Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

University of Turku

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II.

Phosphatidylglycerol (PG) is a ubiquitous component of thylakoid membranes. Experiments with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 defective in biosynthesis of PG have demonstrated an indispensable role of PG in photosynthesis. In the present study, we have investigated the light susceptibility of the pgsA mutant with regard to the maintenance of the photosynthetic machinery. Growth of the mutant cells without PG increased the light susceptibility of the cells and resulted in severe photoinhibition of photosynthesis upon a high-light treatment, whereas the growth in the presence of PG was protected against photoinhibition. Photoinhibition induced by PG deprivation was mainly caused by an impairment of the restoration process. The primary target of the light-induced damage in thylakoid membranes, the D1 protein of photosystem (PS) II was, however, synthesized and degraded with similar rates irrespective of whether the mutant cells were incubated with PG or not. Intriguingly, it was found that instead of the synthesis of the D1 protein, the dimerization of the PSII core monomers was impaired in the PG-deprived mutant cells. Addition of PG to photoinhibited cells restored the dimerization capacity of PSII core monomers. These results suggest that PG plays an important role in the maintenance of the photosynthetic machinery through the dimerization and reactivation of the PSII core complex.

Requirement of phosphatidylglycerol for maintenance of photosynthetic machinery.

The flavum strain of Tobacco mosaic virus (TMV) differs from the wild-type (wt) virus by causing strong yellow and green mosaic in the systemically infected developing leaves, yellowing in the fully expanded leaves, and distinct malformations of chloroplasts in both types of infected tissues. Analysis of the thylakoid proteins of flavum strain-infected tobacco leaves indicated that the chlorosis in mature leaves was accompanied by depletion of the entire photosystem II (PSII) core complexes and the 33-kDa protein of the oxygen evolving complex. The only change observed in the thylakoid proteins of the corresponding wt TMV-infected leaves was a slight reduction of the alpha and beta subunits of the ATP synthase complex. The coat proteins of different yellowing strains of TMV are known to effectively accumulate inside chloroplasts, but in this work, the viral movement protein also was detected in association with the thylakoid membranes of flavum strain-infected leaves. The mRNAs of different enzymes involved in the chlorophyll biosynthesis pathway were not reduced in the mature chlorotic leaves. These results suggest that the chlorosis was not caused by reduction of pigment biosynthesis, but rather, by reduction of specific proteins of the PSII core complexes and by consequent break-down of the pigments.

Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43' protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins assemble together to form a small low CO2-induced protein complex and further demonstrate the presence of a fourth subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement of PSII activity for de novo synthesis of the NDH-1 complexes.

Towards functional proteomics of membrane protein complexes in Synechocystis sp. PCC 6803.

