PPIase activities and interaction partners of FK506-binding proteins in the wheat thylakoid

FK506-binding proteins (FKBPs) and cyclophilins, collectively called immunophilins, conserve peptidyl-prolyl cis/trans isomerase (PPIase) active sites, although many lack PPIase activity. The chloroplast thylakoid contains a large proportion of the plant immunophilin family, but their functions within this compartment are unclear. Some lumenal immunophilins are important for assembly of photosynthetic complexes, implicating them in the maintenance and turnover of the photosynthetic apparatus during acclimation processes. In this investigation into the functions of three FKBPs localized to the thylakoid of Triticum aestivum (wheat), we present the first evidence of PPIase activity in the thylakoid of a cereal plant, and also show that PPIase activity is not conserved in all lumenal FKBPs. Using yeast two-hybrid analysis we found that the PPIase-active FKBP13 interacts with the globular domain of the wheat Rieske protein, with potential impact on photosynthetic electron transfer. Specific interaction partners for PPIase-deficient FKBP16-1 and FKBP16-3 link these isoforms to photosystem assembly.     

Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen

Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membrane-associated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts.     

Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction

Experimental proteome analysis was combined with a genome-wide prediction screen to characterize the protein content of the thylakoid lumen of Arabidopsis chloroplasts. Soluble thylakoid proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry. The identities of 81 proteins were established, and N termini were sequenced to validate localization prediction. Gene annotation of the identified proteins was corrected by experimental data, and an interesting case of alternative splicing was discovered. Expression of a surprising number of paralogs was detected. Expression of five isomerases of different classes suggests strong (un)folding activity in the thylakoid lumen. These isomerases possibly are connected to a network of peripheral and lumenal proteins involved in antioxidative response, including peroxiredoxins, m-type thioredoxins, and a lumenal ascorbate peroxidase. Characteristics of the experimentally identified lumenal proteins and their orthologs were used for a genome-wide prediction of the lumenal proteome. Lumenal proteins with a typical twin-arginine translocation motif were predicted with good accuracy and sensitivity and included additional isomerases and proteases. Thus, prime functions of the lumenal proteome include assistance in the folding and proteolysis of thylakoid proteins as well as protection against oxidative stress. Many of the predicted lumenal proteins must be present at concentrations at least 10,000-fold lower than proteins of the photosynthetic apparatus.     

Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.     

Lumen Thiol Oxidoreductase1, a disulfide bond-forming catalyst, is required for the assembly of photosystem II in Arabidopsis

Here, we identify Arabidopsis thaliana Lumen Thiol Oxidoreductase1 (LTO1) as a disulfide bond-forming enzyme in the thylakoid lumen. Using topological reporters in bacteria, we deduced a lumenal location for the redox active domains of the protein. LTO1 can partially substitute for the proteins catalyzing disulfide bond formation in the bacterial periplasm, which is topologically equivalent to the plastid lumen. An insertional mutation within the LTO1 promoter is associated with a severe photoautotrophic growth defect. Measurements of the photosynthetic activity indicate that the lto1 mutant displays a limitation in the electron flow from photosystem II (PSII). In accordance with these measurements, we noted a severe depletion of the structural subunits of PSII but no change in the accumulation of the cytochrome b(6)f complex or photosystem I. In a yeast two-hybrid assay, the thioredoxin-like domain of LTO1 interacts with PsbO, a lumenal PSII subunit known to be disulfide bonded, and a recombinant form of the molecule can introduce a disulfide bond in PsbO in vitro. The documentation of a sulfhydryl-oxidizing activity in the thylakoid lumen further underscores the importance of catalyzed thiol-disulfide chemistry for the biogenesis of the thylakoid compartment.     

Differential effects of ambient or diminished CO2 and O2 levels on thylakoid membrane structure in light‐stressed plants

Over‐reduction of the photosynthetic electron transport chain may severely damage the photosynthetic apparatus as well as other constituents of the chloroplast and the cell. Here, we exposed Arabidopsis leaves to saturating light either under normal atmospheric conditions or under CO₂‐ and O₂‐limiting conditions, which greatly increase excitation and electron pressures by draining terminal electron acceptors. The two treatments were found to have very different, often opposing, effects on the structure of the thylakoid membranes, including the width of the granal lumenal compartment. Modulation of the latter is proposed to be related to movements of ions across the thylakoid membrane, which alter the relative osmolarity of the lumen and stroma and affect the partitioning of the proton motive force into its electrical and osmotic components. The resulting changes in thylakoid organization and lumenal width should facilitate the repair of photodamaged photosystem II complexes in response to light stress under ambient conditions, but are expected to inhibit the repair cycle when the light stress occurs concurrently with CO₂ and O₂ depletion. Under the latter conditions, the changes in thylakoid structure are predicted to complement other processes that restrict the flow of electrons into the high‐potential chain, thus moderating the production of deleterious reactive oxygen species at photosystem I.     

