A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

Exploring Camelina sativa lipid metabolism regulation by combining gene co-expression and DNA affinity purification analyses.

Camelina (Camelina sativa) is an annual oilseed plant that is gaining momentum as a biofuel cover crop. Understanding gene regulatory networks is essential to deciphering plant metabolic pathways, including lipid metabolism. Here, we take advantage of a growing collection of gene expression datasets to predict transcription factors (TFs) associated with the control of Camelina lipid metabolism. We identified approximately 350 TFs highly co-expressed with lipid-related genes (LRGs). These TFs are highly represented in the MYB, AP2/ERF, bZIP, and bHLH families, including a significant number of homologs of well-known Arabidopsis lipid and seed developmental regulators. After prioritizing the top 22 TFs for further validation, we identified DNA-binding sites and predicted target genes for 16 out of the 22 TFs tested using DNA affinity purification followed by sequencing (DAP-seq). Enrichment analyses of targets supported the co-expression prediction for most TF candidates, and the comparison to Arabidopsis revealed some common themes, but also aspects unique to Camelina. Within the top potential lipid regulators, we identified CsaMYB1, CsaABI3AVP1-2, CsaHB1, CsaNAC2, CsaMYB3, and CsaNAC1 as likely involved in the control of seed fatty acid elongation and CsaABI3AVP1-2 and CsabZIP1 as potential regulators of the synthesis and degradation of triacylglycerols (TAGs), respectively. Altogether, the integration of co-expression data and DNA-binding assays permitted us to generate a high-confidence and short list of Camelina TFs involved in the control of lipid metabolism during seed development.

Molecular Topology of the Transit Peptide during Chloroplast Protein Import.

Chloroplast protein import is directed by the interaction of the targeting signal (transit peptide) of nucleus-encoded preproteins with translocons at the outer (TOC) and inner (TIC) chloroplast envelope membranes. Studies of the energetics and determinants of transit peptide binding have led to the hypothesis that import occurs through sequential recognition of transit peptides by components of TOC and TIC during protein import. To test this hypothesis, we employed a site-specific cross-linking approach to map transit peptide topology in relation to TOC-TIC components at specific stages of import in Arabidopsis thaliana and pea (Pisum sativum). We demonstrate that the transit peptide is in contact with Tic20 at the inner envelope in addition to TOC complex components at the earliest stages of chloroplast binding. Low levels of ATP hydrolysis catalyze the commitment of the preprotein to import by promoting further penetration across the envelope membranes and stabilizing the association of the preprotein with TOC-TIC. GTP hydrolysis at the TOC receptors serves as a checkpoint to regulate the ATP-dependent commitment of the preprotein to import and is not essential to drive preprotein import. Our results demonstrate the close cooperativity of the TOC and TIC machinery at each stage of transit peptide recognition and membrane translocation during protein import.

Increased Cuticle Waxes by Overexpression of WSD1 Improves Osmotic Stress Tolerance in Arabidopsis thaliana and Camelina sativa.

To ensure global food security under the changing climate, there is a strong need for developing 'climate resilient crops' that can thrive and produce better yields under extreme environmental conditions such as drought, salinity, and high temperature. To enhance plant productivity under the adverse conditions, we constitutively overexpressed a bifunctional wax synthase/acyl-CoA:diacylglycerol acyltransferase (WSD1) gene, which plays a critical role in wax ester synthesis in Arabidopsis stem and leaf tissues. The qRT-PCR analysis showed a strong upregulation of WSD1 transcripts by mannitol, NaCl, and abscisic acid (ABA) treatments, particularly in Arabidopsis thaliana shoots. Gas chromatography and electron microscopy analyses of Arabidopsis seedlings overexpressing WSD1 showed higher deposition of epicuticular wax crystals and increased leaf and stem wax loading in WSD1 transgenics compared to wildtype (WT) plants. WSD1 transgenics exhibited enhanced tolerance to ABA, mannitol, drought and salinity, which suggested new physiological roles for WSD1 in stress response aside from its wax synthase activity. Transgenic plants were able to recover from drought and salinity better than the WT plants. Furthermore, transgenics showed reduced cuticular transpirational rates and cuticle permeability, as well as less chlorophyll leaching than the WT. The knowledge from Arabidopsis was translated to the oilseed crop Camelina sativa (L.) Crantz. Similar to Arabidopsis, transgenic Camelina lines overexpressing WSD1 also showed enhanced tolerance to drought stress. Our results clearly show that the manipulation of cuticular waxes will be advantageous for enhancing plant productivity under a changing climate.

Origins, function, and regulation of the TOC-TIC general protein import machinery of plastids.