&#x5317;&#x533A; (NADH &#x918C;&#x6C27;&#x5316;&#x8FD8;&#x539F;&#x9176;&#xFF09;-1 &#x914D;&#x5408;&#x7269;&#x5728;&#x84DD;&#x85FB;&#x4E2D;&#x7684;&#x6709;&#x547C;&#x5438;&#x548C;&#x5FAA;&#x73AF;&#x7684;&#x7535;&#x5B50;&#x6D41;&#x4EE5;&#x53CA;&#x79EF;&#x6781;&#x7684; CO2 &#x5438;&#x6536;&#x91CF;&#x7684;&#x5177;&#x4F53;&#x804C;&#x80FD;&#x3002;&#x4E3A;&#x4E86;&#x9694;&#x79BB;&#x5317;&#x533A; 1 &#x914D;&#x5408;&#x7269;&#xFF0C;&#x7814;&#x7A76;&#x590D;&#x6742;&#x590D;&#x6742;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#xFF0C;&#x901A;&#x8FC7;&#x5C06;&#x4ED6;&#x6807;&#x8BB0; &#xFF08;&#x7EC4;&#x6C28;&#x9178;&#x6807;&#x7B7E;&#xFF09; &#x6DFB;&#x52A0;&#x5230;&#x5317;&#x533A; 1 &#x4E0D;&#x540C;&#x4E9A;&#x57FA;&#x5EFA;&#x9020;&#x4E86; Thermosynechococcus &#x805A;&#x7403;&#x85FB;&#x7684;&#x51E0;&#x682A;&#x3002;&#x4E0E;&#x4ED6;&#x6807;&#x8BB0;&#x4E0A; CupA &#x548C; NdhL &#x7684;&#x4E24;&#x682A;&#x88AB;&#x6210;&#x529F;&#x5730;&#x7528;&#x4E8E;&#x9694;&#x79BB;&#x5317;&#x533A; 1 &#x914D;&#x5408;&#x7269;&#x4E00;&#x6B65;&#x6CD5; Ni2 + &#x67F1;&#x5C42;&#x6790;&#x6CD5;&#x3002;BN &#xFF08;&#x84DD;&#x8272;&#x672C;&#x673A;&#xFF09;/SDS/&#x9875;&#x4ECE; Ni2 + &#x5217;&#x6D17;&#x8131;&#x86CB;&#x767D;&#x8D28;&#x5206;&#x6790;&#x53D1;&#x73B0;&#x5B58;&#x5728;&#x4E09;&#x4E2A;&#x914D;&#x5408;&#x7269;&#x5206;&#x5B50;&#x7684;&#x8D28;&#x91CF;&#x7EA6;&#x4E3A; 450&#x3001; 300 &#x548C; 190 kDa&#xFF0C;&#x786E;&#x5B9A;&#x7531; MS &#x5206;&#x522B;&#x4E3A;&#x5317;&#x533A;-1 L&#x3001; M 1 &#x5317;&#x533A;&#x533B;&#x9662;&#x548C;&#x5317;&#x533A;&#x533B;&#x9662;-1S&#xFF0C;&#x4EE5;&#x524D;&#x53D1;&#x73B0;&#x591A;&#x79CD; sp PCC 6803&#x3002;&#x3002;&#x7EA6; 490 kDa &#x5927;&#x590D;&#x6742;&#x4E5F;&#x662F;&#x5B64;&#x7ACB;&#x5728; NdhL--&#x4ED6;&#x7684;&#x5E94;&#x53D8;&#x3002;&#x6B64;&#x590D;&#x6742;&#x3001; &#x6307;&#x5B9A; &#39; &#x5317;&#x533A;&#x533B;&#x9662;-1MS&#39;&#xFF0C;&#x662F;&#x7531; M 1 &#x5317;&#x533A;&#x533B;&#x9662;&#x53CA;&#x5317;&#x533A;&#x533B;&#x9662;-1S &#x7EC4;&#x6210;&#x3002;&#x5317;&#x533A;-1 L &#x590D;&#x6742;&#x662F; Ni2 + &#x67F1;&#x5C42;&#x6790;&#x6CD5;&#x4ECE; WT (&#x91CE;&#x751F;&#x578B;) T.&#x805A;&#x7403;&#x85FB;&#x7EC6;&#x80DE;&#x6062;&#x590D;&#x7684;&#x3002;&#x76EE;&#x524D;&#x4EC5;&#x5728;&#x5317;&#x533A;-1 L NdhF1 &#x4E9A;&#x57FA;&#x90FD;&#x6709;&#x4E00;&#x4E2A;&#x5E8F;&#x5217;&#x7684;-HHDHHSHH-&#x5185;&#x90E8;&#xFF0C;&#x5176;&#x4E2D;&#x4F3C;&#x4E4E;&#x6709;&#x4EB2;&#x548C;&#x529B;&#x7684; Ni2 + &#x5217;&#x3002;&#x5317;&#x533A;&#x533B;&#x9662;-1S &#x6216;&#x5317;&#x533A;-1 M &#x672A;&#x6062;&#x590D; WT &#x7EC6;&#x80DE;&#x4ECE;&#x8FD9;&#x79CD;&#x8272;&#x8C31;&#x6CD5;&#x3002;&#x819C;&#x7684;&#x4F4E;&#x6D53;&#x5EA6;&#x7684;&#x6D17;&#x6DA4;&#x5242;&#x589E;&#x6EB6; BN SDS &#x5206;&#x6790;&#x8868;&#x660E;&#x5B58;&#x5728;&#x4E30;&#x5BCC;&#x5317;&#x533A;-1 &#x6BEB;&#x79D2;&#xFF0C;&#x4F46;&#x672A;&#x5317;&#x533A;-1 M &#x6216;&#x5317;&#x533A;&#x533B;&#x9662;-1S&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x6E05;&#x695A;&#x5730;&#x8868;&#x660E;&#x5317;&#x533A;&#x533B;&#x9662;-1S &#x662F;&#x5173;&#x8054;&#x4E0E;&#x5317;&#x533A;-1 M &#x4F53;&#x5185;&#x3002;

&#x9694;&#x79BB;&#x3001; &#x4E9A;&#x57FA;&#x7EC4;&#x6210;&#x53CA;&#x5317;&#x533A; 1 &#x914D;&#x5408;&#x7269;&#x805A;&#x7403;&#x85FB; Thermosynechococcus BP 1 &#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3002;

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.

