Recent developments in chloroplast protein transport

Most proteins located in chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and transported into the organelle. The study of protein uptake by chloroplasts has greatly expanded over the past few years. The increased activity in this field is due, in part, to the application of recombinant DNA methodology to the analysis of protein translocation. Added interest has also been gained by the realization that the transport mechanisms that mediate protein uptake by chloroplasts, mitochondria and the endoplasmic reticulum display certain characteristics in common. These include amino terminal sequences that target proteins to particular organelles, a transport process that is mechanistically independent from the events of translation, and an ATP-requiring transport step that is thought to involve partial unfolding of the protein to be translocated. In this review we examine recent studies on the binding of precursors to the chloroplast surface, the energy-dependent uptake of proteins into the stroma, and the targeting of proteins to the thylakoid lumen. These aspects of protein transport into chloroplasts are discussed in the context of recent studies on protein uptake by mitochondria.Abbrevlations CAT chloramphenicol acetyl transferase - CCCP carbonylcyanide m-chlorophenylhydrazone - DHFR dihydrofolate reductase - EPSP 5-enol-pyruvylshikimate-3-phosphate - ER endoplasmic reticulum - LHCP light harvesting chlorophyll a/b apoprotein - NPT neomycin phosphotransferase - oATP adenosine-2,3-dialdehyde-5-triphosphate - P-inorganic phosphate Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - SRP signal recognition particle     

腺苷-磷酸激活的蛋白激酶在脂类营养代谢中的研究及应用

腺苷-磷酸激活的蛋白激酶（AMP-activated protein kinase,AMPK）是公认的重要能量感受酶。其作用与多个代谢途径有关,尤其在脂类营养代谢过程中发挥着关键的调控作用。AMPK对脂质代谢的调控通过多个信号通路进行,涉及到骨骼肌、肝脏、乳腺等多个组织。对AMPK调控脂类营养代谢机理的研究为2型糖尿病、脂肪肝、肥胖症、癌症等多种疾病的治疗提供了靶点,但AMPK在奶牛乳腺组织的研究较少,其在提高奶牛生产性能方面潜能巨大。     

Reconstitution of energy transfer and electron transfer between solubilised pigment-protein complexes from thylakoid membranes. The role of acyl lipids

Solubilisation of thylakoid membranes from young leaves of Pisum sativum in the presence of Triton X-100 resulted in an almost complete loss of quenching of light-harvesting chlorophyll-protein (LHCP) fluorescence, as measured at 77°K. There were concomitant changes in the kinetics of light-saturation curves of electron transport from 2,6-dichlorophenolindophenol/ascorbate to methyl viologen. These effects were accompenied by a physical dissociation of LHCP polypeptides from photosystem I (PSI) and photosystem II (PSII) polypeptides, as determined by polyacrylamide gel-electrophoresis. Detergent-dialysis in the presence of exogenous purified galactolipids, about 80% of which were linoleoyl molecular species, only partially reversed these effects. However, detergent-dialysis using the phospholipids, phosphatidylglycerol and phosphatidylcholine, resulted in the substantial restoration of 77°K fluorescence quenching and the restoration of both emission spectra and electron transport kinetics of both Photosystems I and II that were typical of native membranes.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DGD digalactosyldiacylglycerol - LHCP light-harvesting chlorophyll-protein - MGD monogalactosyldiacylglycerol - PCi phosphatidylcholine — Sigma grade NS - PCii -oleoyl, -palmitoyl phosphalidylcholine - PG phosphatidylglycerol - PSI photosystem I - PSII photosystem II     

The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane,is involved in thylakoid biogenesis

Thylakoid biogenesis is a crucial step for plant development involving the combined action of many cellular actors. CPSAR1 is shown here to be required for the normal organization of mature thylakoid stacks, and ultimately for embryo development. CPSAR1 is a chloroplast protein that has a dual localization in the stroma and the inner envelope membrane, according to microscopy studies and subfractionation analysis. CPSAR1 is close to the Obg nucleotide binding protein subfamily and displays GTPase activity, as demonstrated by in vitro assays. Disruption of the CPSAR1 gene via T‐DNA insertion results in the arrest of embryo development. In addition, transmission electron microscopy analysis indicates that mutant embryos are unable to develop thylakoid membranes, and remain white. Unstacked membrane structures resembling single lamellae accumulate in the stroma, and do not assemble into mature thylakoid stacks. CPSAR1 RNA interference induces partially developed thylakoids leading to pale‐green embryos. Altogether, the presented data demonstrate that CPSAR1 is a protein essential for the formation of normal thylakoid membranes, and suggest a possible involvement in the initiation of vesicles from the inner envelope membrane for the transfer of lipids to the thylakoids.     