The evolution of chloroplasts from the original endosymbiont involved the transfer of thousands of genes from the ancestral bacterial genome to the host nucleus, thereby combining the two genetic systems to facilitate coordination of gene expression and achieve integration of host and organelle functions. A key element of successful endosymbiosis was the evolution of a unique protein import system to selectively and efficiently target nuclear-encoded proteins to their site of function within the chloroplast after synthesis in the cytoplasm. The chloroplast TOC-TIC (translocon at the outer chloroplast envelope-translocon at the inner chloroplast envelope) general protein import system is conserved across the plant kingdom, and is a system of hybrid origin, with core membrane transport components adapted from bacterial protein targeting systems, and additional components adapted from host genes to confer the specificity and directionality of import. In vascular plants, the TOC-TIC system has diversified to mediate the import of specific, functionally related classes of plastid proteins. This functional diversification occurred as the plastid family expanded to fulfill cell- and tissue-specific functions in terrestrial plants. In addition, there is growing evidence that direct regulation of TOC-TIC activities plays an essential role in the dynamic remodeling of the organelle proteome that is required to coordinate plastid biogenesis with developmental and physiological events.

Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus.

HEMERA (HMR) is a nuclear and plastidial dual-targeted protein. While it functions in the nucleus as a transcriptional coactivator in phytochrome signaling to regulate a distinct set of light-responsive, growth-relevant genes, in plastids it is known as pTAC12, which associates with the plastid-encoded RNA polymerase, and is essential for inducing the plastomic photosynthetic genes and initiating chloroplast biogenesis. However, the mechanism of targeting HMR to the nucleus and plastids is still poorly understood. Here, we show that HMR can be directly imported into chloroplasts through a transit peptide residing in the N-terminal 50 amino acids. Upon cleavage of the transit peptide and additional proteolytic processing, mature HMR, which begins from Lys-58, retains its biochemical properties in phytochrome signaling. Unexpectedly, expression of mature HMR failed to rescue not only the plastidial but also the nuclear defects of the hmr mutant. This is because the predicted nuclear localization signals of HMR are nonfunctional, and therefore mature HMR is unable to accumulate in either plastids or the nucleus. Surprisingly, fusing the transit peptide of the small subunit of Rubisco with mature HMR rescues both its plastidial and nuclear localization and functions. These results, combined with the observation that the nuclear form of HMR has the same reduced molecular mass as plastidial HMR, support a retrograde protein translocation mechanism in which HMR is targeted first to plastids, processed to the mature form, and then relocated to the nucleus.

The integration of chloroplast protein targeting with plant developmental and stress responses.

The plastids, including chloroplasts, are a group of interrelated organelles that confer photoautotrophic growth and the unique metabolic capabilities that are characteristic of plant systems. Plastid biogenesis relies on the expression, import, and assembly of thousands of nuclear encoded preproteins. Plastid proteomes undergo rapid remodeling in response to developmental and environmental signals to generate functionally distinct plastid types in specific cells and tissues. In this review, we will highlight the central role of the plastid protein import system in regulating and coordinating the import of functionally related sets of preproteins that are required for plastid-type transitions and maintenance.

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

Chloroplast heat shock protein 90 (Hsp90C) represents a highly conserved subfamily of the Hsp90 family of molecular chaperones whose function has not been defined. We identified Hsp90C as a component that interacts with import intermediates of nuclear-encoded preproteins during posttranslational import into isolated chloroplasts. Hsp90C was specifically coprecipitated with a complex of protein import components, including Tic110, Tic40, Toc75, Tic22, and the stromal chaperones, Hsp93 and Hsp70. Radicicol, an inhibitor of Hsp90 ATPase activity, reversibly inhibited the import of a variety of preproteins during translocation across the inner envelope membrane, indicating that Hsp90C functions in membrane translocation into the organelle. Hsp90C is encoded by a single gene in Arabidopsis thaliana, and insertion mutations in the Hsp90C gene are embryo lethal, indicating an essential function for the chaperone in plant viability. On the basis of these results, we propose that Hsp90C functions within a chaperone complex in the chloroplast stroma to facilitate membrane translocation during protein import into the organelle.

Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments.

The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for the integration of polytopic proteins to the IEM.

Overexpression of the Phosphatidylcholine:DiacylglycerolCholinephosphotransferase (PDCT) gene increases carbon flux toward triacylglycerol (TAG) synthesis in Camelinasativa seeds.

Camelinasativa has considerable promise as a dedicated industrial oilseed crop. Its oil-based blends have been tested and approved as liquid transportation fuels. Previously, we utilized metabolomic and transcriptomic profiling approaches and identified metabolic bottlenecks that control oil production and accumulation in seeds. Accordingly, we selected candidate genes for the metabolic engineering of Camelina. Here we targeted the overexpression of Camelina PDCT gene, which encodes the phosphatidylcholine: diacylglycerol cholinephosphotransferase enzyme. PDCT is proposed as a gatekeeper responsible for the interconversions of diacylglycerol (DAG) and phosphatidylcholine (PC) pools and has the potential to increase the levels of TAG in seeds. To confirm whether increased CsPDCT activity in developing Camelina seeds would enhance carbon flux toward increased levels of TAG and alter oil composition, we overexpressed the CsPDCT gene under the control of the seed-specific phaseolin promoter. Camelina transgenics exhibited significant increases in seed yield (19-56%), seed oil content (9-13%), oil yields per plant (32-76%), and altered polyunsaturated fatty acid (PUFA) content compared to their parental wild-type (WT) plants. Results from [14C] acetate labeling of Camelina developing embryos expressing CsPDCT in culture indicated increased rates of radiolabeled fatty acid incorporation into glycerolipids (up to 64%, 59%, and 43% higher in TAG, DAG, and PC, respectively), relative to WT embryos. We conclude that overexpression of PDCT appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, thereby further increasing oil yields in Camelina.

