Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley etioplasts

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.     

Localisation and processing of the precursor form of photosystem II protein D1 inSynechocystis 6803

Pure plasma membrane and thylakoid membrane fractions from Synechocystis 6803 were isolated to study the localisation and processing of the precursor form of the D1 protein (pD1) of photosystem II (PSII). PSII core proteins (D1, D2 and cytb559) were localised both to plasma and thylakoid membrane fractions, the majority in thylakoids. pD1 was found only in the thylakoid membrane where active PSII is known to function. Membrane fatty acid unsaturation was shown to be critical in processing of pD1 into mature D1 protein. This was concluded from pulse-labelling experiments at low temperature using wild type and a mutant Synechocystis 6803 with a low level of membrane fatty acid unsaturation. Further, pD1 was identified as two distinct bands, an indication of two cleavage sites in the precursor peptide or, alternatively, two different conformations of pD1. Our results provide evidence for thylakoid membranes being a primary synthesis site for D1 protein during its light-activated turnover. The existence of the PSII core proteins in the plasma membrane, on the other hand, may be related to the biosynthesis of new PSII complexes in these membranes.     

Mechanism of photoinhibition in vivo. A reversible light-induced conformational change of reaction center II is related to an irreversible modification of the D1 protein

The light-induced inactivation of the photochemical reaction center II (RCII) of oxygenic chloroplasts (photoinhibition) was investigated in cells and isolated thylakoids of the green alga Chlamydomonas reinhardtii. The process is resolved into a reversible conformational change followed by an irreversible modification of RCII D1 protein. The light-induced changes in vivo persisted in isolated thylakoids. The first step is characterized by (i) destabilization of the secondary acceptor semiquinone anion, Q-B, bound to the D1 protein. This is demonstrated by a reduction in the activation energy of S2,3Q-B charge recombination as measured by the thermoluminescence technique; and (ii) a rise in the intrinsic fluorescence and a decrease of the maximal fluorescence. Unoccupancy of the QB site by plastoquinone partially protected RCII against the light-induced destabilization of Q-B. The extent of charge separation (P+680Q-A) was not affected. However, the slow phase (microsecond) of P+680 dark reduction increased, and the amplitude of signal II was reduced by 20-30%, indicating that in a fraction of RCII, electron donation from Z to P+680 was impaired without losing primary photochemistry. This modification correlates with the irreversible change in D1 protein resulting in the formation of a trypsin-resistant fragment of 16 kDa detected in D1 isolated from light-exposed cells. The change in the Q-B stability could allow charge equilibration with QA and thus explain the rise in the intrinsic fluorescence level and reduction of electron flow to plastoquinone. The change in the lifetime of P+680 can account for further reduction in electron flow (photo-inhibition). The irreversible light-dependent modification of D1 may serve as the signal for its degradation and replacement by a newly synthesized molecule (turnover).     

GTP enhances the degradation of the photosystem II D1 protein irrespective of its conformational heterogeneity at the Q(B) site

The light exposure history and/or binding of different herbicides at the Q(B) site may induce heterogeneity of photosystem II acceptor side conformation that affects D1 protein degradation under photoinhibitory conditions. GTP was recently found to stimulate the D1 protein degradation of photoinactivated photosystem II (Spetea, C. , Hundal, T., Lohmann, F., and Andersson, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6547-6552). Here we report that GTP enhances the cleavage of the D1 protein D-E loop following exposure of thylakoid membranes to either high light, low light, or repetitive single turnover flashes but not to trypsin. GTP does not stimulate D1 protein degradation in the presence of herbicides known to affect the accessibility of the cleavage site to proteolysis. However, GTP stimulates degradation that can be induced even in darkness in some photosystem II conformers following binding of the PNO8 herbicide (Nakajima, Y., Yoshida, S., Inoue, Y., Yoneyama, K., and Ono, T. (1995) Biochim. Biophys. Acta 1230, 38-44). Both the PNO8- and the light-induced primary cleavage of the D1 protein occur in the grana membrane domains. The subsequent migration of photosytem II containing the D1 protein fragments to the stroma domains for secondary proteolysis is light-activated. We conclude that the GTP effect is not confined to a specific photoinactivation pathway nor to the conformational state of the photosystem II acceptor side. Consequently, GTP does not interact with the site of D1 protein cleavage but rather enhances the activity of the endogenous proteolytic system.     

Evidence that D1 processing is required for manganese binding and extrinsic protein assembly into photosystem II

Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated DeltactpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor Y(Z) was accessible to exogenous Mn(2+) ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in DeltactpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis.     