Intracellular transport of lipids

Summary Translocation of lipids inside mammalian cells is considered to be facilitated by a number of low-molecular weight lipid binding proteins. An overview of these proteins is given, with particular reference to the heart. Three distinct phospholipid transfer proteins specifically stimulate the net transfer of individual phospholipid classes between membrane structures. In rat cardiac muscle their content is 15–140 pmol/g ww. Fatty acid-binding proteins (FABP) are abundantly present in tissues actively involved in the uptake or utilization of long-chain fatty acids, such as intestine, liver and heart. The four distinct FABP types now identified show a complex tissue distribution with some tissues containing more than one type. Heart (H-) FABP comprises about 5% of the cytosolic protein mass; its content in rat heart is 100 nmol/g ww. Immunochemical evidence has been obtained for the presence of H-FABP in several other tissues, including red skeletal muscle, mammary gland and kidney. Beside long-chain fatty acids FABP binds with similar affinity also fatty acyl-CoA and acyl-L-carnitines. In heart the latter compound may be the primary ligand, since normoxic acyl-L-carnitine levels are several fold higher than those of fatty acids. In addition, H-FABP was found to modulate cardiac energy production by controlling the transfer of acyl-L-carnitine to the mitochondrial -oxidative system. H-FABP may also protect the heart against the toxic effects of high intracellular levels of fatty acid intermediates that arise during ischemia.     

The role of thylakoid lipids in the photodamage of photosynthetic activity

The effect of excess light at 10 or 30°C under aerobic or low O₂ condition on peroxidation of thylakoid lipids and primary photochemistry of photoinsynthesis was studied in wheat ( Triticum aestivum L. cv. HD 2329). Photoinhibitory damage to photosythesis was directly proportional to the peroxidation of thylakoid lipids. Photoinhibitory treatment given under low O₂ conditions resulted in significantly less peroxidation of the primary photochemistry of photosythesis measured using chlorophyll fluorescence and photosythetic electron trasport. Short term recovery of F_v/F_m ratio was fast while thylakoid lipids did not show much recovry. However, recovery (of F_v/F_m ratio and thylakoid lipids) was almost complete within 12 h after photoinhibition treatment. A possible relationship between peroxidation of thylakoid lipids and photodamage to photosynthesis is discussed.     

The role of lipids in cancer progression and metastasis

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Regulatory factors for the assembly of thylakoid membrane protein complexes

Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.     

Anionic lipids are required for chloroplast structure and function in Arabidopsis

Photosynthetic membranes of plants primarily contain non-phosphorous glycolipids. The exception is phosphatidylglycerol (PG), which is an acidic/anionic phospholipid. A second major anionic lipid in chloroplasts is the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). It is hypothesized that under severe phosphate limitation, SQDG substitutes for PG, ensuring a constant proportion of anionic lipids even under adverse conditions. A newly constructed SQDG and PG-deficient double mutant supports this hypothesis. This mutant, sqd2 pgp1-1, carries a T-DNA insertion in the structural gene for SQDG synthase (SQD2) and a point mutation in the structural gene for phosphatidylglycerolphosphate synthase (PGP1). In the sqd2 pgp1-1 double mutant, the fraction of total anionic lipids is reduced by approximately one-third, resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant is severely compromised, and its photosynthetic capacity is impaired. In particular, photosynthetic electron transfer at the level of photosystem II (PSII) is affected. Besides these physiological changes, the mutant shows altered leaf structure, a reduced number of mesophyll cells, and ultrastructural changes of the chloroplasts. All observations on the sqd2 pgp1-1 mutant lead to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development.     

