Chromoplast differentiation in bell pepper (Capsicum annuum) fruits.

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell pepper (Capsicum annuum) fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b6 f complex, the ATPase complex, and Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts' redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids, such as the oxidative pentose phosphate pathway (OPPP) and amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins decline but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation are consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper that lack a respiratory burst at the onset of fruit ripening.

Working day and night: plastid casein kinase 2 catalyses phosphorylation of proteins with diverse functions in light- and dark-adapted plastids.

Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the in vivo targets of plastid casein kinase 2 (pCK2) in Arabidopsis thaliana. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and pck2 knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the pck2 knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of in vivo pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions.

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis.

Importance of Translocon Subunit Tic56 for rRNA Processing and Chloroplast Ribosome Assembly.

Toc159-containing complexes at the outer chloroplast envelope membrane form stable supercomplexes with a 1-MD translocon at the inner chloroplast envelope membrane of which Tic56 is one essential subunit. While the single mutants tic56-1 and ppi2 (toc159) have an albino phenotype and are able to grow heterotrophically, we find the double mutant to be embryo lethal. Comprehensive quantitative proteome profiling with both single mutants in combination with GeneChip analyses identified a posttranscriptional defect in the accumulation of plastid ribosomal proteins and diminished expression of plastid encoded proteins. In the tic56-1 mutant, the assembly of functional ribosomes is furthermore hampered by a processing defect of the plastid 23S rRNA. Spectinomycin-treatment of wild-type plants phenocopies the molecular phenotype of plastid proteome accumulation in tic56-1 and to a smaller degree also ppi2 plastids, suggesting that a defect in plastid translation is largely responsible for the phenotype of both import mutants. Import experiments with the tic56-3 mutant revealed no significant defect in the import of small ribosomal protein 16 in the absence of full-length Tic56, suggesting that the defect in ribosome assembly in tic56-1 may be independent of a function of Tic56 in protein import. Our data establish a previously unknown link between plastid protein import, the processing of plastid rRNAs, and the assembly of plastid ribosomes and provide further knowledge on the function of the translocon components and the molecular basis for their albino phenotype.

Characterization of chloroplast protein import without Tic56, a component of the 1-megadalton translocon at the inner envelope membrane of chloroplasts.

We report on the characterization of Tic56, a unique component of the recently identified 1-MD translocon at the inner envelope membrane of chloroplasts (TIC) in Arabidopsis (Arabidopsis thaliana) comprising Tic20, Tic100, and Tic214. We isolated Tic56 by copurification with Tandem Affinity Purification-tagged Toc159 in the absence of precursor protein, indicating spontaneous and translocation-independent formation of the translocon at the outer envelope membrane of chloroplasts (TOC) and TIC supercomplexes. Tic56 mutant plants have an albino phenotype and are unable to grow without an external carbon source. Using specific enrichment of protein amino termini, we analyzed the tic56-1 and plastid protein import2 (toc159) mutants to assess the in vivo import capacity of plastids in mutants of an outer and inner envelope component of the anticipated TOC-TIC supercomplex. Inboth mutants, we observed processing of several import substrates belonging to various pathways. Our results suggest that despite the severe developmental defects, protein import into Tic56-deficient plastids is functional to a considerable degree, indicating the existence of alternative translocases at the inner envelope membrane.

Common and specific protein accumulation patterns in different albino/pale-green mutants reveals regulon organization at the proteome level.

Research interest in proteomics is increasingly shifting toward the reverse genetic characterization of gene function at the proteome level. In plants, several distinct gene defects perturb photosynthetic capacity, resulting in the loss of chlorophyll and an albino or pale-green phenotype. Because photosynthesis is interconnected with the entire plant metabolism and its regulation, all albino plants share common characteristics that are determined by the switch from autotrophic to heterotrophic growth. Reverse genetic characterizations of such plants often cannot distinguish between specific consequences of a gene defect from generic effects in response to perturbations in photosynthetic capacity. Here, we set out to define common and specific features of protein accumulation in three different albino/pale-green plant lines. Using quantitative proteomics, we report a common molecular phenotype that connects the loss of photosynthetic capacity with other chloroplast and cellular functions, such as protein folding and stability, plastid protein import, and the expression of stress-related genes. Surprisingly, we do not find significant differences in the expression of key transcriptional regulators, suggesting that substantial regulation occurs at the posttranscriptional level. We examine the influence of different normalization schemes on the quantitative proteomics data and report all identified proteins along with their fold changes and P values in albino plants in comparison with the wild type. Our analysis provides initial guidance for the distinction between general and specific adaptations of the proteome in photosynthesis-impaired plants.