NDH (NADH-quinone oxydor&eacute;ductase)-1 complexes chez les cyanobact&eacute;ries ont des fonctions sp&eacute;cifiques dans la respiration et le flux d&#39;&eacute;lectrons cyclique aussi bien que dans l&#39;absorption de CO2 active. Afin d&#39;isoler les complexes NDH-1 et pour &eacute;tudier les interactions complexes complex, plusieurs souches de Thermosynechococcus elongatus ont &eacute;t&eacute; construits en ajoutant un His-tag (&eacute;tiquette histidine) &agrave; diff&eacute;rentes sous-unit&eacute;s de NDH-1. Deux souches avec His-tag sur CupA et NdhL ont &eacute;t&eacute; utilis&eacute;s avec succ&egrave;s pour isoler les complexes NDH-1 par chromatographie Ni2 + en une seule &eacute;tape. BN (bleu-indig&egrave;ne) / SDS/PAGE analyse des prot&eacute;ines &eacute;lu&eacute;e de la colonne Ni2 + ont r&eacute;v&eacute;l&eacute; la pr&eacute;sence de trois complexes avec des masses mol&eacute;culaires d&#39;environ 450, 300 et 190 kDa, qui ont &eacute;t&eacute; identifi&eacute;s par MS respectivement de NDH - 1 L, NDH - 1 M et NDH-1 s, pr&eacute;c&eacute;demment trouv&eacute; chez Synechocystis sp PCC 6803. Un plus grand complexe d&#39;environ 490 kDa a &eacute;t&eacute; isol&eacute; de la souche NdhL-His. Ce complexe, d&eacute;sign&eacute; &#39;NDH-1MS&#39;, &eacute;tait compos&eacute; de NDH - 1 M et NDH-1 s. Complexe NDH - 1 L a &eacute;t&eacute; r&eacute;cup&eacute;r&eacute; &agrave; partir de cellules (type sauvage) WT de T. elongatus par Ni2 + chromatographie sur colonne. NdhF1 sous-unit&eacute; pr&eacute;sente seulement dans NDH - 1 L a une s&eacute;quence de - HHDHHSHH-en interne, qui semble avoir une affinit&eacute; pour la colonne Ni2 +. NDH-1 s ou NDH - 1 M n&#39;a pas restaur&eacute; de cellules WT par chromatographie de ce genre. L&#39;analyse SDS/BN/PAGE des membranes solubilis&eacute;es par une faible concentration de d&eacute;tergent a r&eacute;v&eacute;l&eacute; la pr&eacute;sence d&#39;abondants NDH-1MS, mais pas de NDH - 1 M ou NDH-1 s. Ces r&eacute;sultats d&eacute;montrent clairement que NDH-1 s est associ&eacute; avec NDH - 1 M in vivo.

Isolement, subunit composition et l&#39;interaction des complexes de Thermosynechococcus elongatus BP-1 NDH-1.

Isolation, Untereinheit Zusammensetzung und Wechselwirkung der NDH-1-Komplexen aus Thermosynechococcus elongatus BP-1.

NDH (ossidoriduttasi NADH-chinone)-1 complessi nei cianobatteri hanno funzioni specifiche nella respirazione e flusso ciclico degli elettroni cos&igrave; come attivo assorbimento di CO2. Al fine di isolare NDH-1 complessi e per studiare le interazioni complex-complesso, furono costruiti diversi ceppi di Thermosynechococcus elongatus aggiungendo un His-tag (tag di istidina) a differenti subunit&agrave; del NDH-1. Due ceppi con sua etichetta su CupA e NdhL con successo sono stati utilizzati per isolare NDH-1 complessi da One Step Ni2 + colonna cromatografica. BN (blu-Natale) / SDS/pagina analisi delle proteine eluite dalla colonna Ni2 + ha rivelato la presenza di tre complessi con masse molecolari di circa 450, 300 e 190 kDa, che sono stati identificati da MS per essere rispettivamente NDH-1 L, NDH-1 M e NDH-1S, precedentemente trovato nella struttura SP. PCC 6803. Un pi&ugrave; vasto complesso del kDa circa 490 &egrave; stato anche isolato dal ceppo, il suo NdhL. Questo complesso, designato &#39;NDH-1MS&#39;, era composto di NDH-1 M e NDH-1S. NDH-1 L complesso &egrave; stato recuperato dalle cellule di WT (wild-type) di T. elongatus di Ni2 + colonna cromatografica. NdhF1 subunit&agrave; presenti solo nel NDH-1 L ha una sequenza di - HHDHHSHH-internamente, che sembra avere un&#39;affinit&agrave; per la colonna Ni2 +. NDH-1S o NDH-1 M non &egrave; stato recuperato dalle cellule WT mediante cromatografia di questo tipo. L&#39;analisi SDS/BN/pagina di membrane solubilizzato da una bassa concentrazione del detersivo indicato la presenza di abbondante NDH-1MS, ma non NDH-1 M o NDH-1S. Questi risultati hanno dimostrato chiaramente che NDH-1S &egrave; associato con M. NDH-1 in vivo.

Isolamento, subunit&agrave; composizione e interazione dei complessi NDH-1 da Thermosynechococcus elongatus BP-1.

