SChloro: directing Viridiplantae proteins to six chloroplastic sub-compartments

Motivation: Chloroplasts are organelles found in plants and involved in several important cell processes. Similarly to other compartments in the cell, chloroplasts have an internal structure comprising several sub-compartments, where different proteins are targeted to perform their functions. Given the relation between protein function and localization, the availability of effective computational tools to predict protein sub-organelle localizations is crucial for large-scale functional studies. Results: In this paper we present SChloro, a novel machine-learning approach to predict protein sub-chloroplastic localization, based on targeting signal detection and membrane protein information. The proposed approach performs multi-label predictions discriminating six chloroplastic sub-compartments that include inner membrane, outer membrane, stroma, thylakoid lumen, plastoglobule and thylakoid membrane. In comparative benchmarks, the proposed method outperforms current state-of-the-art methods in both single-and multi-compartment predictions, with an overall multi-label accuracy of 74%. The results demonstrate the relevance of the approach that is eligible as a good candidate for integration into more general large-scale annotation pipelines of protein subcellular localization

Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes.

The precursor for a Lemna light-harvesting chlorophyll a/b protein (pLHCP) has been synthesized in vitro from a single member of the nuclear LHCP multigene family. We report the sequence of this gene. When incubated with Lemna chloroplasts, the pLHCP is imported and processed into several polypeptides, and the mature form is assembled into the light-harvesting complex of photosystem II (LHC II). The accumulation of the processed LHCP is enhanced by the addition to the chloroplasts of a precursor and a co-factor for chlorophyll biosynthesis. Using a model for the arrangement of the mature polypeptide in the thylakoid membrane as a guide, we have created mutations that lie within the mature coding region. We have studied the processing, the integration into thylakoid membranes, and the assembly into light-harvesting complexes of six of these deletions. Four different mutant LHCPs are found as processed proteins in the thylakoid membrane, but only one appears to have an orientation in the membrane that is similar to that of the wild type. No mutant LHCP appears in LHC II. The other two mutant LHCPs cannot be detected within the chloroplasts. We conclude that stable complex formation is not required for the processing and insertion of altered LHCPs into the thylakoid membrane. We discuss the results in light of our model

Knock-Out of the Genes Coding for the Rieske Protein and the ATP-Synthase δ-Subunit of Arabidopsis

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b6/f (cyt b6/f) complex and the δ-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b6/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor QA in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-δ destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b6/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response

A Sec14 domain protein is required for photoautotrophic growth and chloroplast vesicle formation in Arabidopsis thaliana.

In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans-Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium

Comparison of the α and β isomeric forms of the detergent n-dodecyl-D-maltoside for solubilizing photosynthetic complexes from pea thylakoid membranes

AbstractMild non-ionic detergents are indispensable in the isolation of intact integral membrane proteins and protein-complexes from biological membranes. Dodecylmaltoside (DM) belongs to this class of detergents being a glucoside-based surfactant with a bulky hydrophilic head group composed of two sugar rings and a non-charged alkyl glycoside chain. Two isomers of this molecule exist, differing only in the configuration of the alkyl chain around the anomeric center of the carbohydrate head group, axial in α-DM and equatorial in β-DM. In this paper, we have investigated the solubilizing properties of α-DM and β-DM on the isolation of photosynthetic complexes from pea thylakoids membranes maintaining their native architecture of stacked grana and stroma lamellae. Exposure of these stacked thylakoids to a single step treatment with increasing concentrations (5–100mM) of α-DM or β-DM resulted in a quick partial or complete solubilization of the membranes. Regardless of the isomeric form used: 1) at the lowest DM concentrations only a partial solubilization of thylakoids was achieved, giving rise to the release of mainly small protein complexes mixed with membrane fragments enriched in PSI from stroma lamellae; 2) at concentrations above 30mM a complete solubilization occurred with the further release of high molecular weight protein complexes identified as dimeric PSII, PSI-LHCI and PSII–LHCII supercomplexes. However, at concentrations of detergent which fully solubilized the thylakoids, the α and β isomeric forms of DM exerted a somewhat different solubilizing effect on the membranes: higher abundance of larger sized PSII–LHCII supercomplexes retaining a higher proportion of LHCII and lower amounts of PSI–LHCI intermediates were observed in α-DM treated membranes, reflecting the mildness of α-DM compared with its isomer. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial

Visualizing the mobility and distribution of chlorophyll proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation

The definitive version is available at www.blackwell-synergy.co

Entropy and biological systems: experimentally-investigated entropy-driven stacking of plant photosynthetic membranes

According to the Second Law of thermodynamics, an overall increase of entropy contributes to the driving force for any physicochemical process, but entropy has seldom been investigated in biological systems. Here, for the first time, we apply Isothermal Titration Calorimetry (ITC) to investigate the Mg21-induced spontaneous stacking of photosynthetic membranes isolated from spinach leaves. After subtracting a large endothermic interaction of MgCl2 with membranes, unrelated to stacking, we demonstrate that the enthalpy change (heat change at constant pressure) is zero or marginally positive or negative. This first direct experimental evidence strongly suggests that an entropy increase significantly drives membrane stacking in this ordered biological structure. Possible mechanisms for the entropy increase include: (i) the attraction between discrete oppositely-charged areas, releasing counterions; (ii) the release of loosely-bound water molecules from the inter-membrane gap; (iii) the increased orientational freedom of previously-aligned water dipoles; and (iv) the lateral rearrangement of membrane components.This work was supported consecutively by Australian Research Council grants (DP0664719 and DP 1093927)

A three dimensional model of the photosynthetic membranes of Ectothiorhodospira halochloris

The three dimensional organization of the complete photosynthetic apparatus of the extremely halophilic, bacteriochlorophyll b containing Ectothiorhodospira halochloris has been elaborated by several techniques of electron microscopy. Essentially all thylakoidal sacs are disc shaped and connected to the cytoplasmic membrane by small membraneous ldquobridgesrdquo. In sum, the lumina of all thylakoids (intrathylakoidal space) form one common periplasmic space. Thin sections confirm a paracrystalline arrangement of the photosynthetic complexes in situ. The ontogenic development of the photosynthetic apparatus is discussed based on a structural model derived from serial thin sections

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of -tocopherol and plastoquinone-9 precursors. The biosynthesis of -tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in -tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl-PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of -tocopherol in spinach chloroplasts