Protein- and energy-mediated targeting of chloroplast outer envelope membrane proteins

While the import of nuclear-encoded chloroplast proteins is relatively well studied, the targeting of proteins to the outer membrane of the chloroplast envelope is not. The insertion of most outer membrane proteins (OMP) is generally considered to occur without the utilization of energy or proteinaceous components. Recently, however, proteins have been shown to be involved in the integration of outer envelope protein 14 (OEP14), whose outer membrane insertion was previously thought to be spontaneous. Here we investigate the insertion of two proteins from Physcomitrella patens, PpOEP64-1 and PpOEP64-2 (formerly known as PpToc64-1 and PpToc64-2), into the outer membrane of chloroplasts. The association of PpOEP64-1 with chloroplasts was not affected by chloroplast pre-treatments. Its insertion into the membrane was affected, however, demonstrating the importance of measuring insertion specifically in these types of assays. We found that the insertion of PpOEP64-1, PpOEP64-2 and two other OMPs, OEP14 and digalactosyldiacylglycerol synthase 1 (DGD1), was reduced by either nucleotide depletion or proteolysis of the chloroplasts. Integration was also inhibited in the presence of an excess of an imported precursor protein. In addition, OEP14 competed with the insertion of the OEP64s and DGD1. These data demonstrate that the targeting of several OMPs involves proteins present in chloroplasts and requires nucleotides. Together with previous reports, our data suggest that OMPs in general do not insert spontaneously.     

A systemic proteomic analysis of <Emphasis Type="Italic">Populus</Emphasis> chloroplast by using shotgun method

The chloroplast is one of the most important organelles in plants. Proteomic investigations of chloroplasts have been undertaken for many herb plant species, but to date no such investigation has been reported for woody plant chloroplasts. In the present study we initiated a systematic proteomic study of Populus chloroplasts using a shotgun proteomic method. After isolation of chloroplasts and tryptic digestion of the proteins, the protein fragments were separated via HPLC using an SCX column, and the peptides were analyzed by LC-MS/MS; 119 proteins were successfully identified. Based on annotation information in the UniProtKB/Swiss-Prot database, these proteins were identified as being localized in the chloroplast thylakoid membrane, chloroplast stroma, chloroplast thylakoid lumen, and plastoglobules. Over 50% of all identified proteins were confirmed as chloroplast thylakoid proteins, and 85 are encoded by the chloroplast genome with the remaining proteins encoded by the nuclear genome. Based on functional annotation, these proteins were classified into four functional categories, including photosynthesis, redox regulation and stress, primary and secondary metabolism, transport and signaling. These data provide a valuable basis for further studies on photosynthesis in poplar species.     

A mutant deficient in the plastid lipid DGD is defective in protein import into chloroplasts

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol with N-terminal extensions called transit peptides. Transit peptides function as the import signal to chloroplasts. The import process requires several protein components in the envelope and stroma and also requires the hydrolysis of ATP. Lipids have been implicated in the import process based on theories or experiments with in vitro model systems. We show here that chloroplasts isolated from an Arabidopsis mutant deficient in the plastid lipid digalactosyl diacylglycerol (DGD) were normal in importing a chloroplast outer membrane protein, but were defective in importing precursor proteins targeted to the interior of chloroplasts. The impairment includes the binding, or docking, step of the import process that is supported by 100 μM ATP.     

盐胁迫下苜蓿中盐蛋白的诱导产生

盐胁迫下苜蓿叶片中蛋白质的合成受到抑制,而其离体叶绿体中蛋白质合成增强,ABA阻碍了后者的蛋白质合成。NaCl胁迫下,“松江”和“肇东”两品种的根和叶中均无新多肽出现。在盐敏感的“松江”品种离体叶绿体中,NaGl诱导70,65,60和43kD4种多肽产生,ABA诱导60和17kD两种多肽产生;在较抗盐的“肇东”品种离体叶绿体中,NaGl诱导83,80kD和43kD3种多肽产生,但100mmol/L NaCl并不诱导83kD多肽出现,ABA无明显作用。两品种的43kD多肽和肇东品种的80kD多肽都存在于类囊体膜上,而松江品种的60kD多肽则存在于叶绿体间质中。     