NDH (NADH &#x30AD;&#x30CE;&#x30F3;&#x9178;&#x5316;&#x9084;&#x5143;&#x9175;&#x7D20;)-1 &#x932F;&#x4F53;&#x30B7;&#x30A2;&#x30CE; &#x30D0;&#x30AF;&#x30C6;&#x30EA;&#x30A2;&#x306F;&#x3001;&#x547C;&#x5438;&#x3068;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306E; CO2 &#x5438;&#x53CE;&#x306E;&#x3088;&#x3046;&#x306B;&#x3001;&#x7E70;&#x8FD4;&#x3057;&#x96FB;&#x5B50;&#x6D41;&#x306E;&#x7279;&#x5B9A;&#x6A5F;&#x80FD;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;NDH 1 &#x932F;&#x4F53;&#x3092;&#x9694;&#x96E2;&#x3059;&#x308B;&#x305F;&#x3081;&#x3001;&#x8907;&#x96D1;&#x306A;&#x8907;&#x96D1;&#x306A;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x7814;&#x7A76;&#x3059;&#x308B;&#x3001;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x7CFB;&#x7D71;&#x306E;&#x71B1;&#x6027;&#x85CD;&#x8272;&#x7D30;&#x83CC;&#x306E;&#x6E29;&#x5EA6;&#x306F; NDH 1 &#x306E;&#x7570;&#x306A;&#x308B;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306B; His &#x30BF;&#x30B0; (&#x30D2;&#x30B9;&#x30C1;&#x30B8;&#x30F3; &#x30BF;&#x30B0;) &#x3092;&#x8FFD;&#x52A0;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5EFA;&#x8A2D;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x5F7C;&#x306E;&#x672D;&#x3092; 2 &#x7CFB;&#x7D71;&#x30BF;&#x30EB;&#x30B0;&#x30E2;&#x304A;&#x3088;&#x3073; NdhL &#x306F;&#x3001;&#x6B63;&#x5E38;&#x306B; NDH 1 &#x932F;&#x4F53;&#x306E;&#x5206;&#x96E2;&#x3059;&#x308B;&#x30EF;&#x30F3;&#x30B9;&#x30C6;&#x30C3;&#x30D7; ni 2 + &#x30AB;&#x30E9;&#x30E0;&#x30AF;&#x30ED;&#x30DE;&#x30C8;&#x30B0;&#x30E9;&#x30D5;&#x30A3;&#x30FC;&#x306B;&#x3088;&#x308A;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;BN (&#x9752;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;)/SDS/&#x30DA;&#x30FC;&#x30B8; ni 2 + &#x30AB;&#x30E9;&#x30E0;&#x304B;&#x3089;&#x6EB6;&#x51FA;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x89E3;&#x6790;&#x306E;&#x5B58;&#x5728;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x7D04; 450 &#x306E;&#x5206;&#x5B50;&#x306E;&#x8CEA;&#x91CF;&#x3092;&#x6301;&#x3064; 3 &#x3064;&#x932F;&#x4F53;&#x306E; NDH 1 L&#x3001;NDH 1 M&#x3001;NDH 1S &#x305D;&#x308C;&#x305E;&#x308C;&#x306B; MS &#x306B;&#x3088;&#x3063;&#x3066;&#x8B58;&#x5225;&#x3055;&#x308C;&#x305F;&#x3001;300 &#x3068; 190 &#x306E; kDa &#x4EE5;&#x524D;&#x898B;&#x3064;&#x3051;&#x305F;&#x30B3;&#x30F3;&#x30B9;&#x30C8;&#x30E9;&#x30AF;&#x30C8; PCC 6803&#x3002;&#x7D04; 490 kDa &#x306E;&#x5927;&#x304D;&#x3044;&#x8907;&#x5408;&#x4F53;&#x3082; NdhL &#x5F7C;&#x306E;&#x682A;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3055;&#x308C;&#x305F;.&#x6307;&#x5B9A; &#39; NDH 1 ms &#39;&#x3001;&#x3053;&#x306E;&#x8907;&#x5408;&#x4F53;&#x306F;&#x3001;NDH 1 M &#x3068; NDH 1S &#x306E;&#x4F5C;&#x66F2;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;NDH 1 L &#x30B3;&#x30F3;&#x30D7;&#x30EC;&#x30C3;&#x30AF;&#x30B9; WT &#xFF08;&#x91CE;&#x751F;&#x578B;&#xFF09; &#x7D30;&#x80DE;&#x304B;&#x3089; t. &#x5703;&#x306E; ni 2 + &#x30AB;&#x30E9;&#x30E0; &#x30FB; &#x30AF;&#x30ED;&#x30DE;&#x30C8;&#x30B0;&#x30E9;&#x30D5;&#x30A3;&#x306B;&#x3088;&#x3063;&#x3066;&#x56DE;&#x5FA9;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;NdhF1 &#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3067; NDH 1 L &#x306E;&#x307F;&#x5B58;&#x5728;&#x5217; ni 2 + &#x89AA;&#x548C;&#x6027;&#x3092;&#x6301;&#x3064;&#x3088;&#x3046;&#x306B;&#x898B;&#x3048;&#x307E;&#x3059;&#x3001;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9; - HHDHHSHH-&#x5185;&#x90E8;&#x7684;&#x306B;&#x306F;&#x3001;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;NDH 1 &#x79D2;&#x307E;&#x305F;&#x306F; NDH 1 M WT &#x7D30;&#x80DE;&#x304B;&#x3089;&#x3053;&#x306E;&#x7A2E;&#x306E;&#x30AF;&#x30ED;&#x30DE;&#x30C8;&#x30B0;&#x30E9;&#x30D5;&#x30A3;&#x30FC;&#x306B;&#x3088;&#x308B;&#x56DE;&#x5FA9;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x6D17;&#x5264;&#x306E;&#x4F4E;&#x6FC3;&#x5EA6;&#x3067;&#x53EF;&#x6EB6;&#x5316;&#x819C;&#x306E; BN/SDS/&#x30DA;&#x30FC;&#x30B8;&#x306E;&#x5206;&#x6790;&#x8C4A;&#x5BCC;&#x306A; NDH-1 ms &#x304C;&#x306A;&#x3044; NDH 1 M &#x307E;&#x305F;&#x306F; NDH 1 s &#x306E;&#x5B58;&#x5728;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x660E;&#x3089;&#x304B;&#x306B; NDH 1S &#x304C; in vivo &#x306E; NDH 1 M &#x3068;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;

