Lipid composition determines the effects of arbutin on the stability of membranes.
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Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers. (+info)
Metal ligation by Walker homology B aspartate betaD262 at site 3 of the latent but not activated form of the chloroplast F(1)-ATPase from Chlamydomonas reinhardtii.
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Site-directed mutations D262C, D262H, D262N, and D262T were made to the beta subunit Walker Homology B aspartate of chloroplast F(1)-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3-14% of wild type as were ATPase activities of purified chloroplast F(1) indicating that betaD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F(1) as VO(2+)-ATP gave rise to two EPR species designated B and C in wild type and mutants. (51)V-hyperfine parameters of species C, present exclusively in the activated enzyme state, did not change significantly by the mutations examined indicating that it is not an equatorial ligand to VO(2+), nor is it hydrogen-bonded to a coordinated water at an equatorial position. Every mutation changed the ratio of EPR species C/B and/or the (51)V-hyperfine parameters of species B, the predominant conformation of VO(2+)-nucleotide bound to Site 3 in the latent (down-regulated) state. The results indicate that the Walker Homology B aspartate coordinates the metal of the predominant metal-nucleotide conformation at Site 3 in the latent state but not in the conformation present exclusively upon activation and elucidates one of the specific changes in metal ligation involved with activation. (+info)
Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.
(3/814)
In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed. (+info)
Two distinct translocation intermediates can be distinguished during protein transport by the TAT (Deltaph) pathway across the thylakoid membrane.
(4/814)
During thylakoid transport of the chimeric precursor protein 16/23 which takes place by the twin arginine translocation (TAT) (Deltaph)-dependent pathway, two distinct translocation intermediates can be identified which represent successive steps in the translocation process. Both intermediates are partially inserted into the thylakoid membrane and can be distinguished by specific degradation fragments occurring after thermolysin treatment of the thylakoids. While the formation of the early translocation intermediate does not depend on a functional TAT translocation machinery, the appearance of the late intermediate is strictly coupled to the Deltaph-dependent transport of the 16/23 chimera. Accordingly, this translocation intermediate is found associated with two distinct complexes in the thylakoid membrane having apparent molecular masses of approximately 560 and 620 kDa, respectively. (+info)
Turnover of the aggregates and cross-linked products of the D1 protein generated by acceptor-side photoinhibition of photosystem II.
(5/814)
It is known that the reaction-center binding protein D1 in photosystem (PS) II is degraded significantly during photoinhibition. The D1 protein also cross-links covalently or aggregates non-covalently with the nearby polypeptides in PS II complexes by illumination. In the present study, we detected the adducts between the D1 protein and the other reaction-center binding protein D2 (D1/D2), the alpha-subunit of cyt b(559) (D1/cyt b(559)), and the antenna chlorophyll-binding protein CP43 (D1/CP43) by SDS/urea-polyacrylamide gel electrophoresis and Western blotting with specific antibodies. The adducts were observed by weak and strong illumination (light intensity: 50-5000 microE m(-2) s(-1)) of PS II membranes, thylakoids and intact chloroplasts from spinach, under aerobic conditions. These results indicate that the cross-linking or aggregation of the D1 protein is a general phenomenon which occurs in vivo as well as in vitro with photodamaged D1 proteins. We found that the formation of the D1/D2, D1/cyt b(559) and D1/CP43 adducts is differently dependent on the light intensity; the D1/D2 heterodimers and D1/cyt b(559) were formed even by illumination with weak light, whereas generation of the D1/CP43 aggregates required strong illumination. We also detected that these D1 adducts were efficiently removed by the addition of stromal components, which may contain proteases, molecular chaperones and the associated proteins. By two-dimensional SDS/urea-polyacrylamide gel electrophoresis, we found that several stromal proteins, including a 15-kDa protein are effective in removing the D1/CP43 aggregates, and that their activity is resistant to SDS. (+info)
Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes.
(6/814)
We present an extended analysis of the organization of green plant photosystem II and its associated light-harvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII-LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry 38, 2233-2239], the following results were obtained. The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about -20 degrees and -50 degrees, respectively. The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions. A new type of megacomplex was observed with a characteristic windmill-like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips. Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins. (+info)
Localization of cyanobacterial photosystem II donor-side subunits by electron microscopy and the supramolecular organization ofphotosystem II in the thylakoid membrane.
(7/814)
A large set of electron microscopy projections of photosystem II (PSII) dimers isolated from the cyanobacterium Synechococcus elongatus was characterized by single particle image analysis. In addition to previously published maps at lower resolution [Boekema, E.J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A.F., Barber, J. & Rogner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175-179], the new side-view projections show densities of all three lumenal extrinsic proteins, i.e. the 33-kDa, 12-kDa and the cytochrome c-550 subunit encoded by psbO, psbU and psbV, respectively. Analysis of the size and shape of the top-view projections revealed a small number of photosystem II particles of about double the size of the usual dimers. Size and quantity of these 'double dimers' correlates with a small fraction of 1000-kDa particles found with HPLC-size-exclusion chromatographic analysis. Because many cyanobacteria contain dimeric photosystem II complexes arranged in rows within the membrane, the double dimers can be considered as the breakdown fragments of these rows. Their analysis enabled the detection of the arrangement of photosystem II within the rows, in which the dimers interact with other dimers mostly with their tips, leaving a rather open center at the interfaces of two dimers. The dimers have a repeating distance of only 11.7 nm. As a consequence, the phycobilisomes, located on top of PSII and functioning in light-harvesting, must be closely packed or almost touch each other, in a manner similar to a recently suggested model [Bald, D., Kruip, J. & Rogner, M. (1996) Photosynthesis Res. 49, 103-118]. (+info)
A processing intermediate of a stromal chloroplast import protein in Chlamydomonas.
(8/814)
Proteins synthesized in the cytoplasm and destined for importation into the chloroplast across the double envelope membrane contain an N-terminal transit sequence which upon import is cleaved off by a stromal-processing peptidase. Since for stromal-residing proteins no intermediates have ever been found in vivo, it is assumed that precursor proteins are cleaved to the mature size by one proteolytic event which occurs immediately after translocation across both envelope membranes. During import of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into isolated chloroplasts of Chlamydomonas we identified an intermediate-sized product, called iSS. It might be identical to a previously described iSS obtained in vitro by a partially purified soluble chloroplast protease [Su and Boschetti (1993) Eur. J. Biochem. 217, 1039-1047]. The kinetics of the formation of iSS in chloroplasts suggest that pSS is processed to the mature small subunit (SS) not by one, but by two steps via this intermediate product. Since, after an induction period, the ratio of iSS/SS was constant under various experimental conditions of import, the formation of iSS was considered not to be a side-reaction. The location of iSS in the intermembrane space of the envelope, as suggested by protease treatment of chloroplasts, questions the one-step translocation mechanism of precursor import into chloroplasts. (+info)