Toc64 is not required for import of proteins into chloroplasts in the moss Physcomitrella patens

Toc64 has been suggested to be part of the chloroplast import machinery in Pisum sativum. A role for Toc64 in protein transport has not been established, however. To address this, we generated knockout mutants in the moss Physcomitrella patens using the moss's ability to perform homologous recombination with nuclear DNA. Physcomitrella patens contains two genes that encode Toc64-like proteins. Both of those proteins appear to be localized in the chloroplast. The double-mutant plants were lacking Toc64 protein in the chloroplasts but showed no growth phenotype. In addition, these plants accumulated other plastid proteins at wild-type levels and showed no difference from wild type in in vitro protein import assays. These plants did have a slightly altered chloroplast shape in some tissues, however. The evidence therefore indicates that Toc64 proteins are not required for import of proteins in Physcomitrella, but may point to involvement in the determination of plastid shape.     

Filamentous temperature-sensitive Z (FtsZ) isoforms specifically interact in the chloroplasts and in the cytosol of Physcomitrella patens

Plant filamentous temperature-sensitive Z (FtsZ) proteins have been reported to be involved in biological processes related to plastids. However, the precise functions of distinct isoforms are still elusive. Here, the intracellular localization of the FtsZ1-1 isoform in a moss, Physcomitrella patens, was examined. Furthermore, the in vivo interaction behaviour of four distinct FtsZ isoforms was investigated. Localization studies of green fluorescent protein (GFP)-tagged FtsZ1-1 and fluorescence resonance energy transfer (FRET) analyses employing all dual combinations of four FtsZ isoforms were performed in transient protoplast transformation assays. FtsZ1-1 is localized to network structures inside the chloroplasts and exerts influence on plastid division. Interactions between FtsZ isoforms occur in distinct ordered structures in the chloroplasts as well as in the cytosol. The results expand the view of the involvement of Physcomitrella FtsZ proteins in chloroplast and cell division. It is concluded that duplication and diversification of ftsZ genes during plant evolution were the main prerequisites for the successful remodelling and integration of the prokaryotic FtsZ-dependent division mechanism into the cellular machineries of distinct complex processes in plants.     

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.     

The kinesin-like proteins,KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens

In plants, light determines chloroplast position;these organelles show avoidance and accumulation re-sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light ...     

Ptr ToxA interacts with a chloroplast-localized protein

Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces host-selective toxins that are determinants of pathogenicity or virulence. Ptr ToxA (ToxA), a proteinaceous toxin produced by P. tritici-repentis, is a necrotizing toxin produced by the most common races isolated from infected wheat. Recent studies have shown that ToxA is internalized into the mesophyll cells and localizes to chloroplasts of sensitive wheat cultivars only. We employed a yeast two-hybrid screen in an effort to determine plant proteins that interact with ToxA and found that ToxA interacts with a chloroplast protein, designated ToxA binding protein 1 (ToxABP1). ToxABP1 contains a lysine-rich region within a coiled-coil domain that is similar to phosphotidyl-inositol binding sites present in animal proteins involved in endocytosis. In both ToxA-sensitive and -insensitive cultivars, ToxABP1 is expressed at similar levels and encodes an identical protein. ToxABP1 protein is present in both chloroplast membranes and chloroplast stroma. ToxA appears to interact primarily with a multimeric complex of ToxABP1 protein associated with the chloroplast membrane.     

In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana

Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the DeltapH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the DeltapH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the DeltapH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the DeltapH/TAT pathway.     

A uniquely high number of ftsZ genes in the moss Physcomitrella patens

Plant FtsZ proteins are encoded by two small nuclear gene families (FtsZ1 and FtsZ2) and are involved in chloroplast division. From the moss Physcomitrella patens , four FtsZ proteins, two in each nuclear gene family, have been characterised and described so far. In the recently sequenced P. patens genome, we have now found a fifth fts Z gene. This novel gene has a genomic structure similar to Pp fts Z1-1. According to phylogenetic analysis, the encoded protein is a member of the FtsZ1 family, while PpFtsZ1-2, together with an orthologue from Selaginella moellendorffii , forms a separate clade. Further, this new gene is expressed in different gametophytic tissues and the encoded protein forms filamentous networks in chloroplasts, is found in stromules, and acts in plastid division. Based on all these results, we have renamed the PpFtsZ proteins of family 1 and suggest the existence of a third FtsZ family. No species is known to encode more FtsZ proteins per haploid genome than P. patens .     

