A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane.

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol as larger precursors and transported into the thylakoid lumen via stromal intermediate forms. We have investigated the energetics of protein transport across the thylakoid membrane using import assays that utilize either intact chloroplasts or isolated thylakoids. We have found that the light-driven import of the 23-kDa protein into isolated thylakoids is almost completely inhibited by electron transport inhibitors or by the ionophore nigericin but not by valinomycin. These compounds have similar effects in chloroplast import assays: precursors of both the 33- and 23-kDa proteins are imported and processed to intermediate forms in the stroma, but transport into the thylakoid lumen is blocked when electron transport is inhibited or nigericin is present. These results indicate that the transport of these proteins across the thylakoid membrane requires a protonmotive force and that the dominant component in this respect is the proton gradient and not the electrical potential.     

Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.     

植物叶绿体类囊体膜及膜蛋白研究进展

叶绿体是植物和真核藻类进行光合作用的场所。存在于叶绿体类囊体膜上的蛋白质复合物含有光反应所需的光合色素和电子传递链组分,在光合作用过程中,光化学反应发生在类囊体膜上。因此,类囊体膜是光能向化学能转化的主要场所,因而也一直是光合作用研究的热点。叶绿体类囊体膜的深入研究可以促进光合作用的分子机理研究。该文就叶绿体类囊体膜的三维构象及类囊体膜蛋白的组成和功能研究进行了综述。     

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.     

HCF164 receives reducing equivalents from stromal thioredoxin across the thylakoid membrane and mediates reduction of target proteins in the thylakoid lumen

HCF164 is a membrane-anchored thioredoxin-like protein known to be indispensable for assembly of cytochrome b6 f in the thylakoid membranes. In this study, we report the finding that chloroplast stroma m-type thioredoxin is the source of reducing equivalents for reduction of HCF164 in the thylakoid lumen, providing strong evidence that higher plant chloroplasts possess a trans-membrane reducing equivalent transfer system similar to that found in bacteria. To probe the function of HCF164 in the lumen, a screen to identify the reducing equivalent acceptor proteins of HCF164 was carried out by using a resin-immobilized HCF164 single cysteine mutant, leading to the isolation of putative target thylakoid proteins. Among the newly identified target proteins, the reduction of the PSI-N subunit of photosystem I by HCF164 was confirmed both in vitro and in isolated thylakoids. Two components of the cytochrome b6 f complex, the cytochrome f and Rieske FeS proteins, were also identified as novel potential target proteins. The data presented here suggest that HCF164 serves as an important transducer of reducing equivalents to proteins in the thylakoid lumen.     

Isolation of lumenal proteins from spinach thylakoid membranes by triton X-114 phase partitioning

The proteins present in the thylakoid lumen of higher plant chloroplasts have not been rigorously examined. In this communication we present a simple and rapid procedure for the isolation of the soluble proteins and extrinsic membrane proteins present in the thylakoid lumen from spinach. Our procedure involves extensive washing of the thylakoid membranes followed by Triton X-114 phase partitioning. When analyzed by one-dimensional polyacrylamide gel electrophoresis (PAGE), we obtain results which are very similar to those obtained by Kieselbach et al. using more classical methods [T. Kieselbach, A. Hagman, B. Andersson, W.P. Schroder, J. Biol. Chem. 273 (1998) 6710-6716]. About 25 major proteins are observed upon Coomassie blue staining. Upon two-dimensional isoelectric focusing-sodium dodecyl sulfate-PAGE and either Coomassie blue or silver staining, however, numerous other protein components are resolved. Our findings indicate that the total number of proteins (soluble and extrinsic membrane) present in the lumen may exceed 150.     

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.     

Protein translocation into and across the bacterial plasma membrane and the plant thylakoid membrane

Over the past decade, some familiar themes have emerged on how proteins are inserted into or translocated across the plant chloroplast thylakoid membrane and bacterial inner membranes. In the SecA and signal recognition particle (SRP) pathways, nucleotides and soluble factors are used to translocate proteins across the membrane bilayer in the unfolded state. However, the delta pH-dependent pathway in thylakoids uses a radically different mechanism: transport of proteins across the membrane is driven by the transmembrane pH gradient, and neither stromal factors nor nucleotide triphosphates are needed. In addition, this pathway, which requires the membrane-bound protein Hcf106, appears to translocate proteins in a tightly folded form. Recently, a similar pathway has been shown to operate in eubacteria, and several of its components have been identified.     

