Supramolecular organization and dual function of the IsiA chlorophyll-binding protein in cyanobacteria

A significant part of global primary productivity is provided by cyanobacteria, which are abundant in most marine and freshwater habitats. In many oceanographic regions, however, the concentration of iron can be so low that it limits growth. Cyanobacteria respond to this condition by expressing a number of iron stress inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. It was recently shown that 18 IsiA proteins encircle trimeric photosystem I (PSI) under iron-deficient growth conditions. We report here that after prolonged growth of Synechocystis PCC 6803 in an iron-deficient medium, the number of bound IsiA proteins can be much higher than previously known. The largest complexes bind 12-14 units in an inner ring and 19-21 units in an outer ring around a PSI monomer. Fluorescence excitation spectra indicate an efficient light harvesting function for all PSI-bound chlorophylls. We also find that IsiA accumulates in cyanobacteria in excess of what is needed for functional light harvesting by PSI, and that a significant part of IsiA builds supercomplexes without PSI. Because the further decline of PSI makes photosystem II (PSII) increasingly vulnerable to photooxidation, we postulate that the surplus synthesis of IsiA shields PSII from excess light. We suggest that IsiA plays a surprisingly versatile role in cyanobacteria, by significantly enhancing the light harvesting ability of PSI and providing photoprotection for PSII.     

Aggregates of the chlorophyll-binding protein IsiA (CP43') dissipate energy in cyanobacteria

In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of approximately 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light.     

In vitro ferredoxin-dependent desaturation of fatty acids in cyanobacterial thylakoid membranes.

Thylakoid membranes isolated from the cyanobacterium Synechocystis sp. strain PCC6803 were capable of desaturating the acyl groups in monogalactosyl diacylglycerol. This desaturation reaction required the reduced form of ferredoxin.     

Chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyll-binding protein into thylakoid membranes

Multiple sorting pathways operate in chloroplasts to localize proteins to the thylakoid membrane. The signal recognition particle (SRP) pathway in chloroplasts employs the function of a signal recognition particle (cpSRP) to target light harvesting chlorophyll-binding protein (LHCP) to the thylakoid membrane. In assays that reconstitute stroma-dependent LHCP integration in vitro, the stroma is replaceable by the addition of GTP, cpSRP, and an SRP receptor homolog, cpFtsY. Still lacking is an understanding of events that take place at the thylakoid membrane including the identification of membrane proteins that may function at the level of cpFtsY binding or LHCP integration. The identification of Oxa1p in mitochondria, an inner membrane translocase component homologous to predicted proteins in bacteria and to the albino3 (ALB3) protein in thylakoids, led us to investigate the potential role of ALB3 in LHCP integration. Antibody raised against a 50-amino acid region of ALB3 (ALB3-50aa) identified a single 45-kDa thylakoid protein. Treatment of thylakoids with antibody to ALB3-50aa inhibited LHCP integration, whereas the same antibody treatment performed in the presence of antigen reversed the inhibition. In contrast, transport by the thylakoid Sec or Delta pH pathways was unaffected. These data support a model whereby a distinct translocase containing ALB3 is used to integrate LHCP into thylakoid membranes.     

Light stress photodynamics of chlorophyll-binding proteins in Arabidopsis thaliana thylakoid membranes revealed by high-resolution mass spectrometric studies

In higher plants the light energy is captured by the photosynthetic pigments that are bound to photosystem I and II and their light-harvesting complex (LHC) subunits. In this study, we examined the photodynamic changes within chlorophyll-protein complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to low light and subsequently exposed to light stress. Chlorophyll-protein complexes were isolated using sucrose density gradient centrifugation and blue-native polyacrylamid gel electrophoresis (BN-PAGE). Proteome analysis was performed using SDS-PAGE, HPLC and high resolution mass spectrometry. We identified several rarely expressed and stress-induced chlorophyll-binding proteins, showed changes in localization of early light-induced protein family and LHC protein family members between different photosynthetic complexes and assembled/disassembled subcomplexes under light stress conditions and discuss their role in a variety of light stress-related processes.     