&#x5206;&#x96E2;&#x3001;&#x69CB;&#x6210;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3001;&#x71B1;&#x6027;&#x85CD;&#x8272;&#x7D30;&#x83CC;&#x5703; BP 1 &#x304B;&#x3089; NDH 1 &#x932F;&#x4F53;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3002;

&#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544; (NADH quinone oxidoreductase)-1 &#xB2E8;&#xC9C0; &#xB0A8;&#xC870;&#xB958;&#xC5D0; &#xD638;&#xD761; &#xBC0F; &#xC21C;&#xD658; &#xC804;&#xC790; &#xD750;&#xB984; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xD65C;&#xC131; CO2 &#xAE00;&#xADC0; &#xCC98;&#xB7FC; &#xD2B9;&#xC815; &#xD55C; &#xAE30;&#xB2A5;&#xC774; &#xC788;&#xB2E4;. &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544; 1 &#xB2E8;&#xC9C0; &#xBD84;&#xB9AC; &#xD558; &#xACE0; &#xBCF5;&#xC7A1; &#xBCF5;&#xC7A1; &#xD55C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xACF5;&#xBD80; &#xD558;&#xB824;&#xBA74;, Thermosynechococcus elongatus&#xC758; &#xC5EC;&#xB7EC; &#xBCC0;&#xC885; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1&#xC758; &#xB2E4;&#xB978; &#xD558;&#xC704; &#xB2E8;&#xC704;&#xB97C; &#xADF8;&#xC758; &#xD0DC;&#xADF8; (&#xD788;&#xC2A4;&#xD2F0;&#xB518; &#xD0DC;&#xADF8;)&#xC744; &#xCD94;&#xAC00; &#xD558; &#xC5EC; &#xAC74;&#xC124; &#xB418;&#xC5C8;&#xB2E4;. CupA &#xBC0F; Ndhl&#xC5D0; &#xADF8;&#xC758; &#xD0DC;&#xADF8;&#xC640; &#xD568;&#xAED8; 2 &#xAC1C;&#xC758; &#xAE34;&#xC7A5; &#xD06C;&#xB85C;&#xB9C8;&#xD1A0;&#xADF8;&#xB798;&#xD53C; &#xB2E8;&#xACC4; Ni2 + &#xC5F4; &#xD558; &#xC5EC; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544; 1 &#xB2E8;&#xC9C0; &#xBD84;&#xB9AC;&#xB97C; &#xC131;&#xACF5;&#xC801;&#xC73C;&#xB85C; &#xC4F0;&#xC600;&#xB2E4;. &#xBE44; &#xC5D4; (&#xBE14;&#xB8E8;-&#xAE30;&#xBCF8;) / SDS/&#xD398;&#xC774;&#xC9C0; Ni2 + &#xC5F4;&#xC5D0;&#xC11C; eluted &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xBD84;&#xC11D; &#xACF5;&#xAC1C; &#xC874;&#xC7AC; &#xC57D; 450&#xC758; &#xBD84;&#xC790; &#xC9C8;&#xB7C9;&#xC73C;&#xB85C; 3 &#xAC1C; &#xB2E8;&#xC9C0;&#xC758; &#xC218; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1 L, &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1m &#xBC0F; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1S &#xAC01;&#xAC01; MS&#xC5D0; &#xC758;&#xD574; &#xC2DD;&#xBCC4; &#xB41C; 300&#xACFC; 190 kDa &#xC774;&#xC804;&#xC5D0; sp. Synechocystis PCC 6803&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC;. &#xC57D; 490 Kda&#xC758; &#xD070; &#xBCF5;&#xC7A1; &#xD55C;&#xC740; &#xB610;&#xD55C; NdhL &#xADF8;&#xC758; &#xAE34;&#xC7A5; &#xC73C;&#xB85C;&#xBD80;&#xD130; &#xACA9;&#xB9AC;. &#39; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1MS&#39; &#xC9C0;&#xC815;&#xC774; &#xBCF5;&#xC7A1; &#xD55C; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1m &#xBC0F; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1s&#xB85C; &#xAD6C;&#xC131; &#xB418;&#xC5C8;&#xB2E4;. &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1 L &#xBCF5;&#xC7A1; &#xD55C; Ni2 + &#xCE7C;&#xB7FC; &#xD06C;&#xB85C;&#xB9C8;&#xD1A0;&#xADF8;&#xB798;&#xD53C;&#xC5D0; &#xC758;&#xD574; T. elongatus&#xC758; WT (&#xC57C;&#xC0DD;-&#xD0C0;&#xC785;) &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. NdhF1 &#xC18C; &#xB2E8;&#xC704;&#xB9CC;&#xC5D0; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1 L &#xD604;&#xC7AC;&#xB294; &#xC2DC;&#xD000;&#xC2A4;-HHDHHSHH-&#xB0B4;&#xBD80;&#xC801;&#xC73C;&#xB85C;, Ni2 + &#xC5F4;&#xC5D0; &#xB300; &#xD55C; &#xCE5C;&#xADFC;&#xAC10;&#xC744; &#xAC00;&#xC9C8; &#xAC83;&#xC73C;&#xB85C; &#xBCF4;&#xC778;&#xB2E4;. &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1S &#xB610;&#xB294; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1m &#xD558;&#xC9C0; &#xC774;&#xB7F0;&#xC774; &#xC885;&#xB958;&#xC758; &#xD06C;&#xB85C;&#xB9C8;&#xD1A0;&#xADF8;&#xB798;&#xD53C;&#xC5D0; &#xC758;&#xD574; WT &#xC140;&#xC5D0;&#xC11C; &#xBCF5;&#xAD6C; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. Solubilized &#xC138;&#xC81C;&#xC758; &#xB0AE;&#xC740; &#xB18D;&#xB3C4; &#xC758;&#xD574; &#xC138;&#xD3EC; &#xB9C9;&#xC758; BN/SDS/&#xD398;&#xC774;&#xC9C0; &#xBD84;&#xC11D; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1m &#xB098; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1S &#xC544;&#xB2C8;&#xB77C; &#xD48D;&#xBD80;&#xD55C; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544; 1ms&#xC758; &#xC874;&#xC7AC;&#xB97C; &#xAC00;&#xB9AC;&#xD0A8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xBA85;&#xD655; &#xD558; &#xAC8C; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1s&#xC640; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1m vivo&#xC5D0;&#xC11C; &#xC5F0;&#xACB0; &#xB41C; &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;.