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

Transit peptide cleavage sites of integral thylakoid membrane proteins

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria.     

Synthesis, assembly and degradation of thylakoid membrane proteins

The thylakoid membrane of chloroplasts contains four major protein complexes, involved in the photosynthetic electron transfer chain and in ATP synthesis. These complexes are built from a large number of polypeptide subunits encoded either in the nuclear or in the plastid genome. In this review, we are considering the mechanism that couples assembly (association of the polypeptides with each other and with their cofactors) with the upstream and downstream steps of the biogenetic pathway, translation and proteolytic degradation. We present the contrasting images of assembly that have emerged from a variety of approaches (studies of photosynthesis mutants, developmental studies and direct biochemical analysis of the kinetics of assembly). We develop the concept of control by epistasy of synthesis, through which the translation of certain subunits is controlled by the state of assembly of the complex and address the question of its mechanisms. We describe additional factors that assist in the integration and assembly of thylakoid membrane proteins.     

One signal is enough: Stepwise transport of two distinct passenger proteins by the Tat pathway across the thylakoid membrane

The twin-arginine translocation (Tat) pathway, one of four protein transport pathways operating at the thylakoid membrane of chloroplasts, shows remarkable substrate flexibility. Here, we have analyzed the thylakoid transport of chimeric tandem substrates that are composed of two different passenger proteins fused to a single Tat transport signal. The chimera 23/23-EGFP in which the reporter protein EGFP is connected to the C-terminus of the OEC23 precursor shows that a single Tat transport signal is sufficient to mediate transport of two distinct passenger proteins in a row. Replacing the transit peptide of OEC23 in 23/23-EGFP by its homolog from OEC16 yields the chimera 16/23-EGFP, which can likewise be fully translocated by the Tat pathway across the thylakoid membrane. However, transport of 16/23-EGFP is retarded at specific steps in the transport process leading to the temporary and consecutive accumulation of three translocation intermediates with distinct membrane topology. They are associated with two oligomeric membrane complexes presumably representing TatBC-receptor complexes. The composition of the translocation intermediates as determined by immunoprecipitation experiments suggests that the two passenger proteins are translocated in a stepwise manner across the membrane.     

Temporary suppression of the flash-induced electrical potential across the thylakoid membrane upon energization

Energization of the chloroplast thylakoid membrane causes a temporary decrease in the amplitude of the flash-induced transmembrane electrical potential as monitored by the micro-electrode technique and by the electrochromic absorbance band shift at 518 nm in chloroplasts of Peperomia metallica. This energization-dependent decrease of the flash-induced potential has a relaxation time of recovery in the dark of about 23±4 s. The phenomenon can neither be explained by a decrease of the intrinsic efficiency of photosystem I and II (PSI and PSII) nor by a partial closure of reaction centers of PSI and PSII. This leads us to propose that the energization-dependent decrease of the amplitude of the flash-induced electrical potential is caused by either the formation of a fraction of PSI and/or PSII reaction centers with fast charge recombination or by an increase of the membrane capacitance. The dark recovery after energization of the amplitude of the transmembrane electrical potential and that of non-photochemical fluorescence quenching were found to be comparable, which suggests a common cause for both phenomena.     

ATP-dependent import of a lumenal protein by isolated thylakoid vesicles

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.     

Targeting signals in peroxisomal membrane proteins

Peroxisomal membrane proteins (PMPs) are encoded by the nuclear genome and translated on cytoplasmic ribosomes. Newly synthesized PMPs can be targeted directly from the cytoplasm to peroxisomes or travel to peroxisomes via the endoplasmic reticulum (ER). The mechanisms responsible for the targeting of these proteins to the peroxisomal membrane are still rather poorly understood. However, it is clear that the trafficking of PMPs to peroxisomes depends on the presence of cis-acting targeting signals, called mPTSs. These mPTSs show great variability both in the identity and number of requisite residues. An emerging view is that mPTSs consist of at least two functionally distinct domains: a targeting element, which directs the newly synthesized PMP from the cytoplasm to its target membrane, and a membrane-anchoring sequence, which is required for the permanent insertion of the protein into the peroxisomal membrane. In this review, we summarize our knowledge of the mPTSs currently identified.     

Multiple pathways for protein transport into or across the thylakoid membrane.

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.