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N-terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C-terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.     

Chloroplast proteases: possible regulators of gene expression?

A wide range of proteolytic processes in the chloroplast are well recognized. These include processing of precursor proteins, removal of oxidatively damaged proteins, degradation of proteins missing their prosthetic groups or their partner subunit in a protein complex, and adjustment of the quantity of certain chloroplast proteins in response to changing environmental conditions. To date, several chloroplast proteases have been identified and cloned. The chloroplast processing enzyme is responsible for removing the transit peptides of newly imported proteins. The thylakoid processing peptidase removes the thylakoid-transfer domain from proteins translocated into the thylakoid lumen. Within the lumen, Tsp removes the carboxy-terminal tail of the precursor of the PSII D1 protein. In contrast to these processing peptidases which perform a single endo-proteolytic cut, processive proteases that can completely degrade substrate proteins also exist in chloroplasts. The serine ATP-dependent Clp protease, composed of the proteolytic subunit ClpP and the regulatory subunit ClpC, is located in the stroma, and is involved in the degradation of abnormal soluble and membrane-bound proteins. The ATP-dependent metalloprotease FtsH is bound to the thylakoid membrane, facing the stroma. It degrades unassembled proteins and is involved in the degradation of the D1 protein of PSII following photoinhibition. DegP is a serine protease bound to the lumenal side of the thylakoid membrane that might be involved in the chloroplast response to heat. All these peptidases and proteases are homologues of known bacterial enzymes. Since ATP-dependent bacterial proteases and their mitochondrial homologues are also involved in the regulation of gene expression, via their determining the levels of key regulatory proteins, chloroplast proteases are expected to play a similar role.     

Mechanisms of protein import into thylakoids of chloroplasts

The thylakoid membrane of chloroplasts contains the major photosynthetic complexes, which consist of several either nuclear or chloroplast encoded subunits. The biogenesis of these thylakoid membrane complexes requires coordinated transport and subsequent assembly of the subunits into functional complexes. Nuclear-encoded thylakoid proteins are first imported into the chloroplast and then directed to the thylakoid using different sorting mechanisms. The cpSec pathway and the cpTat pathway are mainly involved in the transport of lumenal proteins, whereas the spontaneous pathway and the cpSRP pathway are used for the insertion of integral membrane proteins into the thylakoid membrane. While cpSec-, cpTat- and cpSRP-mediated targeting can be classified as 'assisted' mechanisms involving numerous components, 'unassisted' spontaneous insertion does not require additional targeting factors. However, even the assisted pathways differ fundamentally with respect to stromal targeting factors, the composition of the translocase and energy requirements.     

Proteome map of the chloroplast lumen of Arabidopsis thaliana.

The thylakoid membrane of the chloroplast is the center of oxygenic photosynthesis. To better understand the function of the luminal compartment within the thylakoid network, we have carried out a systematic characterization of the luminal thylakoid proteins from the model organism Arabidopsis thaliana. Our data show that the thylakoid lumen has its own specific proteome, of which 36 proteins were identified. Besides a large group of peptidyl-prolyl cis-trans isomerases and proteases, a family of novel PsbP domain proteins was found. An analysis of the luminal signal peptides showed that 19 of 36 luminal precursors were marked by a twin-arginine motif for import via the Tat pathway. To compare the model organism Arabidopsis with another typical higher plant, we investigated the proteome from the thylakoid lumen of spinach and found that the luminal proteins from both plants corresponded well. As a complement to our experimental investigation, we made a theoretical prediction of the luminal proteins from the whole Arabidopsis genome and estimated that the thylakoid lumen of the chloroplast contains approximately 80 proteins.     

Role of a novel photosystem II-associated carbonic anhydrase in photosynthetic carbon assimilation in Chlamydomonas reinhardtii

Intracellular carbonic anhydrases (CA) in aquatic photosynthetic organisms are involved in the CO2-concentrating mechanism (CCM), which helps to overcome CO2 limitation in the environment. In the green alga Chlamydomonas reinhardtii, this CCM is initiated and maintained by the pH gradient created across the chloroplast thylakoid membranes by photosystem (PS) II-mediated electron transport. We show here that photosynthesis is stimulated by a novel, intracellular alpha-CA bound to the chloroplast thylakoids. It is associated with PSII on the lumenal side of the thylakoid membranes. We demonstrate that PSII in association with this lumenal CA operates to provide an ample flux of CO2 for carboxylation.     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