The molecular basis for distinct pathways for protein import into Arabidopsis chloroplasts.

The translocons at the outer envelope membrane of chloroplasts (TOCs) initiate the import of thousands of nucleus-encoded proteins into the organelle. The identification of structurally and functionally distinct TOC complexes has led to the hypothesis that the translocons constitute different import pathways that are required to coordinate the import of sets of proteins whose expression varies in response to organelle biogenesis and physiological adaptation. To test this hypothesis, we examined the molecular basis for distinct TOC pathways by analyzing the functional diversification among the Toc159 family of TOC receptors. We demonstrate that the N-terminal A-domains of the Toc159 receptors regulate their selectivity for preprotein binding. Furthermore, the in vivo function of the two major Toc159 family members (atToc159 and atToc132) can be largely switched by swapping their A-domains in transgenic Arabidopsis thaliana. On the basis of these results, we propose that the A-domains of the Toc159 receptors are major determinants of distinct pathways for protein import into chloroplasts.

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33.

GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.

Determinants for stop-transfer and post-import pathways for protein targeting to the chloroplast inner envelope membrane.

The inner envelope membrane (IEM) of the chloroplast plays key roles in controlling metabolite transport between the organelle and cytoplasm and is a major site of lipid and membrane synthesis within the organelle. IEM biogenesis requires the import and integration of nucleus-encoded membrane proteins. Previous reports have led to the conclusion that membrane proteins are inserted into the IEM during protein import from the cytoplasm via a stop-transfer mechanism or are completely imported into the stroma and then inserted into the IEM in a post-import mechanism. In this study, we examined the determinants for each pathway by comparing the targeting of APG1 (albino or pale green mutant 1), an example of a stop-transfer substrate, and atTic40, an example of a post-import substrate. We show that the APG1 transmembrane domain is sufficient to direct stop-transfer insertion. The APG1 transmembrane domain also functions as a topology determinant. We also show that the ability of the post-import signals within atTic40 to target proteins to the IEM is dependent upon their context within the full protein sequence. In the incorrect context, the atTic40 signals can behave as stop-transfer signals or fail to target fusion proteins to the IEM. These data suggest that the post-import pathway signals are complex and have evolved to avoid stop-transfer insertion.

Reconstitution of protein targeting to the inner envelope membrane of chloroplasts.

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a "postimport" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.

Toc receptor dimerization participates in the initiation of membrane translocation during protein import into chloroplasts.

The post-translational import of nucleus-encoded preproteins into chloroplasts occurs through multimeric translocons in the outer (Toc) and inner (Tic) membranes. The high fidelity of the protein import process is maintained by specific recognition of the transit peptide of preproteins by the coordinate activities of two homologous GTPase Toc receptors, Toc34 and Toc159. Structural and biochemical studies suggest that dimerization of the Toc receptors functions as a component of the mechanism to control access of preproteins to the membrane translocation channel of the translocon. We show that specific mutations that disrupted receptor dimerization in vitro reduced the rate of protein import in transgenic Arabidopsis compared with the wild type receptor. The mutations did not affect the GTPase activities of the receptors. Interestingly, these mutations did not decrease the initial preprotein binding at the receptors, but they reduced the efficiency of the transition from preprotein binding to membrane translocation. These data indicate that dimerization of receptors has a direct role in protein import and support a hypothesis in which receptor-receptor interactions participate in the initiation of membrane translocation of chloroplast preproteins as part of the molecular mechanism of GTP-regulated protein import.

Chloroplast protein import: solve the GTPase riddle for entry.

The fidelity of the numerous intracellular protein-trafficking pathways to different organelles is dictated by the interactions between the intrinsic targeting signals of substrate proteins and specific receptors that deliver the substrate to the proper organelle. Recent studies of protein targeting to chloroplasts suggest a novel mechanism in which GTP-dependent substrate recognition is coupled to a GTP-driven motor that initiates the translocation of proteins into the organelle.

The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP.

The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation. Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined. A recent study indicates that Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, raising the possibility that it might cycle between the cytoplasm and chloroplast as a soluble preprotein receptor. In the present study, we examined the mechanism of targeting and insertion of the Arabidopsis thaliana orthologue of Toc159, atToc159, to chloroplasts. Targeting of atToc159 to the outer envelope membrane is strictly dependent only on guanine nucleotides. Although GTP is not required for initial binding, the productive insertion and assembly of atToc159 into the Toc complex requires its intrinsic GTPase activity. Targeting is mediated by direct binding between the GTPase domain of atToc159 and the homologous GTPase domain of atToc33, the Arabidopsis Toc33/34 orthologue. Our findings demonstrate a role for the coordinate action of the Toc GTPases in assembly of the functional Toc complex at the chloroplast outer envelope membrane.

atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins.

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203-207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1-13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93-105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.

Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane.

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.