Glycoengineering of cyanobacterial thylakoid membranes for future studies on the role of glycolipids in photosynthesis

The lipid composition of thylakoid membranes is conserved from cyanobacteria to angiosperms. The predominating components are monogalactosyl- and digalactosyldiacylglycerol. In cyanobacteria, thylakoid membrane biosynthesis starts with the formation of monoglucosyldiacylglycerol which is C4-epimerized to the corresponding galactolipid, whereas in plastids monogalactosyldiacylglycerol is formed at the beginning. This suggests that galactolipids have specific functions in thylakoids. We wanted to investigate whether galactolipids can be replaced by glycosyldiacylglycerols with headgroups differing in their epimeric and anomeric details as well as the attachment point of the terminal hexose in diglycosyldiacylglycerols. For this purpose putative glycosyltransferase sequences were identified in databases to be used for functional expression in various host organisms. From 18 newly identified sequences, four turned out to encode glycosyltransferases catalyzing final steps in glycolipid biosynthesis: two alpha-glucosyltransferases, one beta-galactosyltransferase and one beta-glucosyltransferase. Their functional annotation was based on detailed structural characterization of the new glycolipids formed in the transformant hosts as well as on in vitro enzymatic assays. The expression of alpha-glucosyltransferases in the cyanobacterium Synechococcus resulted in the accumulation of the new alpha-galactosyldiacylglycerol which is ascribed to epimerization of the corresponding glucolipid. The expression of the beta-glucosyltransferase led to a high proportion of new beta-glucosyl-(1-->6)-beta-galactosyldiacylglycerol almost entirely replacing the native digalactosyldiacylglycerol. These results demonstrate that modifications of the glycolipid pattern in thylakoids are possible.     

Integration of a chlorophyll-binding protein into Escherichia coli membranes in the absence of chlorophyll

The mechanism by which a protein integrates posttranslationally into a membrane can involve the composition of the membrane itself, domains within the inserting polypeptide, and a number of associating proteins. Some integral membrane proteins do not accumulate to normal levels when certain pigments are deficient, and this has been interpreted to mean that such proteins may be rapidly degraded when not in a correct complex. Alternatively, pigments could facilitate the movement of some proteins from an aqueous to a lipid environment. To determine whether chlorophyll is absolutely required for the membrane integration of the light-harvesting chlorophyll-binding protein (LHCP) of chloroplast thylakoid membranes, we have expressed LHCP in Escherichia coli that lacks photosynthetic pigments. LHCP is targeted to the bacterial inner membrane by the addition of a bacterial signal peptide and cannot be extracted from these membranes by NaOH, NaBr, or Na2HCO3 but is extracted by 0.2% Triton X-100. Treatment of isolated right-side-out and inside-out bacterial inner membrane vesicles with trypsin reveals that only the amino terminus of LHCP is exposed on the cytoplasmic face, and the remaining portion of the protein is inaccessible. Treatment of the inside-out vesicles with trypsin followed by alkaline extraction shows that LHCP is intrinsic to the membrane and is not anchored solely by the bacterial signal peptide. Chlorophyll, therefore, is not required for LHCP to integrate into a membrane, but in the absence of these pigments this process is observed to be inefficient.     

Quantification of energy-converting protein complexes in plant thylakoid membranes

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b₆f complex (cyt b₆f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b₆f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.     

Movement of newly imported light-harvesting chlorophyll-binding protein from unstacked to stacked thylakoid membranes is not affected by light treatment or absence of amino-terminal threonines

In higher plants and algae, the transduction of captured light energy is highly regulated as excess excitation of photosystem II (PSII) reaction centers can be redirected to photosystem I (PSI) reaction centers. Models that attempt to explain this phenomenon involve light-harvesting chlorophyll-protein complexes (LHCII) that capture light energy and migrate between PSII and PSI. This report shows that in pea chloroplasts, the major protein component of LHCII, light-harvesting chlorophyll-binding protein (LHCP), can indeed migrate within the thylakoid membrane. We show, however, that although newly imported LHCP inserts into both stacked and unstacked thylakoid membranes, it then moves only from the unstacked, PSI-rich membranes to the stacked, PSII-rich membranes. The observed migration is not affected by light treatment that induces a redistribution of captured light energy (state I-state II transition) that previously was thought to induce LHCP to migrate in the opposite direction, from stacked to unstacked membranes. A mutation that removes the site of LHCP phosphorylation, the proposed trigger of state transitions, also has no effect on the integration and movement of LHCP, but does render LHCP more susceptible to proteolytic degradation. These results are not consistent with current models that deal with the short-term change in the distribution of light energy.     

Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana

Thylakoid rhodanase‐like protein (TROL) is a nuclear‐encoded protein of thylakoid membranes required for tethering of ferredoxin:nicotinamide adenine dinucleotide phosphate (NADPH) oxydoreductase (FNR). It has been proposed that the dynamic interaction of TROL with flavoenzyme FNR, influenced by environmental light conditions, regulates the fate of photosynthetic electrons, directing them either to NADPH synthesis or to other acceptors, including reactive oxygen species detoxification pathways. Inside the chloroplasts, TROL has a dual localization: an inner membrane precursor form and a thylakoid membrane mature form, which has been confirmed by several large‐scale chloroplast proteomics studies, as well as protein import experiments. Unlike the localization, the topology of TROL in the membranes, which is a prerequisite for further studies of its properties and function, has not been experimentally confirmed yet. Thermolysin was proven to be a valuable protease to probe the surface of chloroplasts and membranes in general. By treating the total chloroplast membranes using increasing protease concentration, sequential degradation of TROL was observed, indicating protected polypeptides of TROL and possible domain orientation. To further substantiate the obtained results, TROL‐overexpressing Arabidopsis line (OX) and line in which the central rhodanase‐like domain (RHO) has been partially deleted (ΔRHO), were used as well. While OX line showed the same degradation pattern of TROL as the wild‐type, surprisingly, TROL from ΔRHO membranes was not detectable even at the lowest protease concentration applied, indicating the importance of this domain to the integrity of TROL. In conclusion, TROL is a polytopic protein with a stroma‐exposed C‐terminal FNR‐binding region, and the thylakoid lumen‐located RHO domain.     

Glycinebetaine enhances and stabilizes the evolution of oxygen and the synthesis of ATP by cyanobacterial thylakoid membranes.

Glycinebetaine (betaine), an osmoregulant in halophilic plants, stabilized the evolution of oxygen and the synthesis of ATP by thylakoid membranes from the cyanobacterium Synechocystis PCC6803 when it was present during the preparation and incubation of the thylakoid membranes. Moreover, betaine enhanced the evolution of oxygen and the synthesis of ATP when present during assays. When betaine at 1.0 M was present during the preparation of thylakoid membranes and during the measurement of activity, the rate of evolution of oxygen was equivalent to that of intact cells.     

Structure and functional role of supercomplexes of IsiA and Photosystem I in cyanobacterial photosynthesis

Cyanobacteria express large quantities of the iron stress-inducible protein IsiA under iron deficiency. IsiA can assemble into numerous types of single or double rings surrounding Photosystem I. These supercomplexes are functional in light-harvesting, empty IsiA rings are effective energy dissipaters. Electron microscopy studies of these supercomplexes show that Photosystem I trimers bind 18 IsiA copies in a single ring, whereas monomers may bind up to 35 copies in two rings. Work on mutants indicates that the PsaF/J and PsaL subunits facilitate the formation of closed rings around Photosystem I monomers but are not obligatory components in the formation of Photosystem I-IsiA supercomplexes.     

Functions of the water soluble chlorophyll-binding protein in plants

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700-900 μmol photons m⁻² s⁻¹. These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.     

Changes in topography and function of thylakoid membranes following membrane protein phosphorylation

The kinetics of the intracellular redistribution of phytochrome (sequestering) in Avena sativa L. coleoptiles following a brief, saturating actinic pulse of red (R) light have been determined. Immunocytochemical labelling of phytochrome with monoclonal antibodies showed that at 22°C sequestering can occur within 1–2 s from the onset of R irradiation and is dependent upon the continued presence of the far-red-absorbing form of phytochrome (Pfr). The initial rate, but not the final extent, of sequestering is reduced by lowering the temperature of the tissue to 1°C. Sequestering at 22°C appears to involve two distinct stages: (1) a rapid association of Pfr with putative binding sites initiates the sequestered condition, following which (2) these sites of sequestered phytochrome appear to aggregate. Neither of these two processes was affected by the cytoskeletal inhibitors colchicine or cytochalasin B. Phytochrome sequestering therefore resembles R-light-induced phytochrome pelletability with respect to kinetics, temperature sensitivity, and dependence upon the continued presence of Pfr in the cell.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DIC differential interference contrast - FR far-red - Ig immunoglobulin - Pfr, Pr far-red-absorbing and red-absorbing form of phytochrome, respectively - R red     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.