ATP and light regulate D1 protein modification and degradation. Role of D1* in photoinhibition.

We have recently shown that during in vivo photoinhibition the D1 protein is degraded via a modified form, designated D1*. Depending on light conditions, the amount of D1* varies in leaves between 0 and 50% of total D1 content. By isolating thylakoids from leaves acclimated to different light levels, and performing photoinhibition experiments on these thylakoids, the following results on D1 protein degradation were obtained: (i) the protease involved in D1 degradation requires activation by light; (ii) neither acceptor nor donor side photoinhibition of PSII induces formation of D1* in vitro; (iii) in isolated thylakoids, the transformation of D1 to D1* can be induced in low light in the presence of ATP, which suggests that D1* is a phosphorylated form of the D1 protein; (iv) D1*, induced either in vivo or in vitro, is much less susceptible to degradation during illumination of isolated thylakoids than the original D1 protein. We suggest that the modification to D1* is a means to prevent disassembly of photodamaged photosystem II complex in appressed membranes.     

The PQ/PQH2 ratio and occupancy of photosystem II-QB site by plastoquinone control the degradation of D1 protein during photoinhibition in vivo

Photoinactivation of photosystem II (PSII) and light-dependent degradation of the reaction center II (RCII) protein D1 have been investigated in Chlamydomonas reinhardtii mutants D6, AC208, and B4 deficient in cytochrome b6/f, plastocyanin, and photosystem I (PSI) activity, respectively. These mutants possess active PSII and reduce plastoquinone (PQ) but cannot oxidize plastoquinol (PQH2) via light-dependent reduction of NADP. In light-exposed cells a high ratio PQH2/PQ and a low turnover of PQ/PQH2 at the RCII-QB site are maintained. In all mutants photoinactivation of RCII was slower as compared to the wild-type (wt) cells, and D1 degradation was drastically decreased. The degradation of D1 was also lower in the wt cells under anaerobic conditions and presence of ascorbate, while raising the concentration of dissolved oxygen increased the degradation of the D1 protein in the AC208 mutant. Photoinactivation and light-dependent degradation of the D1 protein were drastically increased in the Scenedesmus obliquus LF-1 mutant cells altered in its PSII manganese binding and thus unable to reduce PQ using water as an electron donor. Diuron inhibited the light-dependent degradation of D1 protein in both the LF-1 mutant and wt cells. Based on these results we propose that availability of PQ at the QB site is required for (i) the photoinactivation process of the RCII acceptor side followed by inactivation of the donor side leading to the generation of harmful cation radicals (Z+, P680+, chlz+) which damage the D1 protein, and (ii) the accessibility of the cleavage site of the damaged D1 protein to proteolytic degradation.     

The synechocystis sp PCC 6803 oxa1 homolog is essential for membrane integration of reaction center precursor protein pD1

Synechocystis sp PCC 6803 Slr1471p, an Oxa1p/Alb3/YidC homolog, is an essential protein for cell viability for which functions in thylakoid membrane biogenesis and cell division have been proposed. Using a fusion of green fluorescent protein to the C terminus of Slr1471p, we found that the mutant slr1471-gfp is photochemically inhibited when light intensities increase to 80 micromol x m(-2) x s(-1). We show that photoinhibition correlates with an increased redox potential of the reaction center quinone Q(A)(-) and a decreased redox potential of Q(B)(-). Analysis reveals that membrane integration of the D1 precursor protein is affected, leading to the accumulation of pD1 in the membrane phase. We show that Slr1471p interacts directly with the D1 protein and discuss why the accumulation of pD1 in two reaction center assembly intermediates is dependent on Slr1471p.     

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1_mod and D2_mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1_mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2_mod contained D2/cytochrome b₅₅₉ with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1_mod but not D2_mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Analysis of isolated assembly complexes provides new insights into the early stages of photosystem II biogenesis.     

Regulation of chloroplast membrane function: protein phosphorylation changes the spatial organization of membrane components

A chlorophyll-protein complex of chloroplast membranes, which simultaneously serves as light-harvesting antenna and membrane adhesion factor, undergoes reversible, lateral diffusion between appressed and nonappressed membrane regions under the control of a protein kinase. The phosphorylation-dependent migration process regulates the amount of light energy that is delivered to the reaction centers of photosystems I and II (PS I and PS II), and thereby regulates their rate of turnover. This regulatory mechanism provides a rationale for the finding that the two photosystems are physically separated in chloroplast membranes (PS II in appressed, grana membranes, and PS I in nonappressed, stroma membranes). The feedback system involves the following steps: a membrane-bound kinase senses the rate of PS II vs. PS I turnover via the oxidation-reduction state of the plastoquinone pool, which shuttles electrons from PS II via cytochrome f to PS I. If activated, the kinase adds negative charge (phosphate) to a grana- localized pigment-protein complex. The change in its surface charge at a site critical for promoting membrane adhesion results in increased electrostatic repulsion between the membranes, unstacking, the lateral movement of the complex to adjacent stroma membranes, which differ in their functional composition. The general significance of this type of membrane regulatory mechanism is discussed.     