&#xC808;&#xC5F0;, &#xC18C; &#xB2E8;&#xC704; &#xAD6C;&#xC131; &#xBC0F; &#xD06C;&#xB85C;&#xC544;&#xD2F0;&#xC544;-1 &#xB2E8;&#xC9C0; Thermosynechococcus elongatus BP-1&#xC5D0;&#xC11C;&#xC5D0;&#xC11C;&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9;.

NDH (&oacute;xidorreductasa de la NADH-quinona)-1 complejos en cianobacterias tienen funciones espec&iacute;ficas en la respiraci&oacute;n y el flujo del electr&oacute;n c&iacute;clica, as&iacute; como en la absorci&oacute;n de CO2 activa. Para aislar el NDH-1 complejos y para estudiar las interacciones complejo complejo, se construyeron varias cepas de Thermosynechococcus elongatus a&ntilde;adiendo un su-tag (etiqueta de histidina) a diversas subunidades de NDH-1. Dos cepas con su etiqueta de CupA y NdhL fueron utilizadas con &eacute;xito para aislar NDH-1 complejos por cromatograf&iacute;a de columna de un solo paso Ni2 +. BN (azul nativo) / SDS/p&aacute;gina An&aacute;lisis de las prote&iacute;nas que eluyen de la columna de Ni2 + revel&oacute; la presencia de tres complejos con masas moleculares de unos 450, 300 y 190 kDa, que fueron identificados por MS que NDH - 1 L, NDH - 1 M y NDH-1S respectivamente, encontrado en Synechocystis SP PCC 6803. Un conjunto m&aacute;s amplio de unos 490 kDa tambi&eacute;n fue aislado de la cepa NdhL-His. Este complejo, se&ntilde;alada &#39;NDH-1MS&#39;, estaba compuesta por NDH - 1 M y NDH-1S. Complejo NDH - 1 L se recuper&oacute; de las c&eacute;lulas (tipo salvaje) WT de T. elongatus por Ni2 + cromatograf&iacute;a de columna. NdhF1 subunidad presente s&oacute;lo en NDH - 1 L tiene una secuencia de - HHDHHSHH-internamente, que parece tener una afinidad para la columna de Ni2 +. NDH-1S o NDH - 1 M no fue recuperado de las c&eacute;lulas WT por cromatograf&iacute;a de este tipo. El an&aacute;lisis BN/SDS/PAGE de membranas solubilizado por una baja concentraci&oacute;n de detergente indic&oacute; la presencia de abundantes NDH-1MS, pero no NDH - 1 M o NDH-1S. Estos resultados demostraron claramente que el NDH-1S se asocia con NDH - 1 M in vivo.

Aislamiento, la composici&oacute;n de la subunidad y la interacci&oacute;n de los complejos de NDH-1 de Thermosynechococcus elongatus BP-1.