Mobility of photosynthetic proteins

The mobility of photosynthetic proteins represents an important factor that affects light-energy conversion in photosynthesis. The specific feature of photosynthetic proteins mobility can be currently measured in vivo using advanced microscopic methods, such as fluorescence recovery after photobleaching which allows the direct observation of photosynthetic proteins mobility on a single cell level. The heterogeneous organization of thylakoid membrane proteins results in heterogeneity in protein mobility. The thylakoid membrane contains both, protein-crowded compartments with immobile proteins and fluid areas (less crowded by proteins), allowing restricted diffusion of proteins. This heterogeneity represents an optimal balance as protein crowding is necessary for efficient light-energy conversion, and protein mobility plays an important role in the regulation of photosynthesis. The mobility is required for an optimal light-harvesting process (e.g., during state transitions), and also for transport of proteins during their synthesis or repair. Protein crowding is then a key limiting factor of thylakoid membrane protein mobility; the less thylakoid membranes are crowded by proteins, the higher protein mobility is observed. Mobility of photosynthetic proteins outside the thylakoid membrane (lumen and stroma/cytosol) is less understood. Cyanobacterial phycobilisomes attached to the stromal side of the thylakoid can move relatively fast. Therefore, it seems that stroma with their active enzymes of the Calvin–Benson cycle, are a more fluid compartment in comparison to the rather rigid thylakoid lumen. In conclusion, photosynthetic protein diffusion is generally slower in comparison to similarly sized proteins from other eukaryotic membranes or organelles. Mobility of photosynthetic proteins resembles restricted protein diffusion in bacteria, and has been rationalized by high protein crowding similar to that of thylakoids.     

The chloroplast Tat pathway utilizes the transmembrane electric potential as an energy source

The thylakoid membrane, located inside the chloroplast, requires proteins transported across it for plastid biogenesis and functional photosynthetic electron transport. The chloroplast Tat translocator found on thylakoids transports proteins from the plastid stroma to the thylakoid lumen. Previous studies have shown that the chloroplast Tat pathway is independent of NTP hydrolysis as an energy source and instead depends on the thylakoid transmembrane proton gradient to power protein translocation. Because of its localization on the same membrane as the proton motive force-dependent F(0)F(1) ATPase, we believed that the chloroplast Tat pathway also made use of the thylakoid electric potential for transporting substrates. By adjusting the rate of photosynthetic proton pumping and by utilizing ionophores, we show that the chloroplast Tat pathway can also utilize the transmembrane electric potential for protein transport. Our findings indicate that the chloroplast Tat pathway is likely dependent on the total protonmotive force (PMF) as an energy source. As a protonmotive-dependent device, certain predictions can be made about structural features expected to be found in the Tat translocon, specifically, the presence of a proton well, a device in the membrane that converts electrical potential into chemical potential.     

Multiple fates of non‐mature lumenal proteins in thylakoids

Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N–terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid‐transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling‐lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1‐null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid‐transfer signal removal on protein targeting and accumulation by examining the localization of non‐mature lumenal proteins in the Arabidopsis plsp1‐null mutant and performing a protein import assay using pea chloroplasts. In plsp1‐null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non‐mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid‐transfer signal removal did not disrupt cpSEC‐ and cpTAT‐dependent translocation, but prevented release of proteins from the membrane. Interestingly, non‐mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440‐kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1‐null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.     

Immobility of phycobilins in the thylakoid lumen of a cryptophyte suggests that protein diffusion in the lumen is very restricted

The thylakoid lumen is an important photosynthetic compartment which is the site of key steps in photosynthetic electron transport. The fluidity of the lumen could be a major constraint on photosynthetic electron transport rates. We used Fluorescence Recovery After Photobleaching in cells of the cryptophyte alga Rhodomonas salina to probe the diffusion of phycoerythrin in the lumen and chlorophyll complexes in the thylakoid membrane. In neither case was there any detectable diffusion over a timescale of several minutes. This indicates very restricted phycoerythrin mobility. This may be a general feature of protein diffusion in the thylakoid lumen.     

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen.

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.     

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light‐dependent regulation of stromal enzymes by thioredoxin (Trx)‐catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx‐mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol‐dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx‐linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de‐epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox‐controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.     

Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis

The Arabidopsis genome contains 14 genes encoding the serine protease DegP. Products of four of these genes are located in the chloroplast: three in the thylakoid lumen and one on the stromal side of the membrane. We expressed the gene encoding DegP1 as a His-tagged fusion protein in Escherichia coli, purified the protein by affinity chromatography, and characterized it biochemically. Size-exclusion chromatography suggested that DegP1 eluted from the column as a mixture of monomers and hexamers. Proteolytic activity was characterized using beta-casein as a model substrate. DegP1 demonstrated concentration-dependent activity, a pH optimum of 6.0 and increasing activity at elevated temperatures. DegP1 was capable of degrading two lumenal proteins, plastocyanin and OE33, suggesting a role as a general-purpose protease in the thylakoid lumen. The results of this work are discussed in the context of the recent elucidation of the structure of the E. coli homolog and the possible physiological role of the protease in the chloroplast lumen.