F-actin and myosin II binding domains in supervillin

Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.     

Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids

The psbA multigene family in Synechococcus sp. strain PCC 7942 encodes two forms of the D1 protein; Form I, the product of psbAI, differs from Form II, the product of psbAII and psbAIII, at 25 of 360 amino acid positions. D1 is essential for photosynthesis as a protein component of the photosystem II reaction center. Antisera were raised against purified hybrid proteins encoded by psbAI-lacZ and psbAIII-lacZ translational gene fusions that contain the unique amino termini of Form I and Form II, respectively. Form specificity of each antiserum was verified by Western analysis using thylakoid membranes from mutant strains containing only Form I or Form II. Western analysis of thylakoid membranes from wild-type cells cultured at different light intensities detected both forms of D1 in the membrane and showed changes in the ratio of the two forms. The D1 composition of the membrane matched predicted ratios of the forms based on differential gene expression: psbAI is expressed highest at low light, and both psbAII and psbAIII are expressed highest at high light. Along a gradient of light intensity from 5 microE. m-2.s-1 to 482 microE.m-2.s-1, the relative amount of Form I in thylakoid membranes decreased 58%, while the relative amount of Form II increased 60%. Maximum detection of Form I coupled with minimum detection of Form II in membranes from cells harvested at light intensities below 390 microE.m-2.s-1 suggests a central role for Form I in photosystem II. Increased incorporation of Form II into the thylakoid membrane occurred at light intensities reported by others to be photoinhibitory, suggesting that Form II serves a role in adaptation to high light.     

Phosphorylation of the D1 photosystem II reaction center protein is controlled by an endogenous circadian rhythm

The light dependence of D1 phosphorylation is unique to higher plants, being constitutive in cyanobacteria and algae. In a photoautotrophic higher plant, Spirodela oligorrhiza, grown in greenhouse conditions under natural diurnal cycles of solar irradiation, the ratio of phosphorylated versus total D1 protein (D1-P index: [D1-P]/[D1] + [D1-P]) of photosystem II is shown to undergo reproducible diurnal oscillation. These oscillations were clearly out of phase with the period of maximum in light intensity. The timing of the D1-P index maximum was not affected by changes in temperature, the amount of D1 kinase activity present in the thylakoid membranes, the rate of D1 protein synthesis, or photoinhibition. However, when the dark period in a normal diurnal cycle was cut short artificially by transferring plants to continuous light conditions, the D1-P index timing shifted and reached a maximum within 4 to 5 h of light illumination. The resultant diurnal oscillation persisted for at least two cycles in continuous light, suggesting that the rhythm is endogenous (circadian) and is entrained by an external signal.     

Characterization of the stromal protease(s) degrading the cross-linked products of the D1 protein generated by photoinhibition of photosystem II

When photosystem (PS) II-enriched membranes are exposed to strong light, cross-linking of the intrinsic D1 protein with the surrounding polypeptides and degradation of the D1 protein take place. The cross-linking of the D1 protein with the alpha-subunit of cytochrome b(559) is suggested to be an early event of photoinduced damage to the D1 protein (Barbato et al., FEBS Lett. 309 (1992) 165-169). The relationship between the cross-linking and the degradation of the D1 protein, however, is not yet clear. In the present study, we show that the addition of stromal extract from chloroplasts degrades the 41 kDa cross-linked product of D1/cytochrome b(559) alpha-subunit and enhances the degradation of the D1 protein. Incubation of the preilluminated PS II-enriched membranes with the stromal extract at 25 degrees C causes the degradation of the cross-linked product by more than 70%. The activity of the stromal extract showed a pH optimum at 8.0, and was enhanced by the addition of ATP or GTP. Consistent with the nucleotide effect, this stromal activity was eliminated by the preincubation of the stromal extract with apyrase, which hydrolyzes nucleotides. Also, the stromal activity was nearly fully inhibited by a serine-type protease inhibitor, 3,4-dichloroisocoumarin, which suggests participation of a serine-type protease(s).     