The TOC complex: preprotein gateway to the chloroplast.

Photosynthetic eukaryotes strongly depend on chloroplast metabolic pathways. Most if not all involve nuclear encoded proteins. These are synthesized as cytosolic preproteins with N-terminal, cleavable targeting sequences (transit peptide). Preproteins are imported by a major pathway composed of two proteins complexes: TOC and TIC (Translocon of the Outer and Inner membranes of the Chloroplasts, respectively). These selectively recognize the preproteins and facilitate their transport across the chloroplast envelope. The TOC core complex consists of three types of components, each belonging to a small family: Toc34, Toc75 and Toc159. Toc34 and Toc159 isoforms represent a subfamily of the GTPase superfamily. The members of the Toc34 and Toc159 subfamily act as GTP-dependent receptors at the chloroplast surface and distinct members of each occur in defined, substrate-specific TOC complexes. Toc75, a member of the Omp85 family, is conserved from prokaryotes and functions as the unique protein-conducting channel at the outer membrane. In this review we will describe the current state of knowledge regarding the composition and function of the TOC complex.

Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency.

The atToc33 protein is one of several pre-protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis-related pre-proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild-type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159-deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.

The acidic A-domain of Arabidopsis TOC159 occurs as a hyperphosphorylated protein.

The translocon at the outer membrane of the chloroplast assists the import of a large class of preproteins with amino-terminal transit sequences. The preprotein receptors Toc159 and Toc33 in Arabidopsis (Arabidopsis thaliana) are specific for the accumulation of abundant photosynthetic proteins. The receptors are homologous GTPases known to be regulated by phosphorylation within their GTP-binding domains. In addition to the central GTP-binding domain, Toc159 has an acidic N-terminal domain (A-domain) and a C-terminal membrane-anchoring domain (M-domain). The A-domain of Toc159 is dispensable for its in vivo activity in Arabidopsis and prone to degradation in pea (Pisum sativum). Therefore, it has been suggested to have a regulatory function. Here, we show that in Arabidopsis, the A-domain is not simply degraded but that it accumulates as a soluble, phosphorylated protein separated from Toc159. However, the physiological relevance of this process is unclear. The data show that the A-domain of Toc159 as well as those of its homologs Toc132 and Toc120 are targets of a casein kinase 2-like activity.

In vivo interaction between atToc33 and atToc159 GTP-binding domains demonstrated in a plant split-ubiquitin system.

The GTPases atToc33 and atToc159 are pre-protein receptor components of the translocon complex at the outer chloroplast membrane in Arabidopsis. Despite their participation in the same complex in vivo, evidence for their interaction is still lacking. Here, a split-ubiquitin system is engineered for use in plants, and the in vivo interaction of the Toc GTPases in Arabidopsis and tobacco protoplasts is shown. Using the same method, the self-interaction of the peroxisomal membrane protein atPex11e is demonstrated. The finding suggests a more general suitability of the split-ubiquitin system as a plant in vivo interaction assay.

Consequences of impaired 1-MDa TIC complex assembly for the abundance and composition of chloroplast high-molecular mass protein complexes.

