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.     

Slow degradation of the d1 protein is related to the susceptibility of low-light-grown pumpkin plants to photoinhibition

Photoinhibition of photosystem II (PSII) electron transport and subsequent degradation of the D1 protein were studied in pumpkin (Cucurbita pepo L.) leaves developed under high (1000 μmol m⁻² s⁻¹) and low (80 μmol m⁻² s⁻¹) photon flux densities. The low-light leaves were more susceptible to high light. This difference was greatly diminished when illumination was performed in the presence of chloramphenicol, indicating that a poor capacity to repair photodamaged PSII centers is decisive in the susceptibility of low-light leaves to photoinhibition. In fact, the first phases of the repair cycle, degradation and removal of photodamaged D1 protein from the reaction center complex, occurred slowly in low-light leaves, whereas in high-light leaves the degradation of the D1 protein more readily followed photoinhibition of PSII electron transport. A modified form of the D1 protein, with slightly slower electrophoretic mobility than the original D1, accumulated in the appressed thylakoid membranes of low-light leaves during illumination and was subsequently degraded only slowly.     

Rapid, ATP-dependent degradation of a truncated D1 protein in the chloroplast.

The D1 protein constitutes one of the reaction center subunits of photosystem II and turns over rapidly due to photooxidative damage. Here, we studied the degradation of a truncated D1 protein. A plasmid with a precise deletion in the reading frame of the psbA gene encoding D1 was introduced into the chloroplast of Chlamydomonas reinhardtii. A homoplasmic mutant containing the desired gene was able to synthesize the truncated form of the polypeptide, but could not accumulate significant levels of it. As a consequence, other central photosystem II subunits did not assemble within the thylakoid membrane. In vivo pulse-chase experiments showed that the abnormal D1 protein is rapidly degraded in the light. Degradation was delayed in the light in the presence of an uncoupler, or when cells were incubated in the dark. Pulse-chase experiments performed in vitro indicate that an ATP and metal-dependent protease is responsible for the breakdown process. The paper describes the first in vivo and in vitro functional test for ATP-dependent degradation of a defect polypeptide in chloroplasts. The possible involvement of proteases similar to those removing abnormal proteins in prokaryotic organisms is discussed on the basis of proteases recently identified in chloroplasts.     

Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

Photoinhibition of PSII and turnover of the D1 reaction-centre protein in vivo were studied in pumpkin leaves (Cucurbita pepo L.) acclimated to different growth irradiances and in low-light-grown moss, (Ceratodon purpureus) (Hedw.) Brid. The low-light-acclimated pumpkins were most susceptible to photoinhibition. The production rate of photoinhibited PSII centres (k_PI), determined in the presence of a chloroplast-encoded protein-synthesis inhibitor, showed no marked difference between the high- and low-light-grown pumpkin leaves. On the other hand, the rate constant for the repair cycle (k_REC) of PSII was nearly three times higher in the high-light-grown pumpkin when compared to low-light-grown pumpkin. The slower degradation rate of the damaged D1 protein in the low-light-acclimated leaves, determined by pulsechase experiments with [³⁵S]methionine suggested that the degradation of the Dl protein retards the repair cycle of PSII under photoinhibitory light. Slow degradation of the D1 protein in low-light-grown pumpkin was accompanied by accumulation of a phosphorylated form of the D1 protein, which we postulate as being involved in the regulation of D1-protein degradation and therefore the whole PSII repair cycle. In spite of low growth irradiance the repair cycle of PSII in the moss Ceratodon was rapid under high irradiance. When compared to the high- or low-light-acclimated pumpkin leaves, Ceratodon had the highest rate of D1-protein degradation at 1000 mol photons m^–2 s^–1. In contrast to the higher plants, the D1 protein of Ceratodon was not phosphorylated either under high irradiance in vivo or under in-vitro conditions, which readily phosphorylate the D1 protein of higher plants. This is consistent with the rapid degradation of the D1 protein in Ceratodon. Screening experiments indicated that D1 protein can be phosphorylated in the thylakoid membranes of angiosperms and conifers but not in lower plants. The postulated regulation mechanism of D1-protein degradation involving phosphorylation and the role of thylakoid organization in the function of PSII repair cycle are discussed.Abbreviations Chl Chlorophyll - D1^* phosphorylated form of D1 protein - F_max and F_v maximal and variable fluorescence respectively - k_PJ and k_REC rate constants of photoinhibition and concurrent recovery respectively - LHCII lightharvesting chlorophyll a/bprotein of PSII - PFD photon flux density Dr. R. Barbato (Dipartimento di Biologia, Universita di Padova, Padova, Italy), Prof. P. Böger (Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universität Konstanz, Konstanz, Germany), Prof. A. Melis (Department of Plant Biology, University of California, Berkeley, USA), Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) and Mr. A. Soitamo (Department of Biology, University of Turku, Turku, Finland) are gratefully acknowledged for the D1-protein-specific antibodies. The authors thank Ms. Virpi Paakkarinen for excellent technical assistance. This work was supported by the Academy of Finland and the Foundation of the University of Turku.     