The oxygen-evolving complex of eukaryotic photosystem II (PSII) consists of three extrinsic nuclear-encoded subunits, PsbO (33 kDa), PsbP (23 kDa), and PsbQ (17 kDa). Additionally, the 10-kDa PsbR protein has been found in plant PSII and anticipated to play a role in water oxidation, yet the physiological significance of PsbR has remained obscure. Using the Arabidopsis psbR mutant, we showed that the light-saturated rate of oxygen evolution is strongly reduced in the absence of PsbR, particularly in low light-grown plants. Lack of PsbR also induced a reduction in the content of both the PsbP and the PsbQ proteins, and a near depletion of these proteins was observed under steady state low light conditions. This regulation occurred post-transcriptionally and likely involves a proteolytic degradation of the PsbP and PsbQ proteins in the absence of an assembly partner, proposed to be the PsbR protein. Stable assembly of PsbR in the PSII core complex was, in turn, shown to require a chloroplast-encoded intrinsic low molecular mass PSII subunit PsbJ. Our results provided evidence that PsbR is an important link in the PSII core complex for stable assembly of the oxygen-evolving complex protein PsbP, whereas the effects on the assembly of PsbQ are probably indirect. The physiological role of the PsbR, PsbP, and PsbQ proteins is discussed in light of their peculiar expression in response to growth light conditions.

PsbR, a missing link in the assembly of the oxygen-evolving complex of plant photosystem II.

Chloroplast signaling involves mechanisms to relay information from chloroplasts to the nucleus, to change nuclear gene expression in response to environmental cues. Aside from reactive oxygen species (ROS) produced under stress conditions, changes in the reduction/oxidation state of photosynthetic electron transfer components or coupled compounds in the stroma and the accumulation of photosynthesis-derived metabolites are likely origins of chloroplast signals. We attempted to investigate the origin of the signals from chloroplasts in mature Arabidopsis leaves by differentially modulating the redox states of the plastoquinone pool and components on the reducing side of photosystem I, as well as the rate of CO2 fixation, while avoiding the production of ROS by excess light. Differential expression of several nuclear photosynthesis genes, including a set of Calvin cycle enzymes, was recorded. These responded to the stromal redox conditions under prevailing light conditions but were independent of the redox state of the plastoquinone pool. The steady-state CO2 fixation rate was reflected in the orchestration of the expression of a number of genes encoding cytoplasmic proteins, including several glycolysis genes and the trehalose-6-phosphate synthase gene, and also the chloroplast-targeted chaperone DnaJ. Clearly, in mature leaves, the redox state of the compounds on the reducing side of photosystem I is of greater importance in light-dependent modulation of nuclear gene expression than the redox state of the plastoquinone pool, particularly at early signaling phases. It also became apparent that photosynthesis-mediated generation of metabolites or signaling molecules is involved in the relay of information from chloroplast to nucleus.

Chloroplast-mediated regulation of nuclear genes in Arabidopsis thaliana in the absence of light stress.

A proteome analysis of Arabidopsis thaliana thylakoid-associated polysome nascent chain complexes was performed to find novel proteins involved in the biogenesis, maintenance and turnover of thylakoid protein complexes, in particular the PSII (photosystem II) complex, which exhibits a high turnover rate. Four unknown proteins were identified, of which TLP18.3 (thylakoid lumen protein of 18.3 kDa) was selected for further analysis. The Arabidopsis mutants (SALK_109618 and GABI-Kat 459D12) lacking the TLP18.3 protein showed higher susceptibility of PSII to photoinhibition. The increased susceptibility of DeltaTLP18.3 plants to high light probably originates from an inefficient reassembly of PSII monomers into dimers in the grana stacks, as well as from an impaired turnover of the D1 protein in stroma exposed thylakoids. Such dual function of the TLP18.3 protein is in accordance with its even distribution between the grana and stroma thylakoids. Notably, the lack of the TLP18.3 protein does not lead to a severe collapse of the PSII complexes, suggesting a redundancy of proteins assisting these particular repair steps to assure functional PSII. The DeltaTLP18.3 plants showed no clear visual phenotype under standard growth conditions, but when challenged by fluctuating light during growth, the retarded growth of DeltaTLP18.3 plants was evident.

TLP18.3, a novel thylakoid lumen protein regulating photosystem II repair cycle.

The protein complexes of pea (Pisum sativum L.) etioplasts, etio-chloroplasts and chloroplasts were examined using 2D Blue Native/SDS-PAGE. The most prominent protein complexes in etioplasts were the ATPase and the Clp and FtsH protease complexes which probably have a crucial role in the biogenesis of etioplasts and chloroplasts. Also the cytochrome b(6)f (Cyt b(6)f) complex was assembled in the etioplast membrane, as well as Rubisco, at least partially, in the stroma. These complexes are composed of proteins encoded by both the plastid and nuclear genomes, indicating that a functional cross-talk exists between pea etioplasts and the nucleus. In contrast, the proteins and protein complexes that bind chlorophyll, with the PetD subunit and the entire Cyt b(6)f complex as an exception, did not accumulate in etioplasts. Nevertheless, some PSII core components such as PsbE and the luminal oxygen-evolvong complex (OEC) proteins PsbO and PsbP accumulated efficiently in etioplasts. After 6 h de-etiolation, a complete PSII core complex appeared with 40% of the maximal photochemical efficiency, but a fully functional PSII was recorded only after 24 h illumination. Similarly, the core complex of PSI was assembled after 6 h illumination, whereas the PSI-light-harvesting complex I was stably assembled only in chloroplasts illuminated for 24 h. Moreover, a battery of proteins responsible for defense against oxidative stress accumulated particularly in etioplasts, including the stromal and thylakoidal forms of ascorbate peroxidase, glutathione reductase and PsbS.