The role of chloroplast-encoded protein biosynthesis on the rate of D1 protein degradation in Dunaliella salina

Mechanistic aspects of the Photosystem II (PS II) damage and repair cycle in Dunaliella salina were investigated. The work addressed the role of chloroplast-encoded protein biosynthesis on the rate of the D1 protein (chloroplast psbA gene product) degradation, following photoinhibition of PS II under in vivo conditions. Cells were grown under different light-intensities and the rate of D1 photodamage and degradation was measured via pulse-chase measurements with (³⁵S)sulfate. It is shown that no detectable difference exists in the rate of D1 degradation in D. salina, measured in the presence or absence of lincomycin, a chloroplast protein biosynthesis inhibitor. The results suggest that de novo D1 biosynthesis does not play a role in the regulation of D1 degradation. In low-light (100 mol photons m^–2 s^–1) grown cells, the rate of photodamage to D1 did not exceed the rate of its degradation and replacement. In high-light (2200 mol photons m^–1 s^–1) grown cells, the rate of D1 photodamage was faster than the rate of its degradation, resulting in a significant accumulation of photoinactivated PS II centers in the chloroplast thylakoids (chronic photoinhibition). The latter was coincident with the appearance of a 160 kD complex that contained photodamaged D1. Electron micrographs of D. salina thylakoids revealed extensive grana stacks in the thylakoid membrane of low-light grown cells. Only rudimentary appressions consisting of simple membrane pairings were found in the high-light grown cells. The results are discussed in terms of the regulation of D1 degradation in chloroplasts under in vivo conditions.Abbreviations Chl chlorophyll - D1 the 32 kD reaction center protein of PS II, encoded by the chloroplast psbA gene - D2 the 34 kD reaction center protein of PS II, encoded by the chloroplast psbD gene - HL high light - LL low light - Linc lincomycin     

Protein transport towards the thylakoid lumen: post-translational translocation in tandem

Many proteins found in the chloroplast are synthesized in the cytoplasm as precursor molecules containing transit peptides. Proteins targeted to the stroma must pass through the two envelope membranes to reach their destination. Proteins located in the chloroplast lumen also have to be transferred across the thylakoid membrane. That is, lumen proteins must cross three biological membranes in order to reach their final location. Recent evidence shows that the routing of plastocyanin towards the lumen involves two post-translational transport processes mediated by two different regions of the transit peptide and two different processing proteases. It is postulated that the genetic information for the plastocyanin precursor, which already contained a signal peptide, was transferred from the endosymbiont to the nucleus. Then a chloroplast-specific targeting-peptide was added.     

Arabidopsis chloroplast lipid transport protein TGD2 disrupts membranes and is part of a large complex

In most plants the assembly of the photosynthetic thylakoid membrane requires lipid precursors synthesized at the endoplasmic reticulum (ER). Thus, the transport of lipids from the ER to the chloroplast is essential for biogenesis of the thylakoids. TGD2 is one of four proteins in Arabidopsis required for lipid import into the chloroplast, and was found to bind phosphatidic acid in vitro. However, the significance of phosphatidic acid binding for the function of TGD2 in vivo and TGD2 interaction with membranes remained unclear. Developing three functional assays probing how TGD2 affects lipid bilayers in vitro, we show that it perturbs membranes to the point of fusion, causes liposome leakage and redistributes lipids in the bilayer. By identifying and characterizing five new mutant alleles, we demonstrate that these functions are impaired in specific mutants with lipid phenotypes in vivo. At the structural level, we show that TGD2 is part of a protein complex larger than 500 kDa, the formation of which is disrupted in two mutant alleles, indicative of the biological relevance of this TGD2-containing complex. Based on the data presented, we propose that TGD2, as part of a larger complex, forms a lipid transport conduit between the inner and outer chloroplast envelope membranes, with its N terminus anchored in the inner membrane and its C terminus binding phosphatidic acid in the outer membrane.     