Temperature-sensitivity of D1 protein metabolism in isolated Zea mays chloroplasts

The effects of low temperature on the synthesis and stability of the 32 kDa D1 protein of photosystem II were investigated in chloroplasts isolated from maize (Zea mays cv. LG11) leaves. The synthesis of D1 by intact chloroplasts in vitro was strongly dependent on temperature; the Q₁₀ for the initial rate of incorporation of [³⁵S]-methionine into D1 was ca. 2.6 over the range 13–25°C. The synthesis of other thylakoid polypeptides exhibited a similar temperature dependence, whilst synthesis of stromal proteins was considerably less temperature-dependent, with the exception of two polypeptides of ca. 56 and 59.5 kDa. The stability of newly-synthesized D1 in the thylakoid membranes was dependent both on the temperature at which the plants were grown and on the temperature during the pulse-labelling period when the protein was synthesized. In chloroplasts isolated from maize leaves grown at 25°C, D1 that was synthesized and assembled at 25 °C in vitro was rapidly degraded during the chase period. At lower chase temperatures the protein was more stable. When chloroplasts from 25°C-grown leaves were pulse-labelled at 13°C, the stability of D1 was markedly enhanced at all temperatures during the chase period. This effect was even more pronounced in chloroplasts isolated from plants grown at 14°C. The implications of these results are discussed with regard to the ability of maize to recover from photoinhibitory damage at low temperatures.     

Light-dependent D1 protein synthesis and translocation is regulated by reaction center II. Reaction center II serves as an acceptor for the D1 precursor

Light induces an irreversible modification of the photosystem II reaction center (RCII) affecting specifically one of its major components, the D1 protein (Ohad, I., Adir, N., Koike, H., Kyle, D. J., and Inoue, Y. I. (1990) J. Biol. Chem. 265, 1972-1979) which is degraded and replaced continuously (turnover). The turnover rate of D1 is related to light intensity. Evidence is presented showing that RCII translocates from the site of damage in the grana (appressed) domain of the chloroplast membranes to unappressed membrane domains where the D1 precursor protein (pD1) is translated and becomes integrated into RCII. Several forms of RCII (a, a*, and b) were identified on the basis of their electrophoretic mobility. pD1 was found only in the a and b forms in the unappressed membranes. Processing of pD1 occurs after its integration into RCII. Mature D1 appeared mostly in the a form of RCII and following its translocation to the appressed membrane domains also in the a* form. Thus the light intensity-dependent synthesis of D1 protein is related to the availability of modified RCII which serves as an acceptor for pD1. The shuttling of RCII between the two membrane domains may represent a control mechanism of thylakoid membrane protein synthesis.     

