spokes that generate and control axoneme bending (31). The flagellum also contains matrix proteins that are not tightly associated with the flagellar membrane or the axoneme. They serve diverse functions and can be involved in intraflagellar transport (IFT) (37).Proteome analyses of cilia, including, for example, a human cilium, a mouse photoreceptor sensory cilium, and the flagella of the green alga Chlamydomonas reinhardtii, have unraveled hundreds of so far unknown proteins of this organelle (18, 29, 33) and have paved the way to further study the functions of these proteins. Several kinases and phosphatases were found in these proteomes, suggesting that reversible protein phosphorylation plays an important role in signaling in this organelle. This is underlined by earlier studies showing that phosphorylation and dephosphorylation control flagellar motility (35), signaling (30), length, and assembly (37, 53) in C. reinhardtii. Some phosphoproteins known or assumed to be involved in these processes, such as outer dynein arm heavy chain alpha (13), inner dynein arm intermediate chain protein IC138 (7), and central pair kinesin KLP1 (61), were characterized, but the exact in vivo phosphorylation sites were not determined. From earlier studies, it is known that >80 protein spots, representing axonemal components, are labeled by ³²P by two-dimensional electrophoretic techniques (34), but many of them have not been identified so far. In the past years, the relevance of some of the flagellar kinases has been shown. For example, silencing of casein kinase 1 (CK1) disturbs flagellum formation, among several other effects (41). One of its targets is IC138 (54). Glycogen synthase kinase 3 was suggested to regulate the assembly and length of flagella (53). Also, in mammalian cilia, reversible protein phosphorylation plays an important role in ciliary beating. Second messengers such as cyclic AMP (cAMP) and cGMP, which activate special kinases, are known to be relevant there (39).An understanding of how reversible protein phosphorylation influences the function of cilia and their role in diseases will require increased information not only about the nature of the phosphoproteins but also on their in vivo phosphorylation sites. In order to gain insight into the phosphoproteome of a eukaryotic cilium, we used the green alga C. reinhardtii, whose entire genome has been sequenced, as a model (23). This organism has many advantages for biochemical and molecular genetic studies of the flagellum. Importantly, as mentioned before, its flagellar proteome is known (33), and in addition, the proteome of the centriole that anchors the flagella is also known (11, 12).For the identification of the targets of the kinases and phosphatases in the flagella, phosphoproteomics can be applied. However, phosphoproteome analysis has been and still is a challenging task (19, 36, 47). This is due to a few facts, as follows. (i) Phosphoproteins can have more than one phosphorylation site, and the phosphorylation status of these sites can fluctuate depending on the physiological conditions of the cell. (ii) Only a small portion of a given protein in the cell can be phosphorylated. (iii) Furthermore, phosphoproteins, especially those of signaling pathways, are often proteins found in low abundance. Therefore, it is necessary to enrich the phosphopeptides. Among different methods, immobilized metal affinity chromatography (IMAC) is frequently used for phosphopeptide enrichment. In C. reinhardtii, phosphopeptides from proteins of the cellular, thylakoid, and eyespot phosphoproteomes were identified by this way (49, 50, 51, 52). Thereby, it became obvious that biochemical enrichment of subcellular fractions as it was done with the eyespot apparatus results in an increase of phosphopeptide identification (52). In this study, we used IMAC and tandem mass spectrometry (MS/MS) along with the acquisition of data-dependent neutral loss (MS/MS/MS spectra) to identify phosphopeptides from isolated flagella of C. reinhardtii. In this way, we identified 32 flagellar phosphoproteins, including different functional categories, along with 126 in vivo phosphorylation sites. In many cases, a dynamic phosphorylation pattern within one peptide was observed.     

Inorganic Carbon Uptake by Chlamydomonas reinhardtii

The rates of CO₂-dependent O₂ evolution by Chlamydomonas reinhardtii, grown with either air levels of CO₂ or air with 5% CO₂, were measured at varying external pH. Over a pH range of 4.5 to 8.5, the external concentration of CO₂ required for half-maximal rates of photosynthesis was constant, averaging 25 micromolar for cells grown with 5% CO₂. This is consistent with the hypothesis that these cells take up CO₂ but not HCO₃⁻ from the medium and that their CO₂ requirement for photosynthesis reflects the K_m(CO₂) of ribulose bisphosphate carboxylase. Over a pH range of 4.5 to 9.5, cells grown with air required an external CO₂ concentration of only 0.4 to 3 micromolar for half-maximal rates of photosynthesis, consistent with a mechanism to accumulate external inorganic carbon in these cells. Air-grown cells can utilize external inorganic carbon efficiently even at pH 4.5 where the HCO₃⁻ concentration is very low (40 nanomolar). However, at high external pH, where HCO₃⁻ predominates, these cells cannot accumulate inorganic carbon as efficiently and require higher concentrations of NaHCO₃ to maintain their photosynthetic activity. These results imply that, at the plasma membrane, CO₂ is the permeant inorganic carbon species in air-grown cells as well as in cells grown on 5% CO₂. If active HCO₃⁻ accumulation is a step in CO₂ concentration by air-grown Chlamydomonas, it probably takes place in internal compartments of the cell and not at the plasmalemma.     

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-113, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.     

Molecular remodeling of photosystem II during state transitions in Chlamydomonas reinhardtii

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.     

Portrait of a species: Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, the first alga subject to a genome project, has been the object of numerous morphological, physiological, and genetic studies. The organism has two genetically determined mating types (plus and minus) and all stages of the simple life cycle can be evoked in culture. In the nearly 60 years since the first standard laboratory strains were isolated, numerous crosses and exchanges among laboratories have led to some confusion concerning strain genealogy. Here we use analyses of the nuclear internal transcribed spacer regions and other genetic traits to resolve these issues, correctly identify strains currently available, and analyze phylogenetic relationships with all other available similar chlamydomonad types. The presence of a 10-bp indel in ITS2 in some but not all copies of the nuclear ribosomal cistrons of an individual organism, and the changing ratios of these in crosses, provide a tool to investigate mechanisms of concerted evolution. The standard C. reinhardtii strains, plus C. smithii +, plus the new eastern North American C. reinhardtii isolates, comprise one morphological species, one biological species of high sexual intercompatibility, and essentially identical ITS sequences (except the tip of helix I of ITS2). However, variant RFLP patterns characterize strains from each geographic site.