Competitive inhibition analysis of the enzyme-substrate interaction in the carboxy-terminal processing of the precursor D1 protein of photosystem II reaction center using substituted oligopeptides

A clear parallelism was demonstrated between the efficiency as substrate of the substituted oligopeptides corresponding to the carboxy-terminal (C-terminal) sequence of the precursor D1 protein (pD1) in the in vitro enzymatic assay and their competitive inhibitory capacity toward the proteolytic C-terminal processing of the full-length pD1 integrated in the intact photosystem II complex embedded in the thylakoid membrane of Scenedesmus obliquus LF-1 mutant, as shown e.g. by the influence of L343A, A345G and A345V substitutions and the effect of C-terminal fragments. This suggests that the basic mechanism for substrate recognition by the processing protease elucidated in the enzymatic analysis using synthetic oligopeptides is also effective in vivo, although it can sometimes be difficult to detect the consequence of amino acid substitution in the integrated systems.     

Turnover of the aggregates and cross-linked products of the D1 protein generated by acceptor-side photoinhibition of photosystem II.

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.     

Photoinhibition and D1 Protein Degradation in Peas Acclimated to Different Growth Irradiances

The relationship between the susceptibility of photosystem II (PSII) to photoinhibition in vivo and the rate of degradation of the D1 protein of the PSII reaction center heterodimer was investigated in leaves from pea plants (Pisum sativum L. cv Greenfeast) grown under widely contrasting irradiances. There was an inverse linear relationship between the extent of photoinhibition and chlorophyll (Chl) a/b ratios, with low-light leaves being more susceptible to high light. In the presence of the chloroplast-encoded protein synthesis inhibitor lincomycin, the differential sensitivity of the various light-acclimated pea leaves to photoinhibition was largely removed, demonstrating the importance of D1 protein turnover as the most crucial mechanism to protect against photoinhibition. In the differently light-acclimated pea leaves, the rate of D1 protein degradation (measured from [35S]methionine pulse-chase experiments) increased with increasing incident light intensities only if the light was not high enough to cause photoinhibition in vivo. Under moderate illumination, the rate constant for D1 protein degradation corresponded to the rate constant for photoinhibition in the presence of lincomycin, demonstrating a balance between photodamage to D1 protein and subsequent recovery, via D1 protein degradation, de novo synthesis of precursor D1 protein, and reassembly of functional PSII. In marked contrast, in light sufficiently high to cause photoinhibition in vivo, the rate of D1 protein degradation no longer increased concomitantly with increasing photoinhibition, suggesting that the rate of D1 protein degradation is playing a regulatory role. The extent of thylakoid stacking, indicated by the Chl a/b ratios of the differently light-acclimated pea leaves, was linearly related to the half-life of the D1 protein in strong light. We conclude that photoinhibition in vivo occurs under conditions in which the rate of D1 protein degradation can no longer be enhanced to rapidly remove irreversibly damaged D1 protein. We suggest that low-light pea leaves, with more stacked membranes and less stroma-exposed thylakoids, are more susceptible to photoinhibition in vivo mainly due to their slower rate of D1 protein degradation under sustained high light and their slower repair cycle of the photodamaged PSII centers.     

LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana

To gain insight into the processes involved in photosystem II (PSII) biogenesis and maintenance, we characterized the low psii accumulation1 (lpa1) mutant of Arabidopsis thaliana, which generally accumulates lower than wild-type levels of the PSII complex. In vivo protein labeling experiments showed that synthesis of the D1 and D2 proteins was greatly reduced in the lpa1 mutant, while other plastid-encoded proteins were translated at rates similar to the wild type. In addition, turnover rates of the PSII core proteins CP47, CP43, D1, and D2 were higher in lpa1 than in wild-type plants. The newly synthesized PSII proteins were assembled into functional protein complexes, but the assembly was less efficient in the mutant. LPA1 encodes a chloroplast protein that contains two tetratricopeptide repeat domains and is an intrinsic membrane protein but not an integral subunit of PSII. Yeast two-hybrid studies revealed that LPA1 interacts with D1 but not with D2, cytochrome b6, or Alb3. Thus, LPA1 appears to be an integral membrane chaperone that is required for efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1.     

Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles

Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immunostaining using the monoclonal antibody R24 confirmed G(D3) expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of G(D3) and caveolin-1, we used immunogold electron microscopy. This revealed localization of G(D3) in small clusters in the plasma membrane as well as enrichment and localization of ganglioside G(D3) and caveolin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both G(D3) and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1.     

The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.