Effects of salicylic acid on protein kinase activity and chloroplast D1 protein degradation in wheat leaves subjected to heat and high light stress

In the north of China, wheat plants are often stressed by heat and high light during grain-filling stage, which leads to injury in photosynthetic apparatus and decline in photosynthetic rate. In order to develop a method to protect photosynthetic apparatus in wheat leaves subjected to heat and high light stress, the effects of SA (salicylic acid) and FSBA (5′-p-fluorosulfonylbenzoyl adenosine) on PK (protein kinase) activity, D1 protein degradation and the performance of PSII were investigated in present work. Our results showed that PK activity enhanced under heat and high light stress and declined when stress was removed. FSBA pretreatment resulted in marked decreases in PK activity and D1 protein level, suggesting a correlationship between degradation of D1 protein and phosphorylation. After 2 h of stress, D1 protein level in water-pretreated leaves decreased to 79% of control and then recovered to 81% after 3 h of recovery. This clearly indicated that the damage of D1 protein induced by heat and high light stress was reversible. Compared to the control, SA pretreatment could not only increase PK activity, retard the degradation of D1 protein during heat and high light stress, but also accelerate the recovery of D1 protein level when the stress was removed. Correspondingly, F_v/F_m (maximum photochemical efficiency of PSII), Φ_PSII (actual photochemical efficiency of PSII), ETR (electron transfer rate) and Pn (net photosynthetic rate) in SA-treated leaves were higher than that in leaves of control under both stress and non-stress conditions. Taken together, our results revealed that SA pretreatment could significantly alleviate damages of heat and high light stress on D1 protein and PSII of wheat leaves, and accelerate restoration of photosynthetic function.     

Reversion of temperature-sensitive mutation by inhibition of proteasome-mediated degradation of mutated D123 protein.

A temperature-sensitive cell-cycle mutant of the 3Y1 rat fibroblast cell line, 3Y1tsD123 has in the D123 gene coding region a point mutation which causes instability of the D123 protein. Temperature-sensitive G1 arrest of the mutant is caused by increased degradation of the D123 protein at restrictive temperature. In this study we found that the selective proteasome inhibitors lactacystin and MG132 inhibited degradation of the mutated D123 protein in cell lines overexpressing the mutated D123 protein, followed by accumulation of a modified form (increased molecular weight other than by ubiquitination) of the D123 protein. Although a temperature-resistant revertant of the mutant had no further mutation in the D123 gene coding region, the modification of the mutated D123 protein was inhibited and the mutated D123 protein was rendered stable. The modification was also inhibited in the hybrid cell lines between the revertant and the cell line overexpressing the mutated D123 protein. These facts imply that the mutated D123 protein receives unidentified modification before degradation in the proteasome, and that the revertant expresses a gene inhibiting this modification.     

Extensive degradation of mutant-type D123 protein is responsible for temperature-sensitive proliferation inhibition in 3Y1tsD123 cells

A temperature-sensitive mutant of 3Y1, 3Y1tsD123, reversibly arrested in G1 phase of cell cycle at the restrictive temperature of 39.8 degrees C, shows a single amino acid exchange in the D123 protein. In this study, we found that the D123 protein level in 3Y1tsD123, which was 1/8 of that in 3Y1 compared at the permissive temperature of 33.9 degrees C, lowered to 1/4 after a shift to the restrictive temperature. During inhibition of protein synthesis with cycloheximide, the D123 protein level in 3Y1tsD123 decreased markedly depending on the incubation temperature, compared with that in 3Y1, indicating that the lowered levels of D123 protein in 3Y1tsD123 are due to its degradation. Unexpectedly, 2 stably temperature-resistant clones were isolated after transfection of SV-3Y1tsD123 (SV40-transformed 3Y1tsD123, which shows cell death instead of G1 arrest at the restrictive temperature) with the cDNA of the mutant-type (3Y1tsD123-derived) D123 protein. The D123 protein in both clones degraded extensively at both temperatures, suggesting that the overexpression of the mutant-type D123 protein exceeds its degradation. Both temperature-resistant clones contained higher levels of D123 protein at the restrictive temperature than did SV-3Y1tsD123 at the permissive temperature. We concluded that the lowered D123 protein level at the restrictive temperature induces the temperature-sensitive characteristics of 3Y1tsD123 and SV-3Y1tsD123.     

The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein

The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.     