Bacterial protein toxins and lipids: role in toxin targeting and activity

All bacterial toxins, which globally are hydrophilic proteins, interact first with their target cells by recognizing a surface receptor, which is either a lipid or a lipid derivative, or another compound but in a lipid environment. Intracellular active toxins follow various trafficking pathways, the sorting of which is greatly dependent on the nature of the receptor, notably lipidic receptor or receptor embedded into a distinct environment such as lipid microdomains. Numerous other toxins act locally on cell membrane. Indeed, phospholipase activity is a common mechanism shared by several membrane-damaging toxins. In addition, many toxins active intracellularly or on cell membrane modulate host cell phospholipid pathways. Unusually, a few bacterial toxins require a lipid post-translational modification to be active. Thereby, lipids are obligate partners of bacterial toxins.     

The phase behavior of lipids in photosynthetic membranes

The phase behavior of the main classes of polar lipids found in the photosynthetic membranes of higher plants and algae is reviewed and compared to that of binary lipid mixtures and total lipid extracts of such membranes. Particular interest is paid to the way in which factors such as temperature and acyl chain saturation influence the phase behavior of these lipids and the implications this has in terms of the ability of photosynthetic membranes to resist environmental stress.     

Identification and characterization of a stable intermediate in photosystem I assembly in tobacco

Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co‐factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF‐deficient mutant plants, and we show that re‐initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.     

Molecular dynamics simulation of the membrane binding and disruption mechanisms by antimicrobial scorpion venom-derived peptides

Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from venom of the African scorpion Pandinus imperator with high antimicrobial activity against Gram-positive bacteria and less active against Gram-negative bacteria, however it has demonstrated strong hemolytic activity against sheep red blood cells. In the chemically synthesized Pin2GVG analog, the GVG motif grants it low hemolytic activity while keeping its antimicrobial activity. In this work, we performed 12 μs all-atom molecular dynamics simulation of the antimicrobial peptides (AMPs) Pin2 and Pin2GVG to explore their adsorption mechanism and the role of their constituent amino acid residues when interacting with pure POPC and pure POPG membrane bilayers. Starting from an α-helical conformation, both AMPs are attracted at different rates to the POPC and POPG bilayer surfaces due to the electrostatic interaction between the positively charged amino acid residues and the charged moieties of the membranes. Since POPG is an anionic membrane, the PAMs adhesion is stronger to the POPG membrane than to the POPC membrane and they are stabilized more rapidly. This study reveals that, before the insertion begins, Pin2 and Pin2GVG remained partially folded in the POPC surface during the first 300 and 600 ns, respectively, while they are mostly unfolded in the POPG surface during most of the simulation time. The unfolded structures provide for a large number of intermolecular hydrogen bonds and stronger electrostatic interactions with the POPG surface. The results show that the aromatic residues at the N-terminus of Pin2 initiate the insertion process in both POPC and POPG bilayers. As for Pin2GVG in POPC the C-terminus residues seem to initiate the insertion process while in POPG this process seems to be slowed down due to a strong electrostatic attraction. The membrane conformational effects upon PAMs binding are measured in terms of the area per lipid and the contact surface area. Several replicas of the systems lead to the same observations.     

Localization and integration of thylakoid protein translocase subunit cpTatC

Thylakoid membranes have a unique complement of proteins, most of which are nuclear encoded synthesized in the cytosol, imported into the stroma and translocated into thylakoid membranes by specific thylakoid translocases. Known thylakoid translocases contain core multi-spanning, membrane-integrated subunits that are also nuclear-encoded and imported into chloroplasts before being integrated into thylakoid membranes. Thylakoid translocases play a central role in determining the composition of thylakoids, yet the manner by which the core translocase subunits are integrated into the membrane is not known. We used biochemical and genetic approaches to investigate the integration of the core subunit of the chloroplast Tat translocase, cpTatC, into thylakoid membranes. In vitro import assays show that cpTatC correctly localizes to thylakoids if imported into intact chloroplasts, but that it does not integrate into isolated thylakoids. In vitro transit peptide processing and chimeric precursor import experiments suggest that cpTatC possesses a stroma-targeting transit peptide. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate, suggesting that it might integrate through one of the known thylakoid translocation pathways. However, chemical inhibitors to the cpSecA-cpSecY and cpTat pathways did not impede cpTatC localization to thylakoids when used in import assays. Analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration or assembly into the cpTat receptor complex. These data suggest the existence of another translocase, possibly one dedicated to the integration of chloroplast translocases.     