We report a systematic analysis of chloroplast high-molecular mass protein complexes using a combination of native gel electrophoresis and absolute protein quantification by MSE. With this experimental setup, we characterized the effect of the tic56-3 mutation in the 1-MDa inner envelope translocase (TIC) on the assembly of the chloroplast proteome. We show that the tic56-3 mutation results in a reduction of the 1-MDa TIC complex to approximately 10% of wildtype levels. Hierarchical clustering confirmed the association of malate dehydrogenase (MDH) with an envelope-associated FtsH/FtsHi complex and suggested the association of a glycine-rich protein with the 1-MDa TIC complex. Depletion of this complex leads to a reduction of chloroplast ATPase to approx. 75% of wildtype levels, while the abundance of the FtsH/FtsHi complex is increased to approx. 140% of wildtype. The accumulation of the major photosynthetic complexes is not affected by the mutation, suggesting that tic56-3 plants can sustain a functional photosynthetic machinery despite a significant reduction of the 1-MDa TIC complex. Together our analysis expands recent efforts to catalogue the native molecular masses of chloroplast proteins and provides information on the consequences of impaired accumulation of the 1-MDa TIC translocase for chloroplast proteome assembly.

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

Protein import-independent functions of Tic56, a component of the 1-MDa translocase at the inner chloroplast envelope membrane.

Tic56 is an essential subunit of a 1-MDa protein complex at the inner chloroplast envelope membrane that comprises Tic100, Tic214 and the protein conducting channel protein Tic20-I. The complex was characterized as the "general protein import translocase" because mutants in either of its subunits have a severe growth phenotype and fail to assemble a photosynthetic machinery. In a recent publication we show that the albino phenotype of tic56-1 mutants results at least in part from a defect in ribosome assembly and a deficiency in plastid translation. We furthermore could not detect any impairment of protein import activity with plastids from tic56-3 mutants, despite a lack of full-length Tic56 and a decreased abundance of other 1-MDa complex subunits. These findings suggest that the 1-MDa complex consists of subunits that have functions other than protein import.

Arabidopsis proteomics: a simple and standardizable workflow for quantitative proteome characterization.

Arabidopsis is the model plant of choice for large-scale proteome analyses, because its genome is well annotated, essentially free of sequencing errors, and relatively small with little redundancy. Furthermore, most Arabidopsis organs are susceptible to standard protein solubilization protocols making protein extraction relatively simple. Many different facets of functional plant proteomics were established with Arabidopsis such as mapping the subcellular proteomes of organelles, proteo-genomic peptide mapping, and numerous studies on the dynamic changes in protein modification and protein abundances. As most standard proteomics technologies are now routinely applied, research interest is increasingly shifting towards the reverse genetic characterization of gene function at the proteome level, i.e., by profiling the quantitative proteome of wild type in comparison with mutant plant tissue. We report here a simple, standardizable protocol for the large-scale comparative quantitative proteome characterization of different Arabidopsis organs based on normalized spectral counting and suggest a statistical framework for data interpretation. Based on existing organellar proteome maps, proteins can be assigned to organelles, thus allowing the identification of organelle-specific responses.

Protein identification and quantification by data-independent acquisition and multi-parallel collision-induced dissociation mass spectrometry (MS(E)) in the chloroplast stroma proteome.

We report here a systematic evaluation of a multiplex mass spectrometry method coupled with ion mobility separation (HD-MS(E)) for the identification and quantification of proteins in the chloroplast stroma. We show that this method allows the robust quantification of reference proteins in mixtures, and it detects concentration differences with high sensitivity when three replicas are performed. Applied to the analysis of the chloroplast stroma proteome, HD-MS(E) identified and quantified many chloroplast proteins that were not previously identified in large-scale proteome analyses, suggesting HD-MS(E) as a suitable complementary tool for discovery proteomics. We find that HD-MS(E) tends to underestimate protein abundances at concentrations above 25fmol, which is likely due to ion transmission loss and detector saturation. This limitation can be circumvented by omitting the ion mobility separation step in the HD-MS(E) workflow. The robustness of protein quantification is influenced by the selection of peptides and their intensity distribution, therefore critical scrutiny of quantification results is required. Based on the HD-MS(E) quantification of chloroplast stroma proteins we performed a meta-analysis and compared published quantitative data with our results, using a parts per million normalization scheme. Important pathways in the chloroplast stroma show quantitative stability against different experimental conditions and quantification strategies. BIOLOGICAL SIGNIFICANCE: Our analysis establishes MS(E)-based Hi3 quantification as a tool for the absolute quantification of proteins in the chloroplast stroma. The meta-analysis performed with a parts per million normalization scheme shows that quantitative proteomics data acquired in different labs and with different quantification strategies yield comparable results for some metabolic pathways, while others show a higher variability. Our data therefore indicate that such meta-analyses allow distinguishing robust from fine-controlled metabolic pathways.