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.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

Breakdown of the photosystem II reaction center D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products

Illumination of a suspension of thylakoids with light at high intensity causes inhibition of the photosystem II electron transport activity and loss from the membrane of the D1 protein of the photosystem II reaction center. Impairment of the electron transport activity and depletion of D1 protein from the thylakoid membrane of pea were investigated with reference to the presence or absence of oxygen in the suspension. The breakdown products of the D1 protein were identified by immunoblotting with anti-D1 polyclonal antibodies which were proven to recognize mainly the C-terminal region of the protein. The results obtained show that (i) the light-induced inactivation of the photosystem II electron transport activity under anaerobic conditions is faster than in the presence of oxygen; (ii) depletion of D1 protein is observed on a longer time scale with respect to loss of electron transport activity and is faster when photoinhibition is performed in the presence of oxygen; (iii) C-terminal fragments of D1 are only observed when photoinhibition is carried out anaerobically and are mainly localized in the stroma-exposed regions; and (iv) the fragments observed after anaerobic photoinhibition are quickly degraded on further illumination of the thylakoid suspension in the presence of oxygen.     

Stress-induced degradation of the photosynthetic apparatus is accompanied by changes in thylakoid protein turnover and phosphorylation

Development of chlorosis and loss of PSII were compared in young spinach plants suffering under a combined magnesium and sulphur deficiency. Loss of chlorophyll could be detected already after the first week of deficiency and preceded any permanent functional inhibition of PSII as detected by changes in the chlorophyll fluorescence parameter F_v/F_m. A substantial decrease in F_v/F_m was observed only after the second week of deficiency. After 4 weeks, the plants had lost about 70% of their original chlorophyll content, but fluorescence data indicated that 80% of the existing PSII centers were still capable of initiating photosynthetic electron transport. The degradation of the photosynthetic apparatus without loss of PSII activity was due to changes in protein turnover, especially of the PSII D1 reaction center protein. Already by day 7 of deficiency, a 1.4-fold increase in D1 protein synthesis was observed measured as incorporation of ¹⁴C-leucine. Immunological determination by western-blotting did not reveal a change in D1 protein content. Thus, D1 protein was also degraded more rapidly. The increased turnover was high enough to prevent any loss or inhibition of PSII. After 3 weeks, D1 protein synthesis on a chlorophyll basis was further increased by a factor of 2. However, this was not enough to prevent a net loss of D1 protein of about 70%. Immunological determination revealed that together with the D1 protein also other polypeptides of PSII became degraded. This process prevented a large accumulation of photo-inactivated PSII centers. However, it initiated the breakdown of the other thylakoid proteins, especially of LHCII, resulting in the observed chlorosis. Together with the change in protein turnover and stability, a characteristic change in thylakoid protein phosphorylation was observed. In the deficient plants steady state phosphorylation of both LHCII and PSII proteins was increased in the dark. In the light phosphorylation of PSII proteins was stimulated and after 3 weeks of deficiency was even higher in the deficient leaves than in the control plants. In contrast, the phosphorylation level of LHCII decreased in the light and could hardly be detected after 3 weeks of deficiency. Phosphorylation of the reaction center polypeptides presumably increased their stability against proteolytic attack, whereas phosphorylated LHCII seems to be the substrate for proteolysis.     

Low threshold levels of ultraviolet-B in a background of photosynthetically active radiation trigger rapid degradation of the D2 protein of photosystem-II

The photosystem II reaction centre has at its core a heterodimer made up of two proteins, D1 and D2. The D1 protein is known to be rapidly degraded by photosyn-thetically active radiation while the D2 protein is relatively stable. This paper reports that when the aquatic higher plant, Spirodela was exposed to ultraviolet-B radiation, D2 degradation accelerated markedly and half life times approached those of the D1 protein. Moreover, in the presence of an environmentally relevant background of photosynthetically active radiation, low fluxes of ultraviolet-B (but not ultraviolet-A) radiation synergistically stimulated degradation of the D2 protein within functional reaction centres. Thus, above a critical threshold, ultraviolet-B specifically targets the D1–D2 heterodimer for accelerated degradation.     

Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation

Caveolae are long-lived plasma membrane microdomains composed of caveolins, cavins, and a cholesterol-rich membrane. Little is known about how caveolae disassemble and how their coat components are degraded. We studied the degradation of caveolin-1 (CAV1), a major caveolar protein, in CV1 cells. CAV1 was degraded very slowly, but turnover could be accelerated by compromising caveolae assembly. Now, CAV1 became detectable in late endosomes (LE) and lysosomes where it was degraded. Targeting to the degradative pathway required ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery for inclusion into intralumenal vesicles in endosomes. A dual-tag strategy allowed us to monitor exposure of CAV1 to the acidic lumen of individual, maturing LE in living cells. Importantly, we found that "caveosomes," previously described by our group as independent organelles distinct from endosomes, actually correspond to late endosomal compartments modified by the accumulation of overexpressed CAV1 awaiting degradation. The findings led us to a revised model for endocytic trafficking of CAV1.     

Role of two forms of the D1 protein in the recovery from photoinhibition of photosystem II in the cyanobacterium Synechococcus PCC 7942

The study of turnover of two distinct forms of the photosystem II (PSII) D1 protein in cells of the cyanobacterium Synechococcus PCC 7942 showed that the 'high-light' form D1:2 is degraded significantly faster at 500 microE m(-2) s(-1) as compared with 50 microE m(-2) s(-1) while the degradation rates of the 'low-light' form D1:1 under low and high irradiance are not substantially different. Consequently, the D1:1 turnover does not match photoinactivation of PSII under increased irradiance and therefore the cells containing this D1 form exhibit a decrease in the PSII activity. Monitoring of the content of each D1 form during a recovery from growth-temperature photoinhibition showed a good correlation between the synthesis of D1:2 and restoration of the PSII activity. In contrast, when photoinhibitory treatment was conducted at low temperature, a fast recovery was not accompanied by the D1:2 accumulation. The data suggest that photoinactivation at growth temperature results in a modification of PSII that inhibits insertion of D1:1 and, therefore, for restoration of the photochemical activity in the photoinactivated PSII complexes the D1:2 synthesis is needed. This may represent the primary reason for the requirement of psbAII/psbAIII expression under increased irradiance.     

Overexpression of Man2C1 leads to protein underglycosylation and upregulation of endoplasmic reticulum-associated degradation pathway

Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.     

On the molecular mechanism of light-induced D1 protein degradation in photosystem II core particles.

The mechanism of D1 protein degradation was investigated during photoinhibitory illumination of isolated photosystem II core preparations. The studies revealed that a proteolytic activity resides within the photosystem II core complex. A relationship between the inhibition of D1 protein degradation and the binding of the highly specific serine protease inhibitor diisopropyl fluorophosphate to isolated complexes of photosystem II was observed, evidence that this protease is of the serine type. Using radiolabeled inhibitor, it was shown that the binding site, representing the active serine of the catalytic site, is located on a 43-kDa polypeptide, probably the chlorophyll a protein CP43. The protease is apparently active in darkness, with the initiation of breakdown being dependent on high light-induced substrate activation. The proteolysis, which has an optimum at pH 7.5, gives rise to primary degradation fragments of 23 and 16 kDa. In addition, D1 protein fragments of 14, 13, and 10 kDa were identified. Experiments with phosphate-labeled D1 protein and sequence-specific antisera showed that the 23- and 16-kDa fragments originate from the N- and C-termini, respectively, suggesting a primary cleavage of the D1 protein at the outer thylakoid surface in the region between transmembrane helices D and E.     

Photodamaged D1 protein is degraded in Arabidopsis mutants lacking the Deg2 protease

In plants exposed to high irradiances of visible light, the D1 protein in the reaction center of photosystem II is oxidatively damaged and rapidly degraded. Earlier work in our laboratory showed that the serine protease Deg2 performs the primary cleavage of photodamaged D1 protein in vitro. Here, we demonstrate that the rate of D1 protein degradation under light stress conditions in Arabidopsis mutants lacking the Deg2 protease is similar to those in wild-type plants. Therefore, we propose that several redundant D1 protein degradation pathways might exist in vivo.