Expression of protein complexes and individual proteins upon transition of etioplasts to chloroplasts in pea (Pisum sativum).

Exposure of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the plasma membrane. By confocal laser scanning microscopy and subcellular fractionation, it was demonstrated that CaS localizes to the chloroplasts and is enriched in stroma thylakoids. The phosphorylation level of CaS responded strongly to light intensity. The light-dependent thylakoid protein kinase STN8 is required for CaS phosphorylation. The phosphorylation site was mapped to the stroma-exposed Thr380, located in a motif for interaction with 14-3-3 proteins and proteins with forkhead-associated domains, which suggests the involvement of CaS in stress responses and signaling pathways. The knockout Arabidopsis lines revealed a significant role for CaS in plant growth and development.

Light regulation of CaS, a novel phosphoprotein in the thylakoid membrane of Arabidopsis thaliana.

Gel-based analysis of thylakoid membrane protein complexes represents a valuable tool to monitor the dynamics of the photosynthetic machinery. Native-PAGE preserves the components and often also the conformation of the protein complexes, thus enabling the analysis of their subunit composition. Nevertheless, the literature and practical experimentation in the field sometimes raise confusion owing to a great variety of native-PAGE and thylakoid-solubilization systems. In the present paper, we describe optimized methods for separation of higher plant thylakoid membrane protein complexes by native-PAGE addressing particularly: (i) the use of detergent; (ii) the use of solubilization buffer; and (iii) the gel electrophoresis method. Special attention is paid to separation of high-molecular-mass thylakoid membrane super- and mega-complexes from Arabidopsis thaliana leaves. Several novel super- and mega-complexes including PS (photosystem) I, PSII and LHCs (light-harvesting complexes) in various combinations are reported.

Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes.

In nature, plants are challenged by constantly changing light conditions. To reveal the molecular mechanisms behind acclimation to sometimes drastic and frequent changes in light intensity, we grew Arabidopsis thaliana under fluctuating light conditions, in which the low light periods were repeatedly interrupted with high light peaks. Such conditions had only marginal effect on photosystem II but induced damage to photosystem I (PSI), the damage being most severe during the early developmental stages. We showed that PROTON GRADIENT REGULATION5 (PGR5)-dependent regulation of electron transfer and proton motive force is crucial for protection of PSI against photodamage, which occurred particularly during the high light phases of fluctuating light cycles. Contrary to PGR5, the NAD(P)H dehydrogenase complex, which mediates cyclic electron flow around PSI, did not contribute to acclimation of the photosynthetic apparatus, particularly PSI, to rapidly changing light intensities. Likewise, the Arabidopsis pgr5 mutant exhibited a significantly higher mortality rate compared with the wild type under outdoor field conditions. This shows not only that regulation of PSI under natural growth conditions is crucial but also the importance of PGR5 in PSI protection.

PROTON GRADIENT REGULATION5 is essential for proper acclimation of Arabidopsis photosystem I to naturally and artificially fluctuating light conditions.

According to the "state transitions" theory, the light-harvesting complex II (LHCII) phosphorylation in plant chloroplasts is essential to adjust the relative absorption cross section of photosystem II (PSII) and PSI upon changes in light quality. The role of LHCII phosphorylation upon changes in light intensity is less thoroughly investigated, particularly when changes in light intensity are too fast to allow the phosphorylation/dephosphorylation processes to occur. Here, we demonstrate that the Arabidopsis (Arabidopsis thaliana) stn7 (for state transition7) mutant, devoid of the STN7 kinase and LHCII phosphorylation, shows a growth penalty only under fluctuating white light due to a low amount of PSI. Under constant growth light conditions, stn7 acquires chloroplast redox homeostasis by increasing the relative amount of PSI centers. Thus, in plant chloroplasts, the steady-state LHCII phosphorylation plays a major role in preserving PSI upon rapid fluctuations in white light intensity. Such protection of PSI results from LHCII phosphorylation-dependent equal distribution of excitation energy to both PSII and PSI from the shared LHCII antenna and occurs in cooperation with nonphotochemical quenching and the proton gradient regulation5-dependent control of electron flow, which are likewise strictly regulated by white light intensity. LHCII phosphorylation is concluded to function both as a stabilizer (in time scales of seconds to minutes) and a dynamic regulator (in time scales from tens of minutes to hours and days) of redox homeostasis in chloroplasts, subject to modifications by both environmental and metabolic cues. Exceeding the capacity of LHCII phosphorylation/dephosphorylation to balance the distribution of excitation energy between PSII and PSI results in readjustment of photosystem stoichiometry.

Steady-state phosphorylation of light-harvesting complex II proteins preserves photosystem I under fluctuating white light.

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Virpi Paakkarinen

Molecular Plant Biology,
Department of Biochemistry,
Molecular Plant Biology, Department of Biochemistry

University of Turku

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Virpi Paakkarinen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Molecular Plant Biology,Department of Biochemistry,Molecular Plant Biology, Department of Biochemistry

University of Turku

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Molecular Plant Biology,
Department of Biochemistry,
Molecular Plant Biology, Department of Biochemistry