The peptide microarray "ChloroPhos1.0" identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana.

We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.

Preparation of multiprotein complexes from Arabidopsis chloroplasts using tandem affinity purification.

Since its first description in 1998 (Rigaut et al., Nat Biotech 17:1030-1032, 1999), the TAP method, for Tandem Affinity Purification, has become one of the most popular methods for the purification of in vivo protein complexes and the identification of their composition by subsequent mass spectrometry analysis. The TAP method is based on the use of a tripartite tag fused to a target protein expressed in the organism of interest. A TAP tag has two independent binding regions separated by a protease cleavage site, and therefore allows two successive affinity purification steps. The most common TAP tag consists of two IgG binding repeats of Protein A from Staphylococcus aureus (ProtA) separated from a calmodulin-binding peptide by a Tobacco Etch Virus (TEV) protease cleavage site. Using the TAP method, native protein complexes can be purified efficiently with a reduced contaminant background when compared to single step purification methods. Initially developed in the yeast model system, the TAP method has been adapted to most common model organisms. The first report of the purification of protein complexes from plant tissue by the TAP method was published in 2004 by Rohila et al. (Plant J 38:172-181, 2004). The synthetic TAP tag gene described in this study has been optimized for use in plants, and since then, has been successfully used from single gene analyses to high-throughput studies of whole protein families (Rohila et al., PLoS ONE 4:e6685, 2009). Here, we describe a TAP tag purification method for the purification of protein complexes from total Arabidopsis extracts, that we employed successfully using a TAP-tagged chloroplast outer envelope protein.

The chloroplast import receptor Toc90 partially restores the accumulation of Toc159 client proteins in the Arabidopsis thaliana ppi2 mutant.

Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- (translocon at the outer chloroplast membrane) and Tic- (translocon at the inner chloroplast membrane) complexes. In Arabidopsis thaliana, precursor recognition is mainly mediated by outer membrane receptors belonging to two gene families: Toc34/33 and Toc159/132/120/90. The role in import and precursor selectivity of these receptors has been intensively studied, but the function of Toc90 still remains unclear. Here, we report the ability of Toc90 to support the import of Toc159 client proteins. We show that the overexpression of Toc90 partially complements the albino knockout of Toc159 and restores photoautotrophic growth. Several lines of evidence including proteome profiling demonstrate the import and accumulation of proteins essential for chloroplast biogenesis and functionality.

Modifications at the A-domain of the chloroplast import receptor Toc159.

Two families of GTPases, the Toc34 and Toc159 GTPase families, take on the task of preprotein recognition at the translocon at the outer membrane of chloroplasts (TOC translocon). The major Toc159 family members have highly acidic N-terminal domains (A-domains) that are non-essential and so far have escaped functional characterization. But recently, interest in the role of the A-domain has strongly increased. The new data of three independent studies provide evidence that the Toc159 A-domain I) participates in preprotein selectivity, II) has typical features of intrinsically unfolded proteins and III) is highly phosphorylated and possibly released from the rest of the protein by a proteolytic event. This hints to a complex regulation of A-domain function that is important for the maintenance of the preprotein selectivity at the TOC translocons.

Protein transport in organelles: The Toc complex way of preprotein import.

Most of the estimated 1000 or so chloroplast proteins are synthesized as cytosolic preproteins with N-terminal cleavable targeting sequences (transit peptide). Translocon complexes at the outer (Toc) and inner chloroplast envelope membrane (Tic) concertedly facilitate post-translational import of preproteins into the chloroplast. Three components, the Toc34 and Toc159 GTPases together with the Toc75 channel, form the core of the Toc complex. The two GTPases act as GTP-dependent receptors at the chloroplast surface and promote insertion of the preprotein across the Toc75 channel. Additional factors guide preproteins to the Toc complex or support their stable ATP-dependent binding to the chloroplast. This minireview describes the components of the Toc complex and their function during the initial steps of preprotein translocation across the chloroplast envelope.