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. VI. Biochemical and genomic comparison of galactolipid biosynthesis between Chromera velia (Chromerida), a photosynthetic alveolate with red algal plastid ancestry,and the dinoflagellate,Lingulodinium polyedrum

Chromera velia is a recently discovered, photosynthetic, free-living alveolate that is the closest free-living relative to non-photosynthetic apicomplexan parasites. Most plastids, regardless of their origin, have membranes composed chiefly of two galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Because of the hypothesized shared red algal origin between the plastids of C. velia and dinoflagellates, our primary objectives were to examine how growth temperature affects MGDG and DGDG composition via positive-ion electrospray/mass spectrometry (ESI/MS) and positive ion/electrospray/mass spectrometry/mass spectrometry (ESI/MS/MS), and to examine galactolipid biosynthetic genes to determine if shared ancestry translates into shared MGDG and DGDG composition. When growing at 20°C, C. velia produces eicosapentaenoic acid-rich 20:5(n-3)/20:5(n-3) (sn-1/sn-2) MGDG and 20:5(n-3)/20:5(n-3) DGDG as its primary galactolipids, with relative percentage compositions of approximately 35 and 60%, respectively. At 30°C these are lessened by approximately 5 and 8%, respectively, by the corresponding production of 20:5/20:4 forms of these lipids. The presence of 20:5 at the sn-1 position is similar to what has been observed previously in a cluster of peridinin-containing dinoflagellates, but the presence of 20:5(n-3) at the sn-2 position is extremely rare. Thus, the forms of MGDG and DGDG in C. velia displayed similarities and differences to what has been observed in peridinin-containing dinoflagellates, such as Lingulodinium polyedrum, which produces 20:5/18:5 and 20:5/18:4 as the major forms of MGDG and DGDG. We develop conceptual models from the galactolipids observed and galactolipid-relevant gene annotations to explain the presence of polyunsaturated fatty acid-containing MGDG and DGDG in both L. polyedrum and C. velia.     

Computational study of lipid-destabilizing protein fragments: towards a comprehensive view of tilted peptides

Tilted peptides are short sequence fragments (10-20 residues long) that possess an asymmetric hydrophobicity gradient along their sequence when they are helical. Due to this gradient, they adopt a tilted orientation towards a single lipid/water interface and destabilize the lipids. We have detected those peptides in many different proteins with various functions. While being all tilted-oriented at a single lipid/water interface, no consensus sequence can be evidenced. In order to better understand the relationships between their lipid-destabilizing activity and their properties, we used IMPALA to classify the tilted peptides. This method allows the study of interactions between a peptide and a modeled lipid bilayer using simple restraint functions designed to mimic some of the membrane properties. We predict that tilted peptides have access to a wide conformational space in membranes, in contrast to transmembrane and amphipathic helices. In agreement with previous studies, we suggest that those metastable configurations could lead to the perturbation of the acyl chains organization and could be a general mechanism for lipid destabilization. Our results further suggest that tilted peptides fall into two classes: those from proteins acting on membrane behave differently than destabilizing fragments from interfacial proteins. While the former have equal access to the two layers of the membrane, the latter are confined within a single lipid layer. This could be in relation with the organization of lipid substrate on which the peptides physiologically act.     

Peroxisome biogenesis and the role of protein import

Peroxisomes are metabolic organelles with enzymatic content that are found in virtually all cells and are involved in β-oxidation of fatty acids, hydrogen peroxide-based respiration and defence against oxidative stress. The steps of their biogenesis involves "peroxins", proteins encoded by PEX genes. Peroxins are involved in three key stages of peroxisome development: (1) import of peroxisomal membrane proteins; (2) import of peroxisomal matrix proteins and (3) peroxisome proliferation. Of these three areas, peroxisomal matrix-protein import is by far the best understood and accounts for most of the available published data on peroxisome biogenesis. Defects in peroxisome biogenesis result in peroxisome biogenesis disorders (PBDs), which although rare, have no known cure to-date. This review explores current understanding of each key area in peroxisome biogenesis, paying particular attention to the role of protein import.