Targeting of EGFP chimeras within chloroplasts

We have tested the potential of EGFP, a derivative of the green fluorescent protein (GFP), as a passenger protein for the analysis of protein transport processes across the thylakoid membranes in chloroplasts. In contrast to the majority of fusion proteins commonly used in such studies, EGFP is not of plant origin and can therefore be assumed to behave like a "neutral" passenger protein that is unaffected by any internal plant regulatory circuits. Our in vitro transport experiments clearly demonstrate that EGFP is a suitable passenger protein that can be correctly targeted either to the stroma or to the thylakoid lumen if fused to the appropriate transit peptide. The transport of EGFP across the thylakoid membrane shows, however, a clear pathway preference. While the protein is efficiently targeted by the deltapH/TAT pathway, transport by the Sec pathway is barely detectable, either with isolated thylakoids or with intact chloroplasts. This pathway specificity suggests that EGFP is folded immediately after import into the chloroplast stroma, thus preventing further translocation across the thylakoid membrane by the Sec translocase. The data obtained provide a good basis for the development of molecular tools for transport studies using EGFP as a passenger protein. Furthermore, plant lines expressing corresponding EGFP chimeras are expected to allow in vivo studies on the transport and sorting mechanisms involved in the biogenesis of the chloroplast.     

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.     

A 38-amino-acid sequence encompassing the arm domain of the cucumber necrosis virus coat protein functions as a chloroplast transit Peptide in infected plants

Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed.     

An integrated physiological and genetic approach to the dynamics of FtsZ targeting and organisation in a moss, Physcomitrella patens

Plant FtsZ (filamentous temperature-sensitive Z) proteins are regarded as descendants of prokaryotic cell division proteins. We could show previously that four FtsZ isoforms of the moss Physcomitrella patens assemble into, and interact in, distinct structures inside the chloroplasts and in the cytosol. Their organisation and localisation patterns indicate an involvement in chloroplast and cell division and in the maintenance of chloroplast shape and integrity. The cellular processes of chloroplast division and maintenance of chloroplast shape were disturbed either by application of the beta-lactam antibiotic ampicillin or by a mutation that presumably affects signal transduction of the plant hormone cytokinin. When cells of these plants were analysed microscopically, there was no indication that cytosolic functions of FtsZ proteins were affected. Furthermore, FtsZ proteins continued to build three-dimensional plastoskeleton networks, even in considerably enlarged or malformed chloroplasts. On the other hand, macrochloroplast formation promoted the localisation of FtsZ proteins in filaments that emanate from the plastids and, therefore, most likely represent stromules. Annular FtsZ structures that are regarded as essential components of the division apparatus were absent from macrochloroplasts of ampicillin-treated cells. Thus, the distribution of FtsZ proteins after inhibition of chloroplast division further strengthens our hypothesis on the functions of distinct isoforms. In addition, the results provide further insight into the regulation of protein targeting and dynamics of plastoskeletal elements.     

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane.     

The effect of amino acid-modifying reagents on chloroplast protein import and the formation of early import intermediates

In order to identify functionally important amino acid residues in the chloroplast protein import machinery, chloroplasts were preincubated with amino-acid-modifying reagents and then allowed to import or form early import intermediates with precursor proteins. Incubation of chloroplasts with N-ethyl maleimide, diethyl pyrocarbonate, phenylglyoxal, 4,4'-di-isothiocyanatostilbene 2,2'-disulphonic acid (DIDS), dicyclohexylcarbodiimide (DCCD), and 1-ethyl- 3-dimethylaminopropylcarbodiimide (EDC) inhibited both import and formation of early import intermediates with precursor proteins by chloroplasts. This suggests that one or more of the binding components of the chloroplast protein import machinery contains functionally important solvent-exposed cysteine, histidine, arginine, and aspartate/glutamate residues, as well as functionally important lysine and aspartate/ glutamate residues in a hydrophobic environment.     

Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.     

Cytosolic events involved in chloroplast protein targeting

Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.