Immunological similarities between specific chloroplast ribosomal proteins from Chlamydomonas reinhardtii and ribosomal proteins from Escherichia coli

Polyclonal antibodies were elicited against seven of the 33 different proteins of the large subunit of the chloroplast ribosome from Chlamydomonas reinhardtii. Three of these proteins are synthesized in the chloroplast and four are made in the cytoplasm and imported. In western blots, six of the seven antisera are monospecific for their respective large subunit ribosomal proteins, and none of these antisera cross-reacted with any chloroplast small subunit proteins from C. reinhardtii. Antisera to the three chloroplast-synthesized ribosomal proteins cross-reacted with specific Escherichia coli large subunit proteins of comparable charge and molecular weight. Only one of the four antisera to the chloroplast ribosomal proteins synthesized in the cytoplasm cross-reacted with an E. coli large subunit protein. None of the antisera cross-reacted with any E. coli small subunit proteins. On the assumption of a procaryotic, endosymbiotic origin for the chloroplast, those chloroplast ribosomal proteins still synthesized within the organelle appear to have retained more antigenic sites in common with E. coli ribosomal proteins than have those which are now the products of cytoplasmic protein synthesis. Antisera to this cytoplasmically synthesized group of chloroplast ribosomal proteins did not recognize any antigenic sites among C. reinhardtii cytoplasmic ribosomal proteins, suggesting that the genes for the cytoplasmically synthesized chloroplast ribosomal proteins either are not derived from the cytoplasmic ribosomal protein genes or have evolved to a point where no antigenic similarities remain.      

Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts

The chloroplast is an organelle of prokaryotic origin that is situated in an eukaryotic cellular environment. As a result of this formerly endosymbiotic situation, the chloroplast houses a unique set of protein transport machineries. Among those are evolutionarily young transport pathways which are responsible for the import of the nuclear-encoded proteins into the organelle as well as ancient pathways operating in the 'export' of proteins from the stroma (the former cyanobacterial cytosol) across the thylakoid membrane into the thylakoid lumen. In this review, we have tried to address the main features of these various transport pathways.     

Protein import into chloroplasts

Most chloroplastic proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and subsequently imported into the organelle. In general, proteins destined for the chloroplast are synthesized as precursor proteins with a cleavable N-terminal presequence that mediates routing to the inside of the chloroplast. These precursor proteins have to be targeted to the correct organellar membrane surface after their release from the ribosome and furthermore they have to be maintained in a conformation suitable for translocation across the two envelope membranes. Recognition and import of most chloroplastic precursor proteins are accomplished by a jointly used translocation apparatus. Different but complementary studies of several groups converged recently in the identification of the outer envelope proteins OEP86, OEP75, OEP70 (a Hsp 70-related protein), OEP34, and of the inner envelope protein IEP110 as components of this translocation machinery. None of these proteins, except for OEP70, shows any homology to components of other protein translocases. The plastid import machinery thus seems to be an original development in evolution. Following translocation into the organelle, chloroplastic proteins are sorted to their suborganellar destination, i.e., the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen. This structural and evolutionary complexity of chloroplasts is reflected by a variety of routing mechanisms by which proteins reach their final location once inside the organelle. This review will focus on recent advances in the identification of components of the chloroplastic protein import machinery, and new insights into the pathways of inter-and intraorganellar sorting.     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

Evolution of Chloroplast J Proteins

Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance.     

Noncoding RNA Mediated Traffic of Foreign mRNA into Chloroplasts Reveals a Novel Signaling Mechanism in Plants

Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs) are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5′UTR-end mediates the functional import of Green Fluorescent Protein (GFP) mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5′UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells.     

Mechanisms of protein import and routing in chloroplasts

The vast majority of the approximately 3000 different proteins required to build a fully functional chloroplast are encoded by the nuclear genome and translated on cytosolic ribosomes. As chloroplasts are each surrounded by a double-membrane system, or envelope, sophisticated mechanisms are necessary to mediate the import of these nucleus-encoded proteins into chloroplasts. Once inside the organelle, many chloroplast proteins engage one of four additional protein sorting mechanisms that direct targeting to the internal thylakoid membrane system.     

Spectral properties of a pigmented body in Hymenomonas sp.: an extra-chloroplast organelle containing chlorophyll

In the chrysomonad Hymenomonas, a lamellar organelle of undefined function described previously in electron micrographs, is shown to be highly pigmented by absorption and fluorescence light microscopy. Absorption spectra of the lamellar organelle and the chloroplasts of Hymenomonas are presented. In comparison with the chloroplast the lamellar body appears to have an equal concentration of chlorophyll α and nearly three times the concentration of the 490 nm absorbing carotenoid. Fluorescence in the organelle is initially red as in the chloroplast; this is quickly replaced by an intense yellow emission. The rate at which the red fluorescence is replaced by the yellow is oxidation dependent and is quite rapid in the high intensity of the exciting light required for fluorescence micrographs. Possible roles of the organelle in cell metabolism or photochemistry which are considered and evaluated include: photosynthetic organelle, coccolithogenic organelle, symbiont and lysosome.     

Effects of osmotic preconditioning on nuclear replication activity in seeds of pepper (Capsicum annuum)

Routing of cytosolically synthesized precursor proteins into chloroplasts is a specific process which involves a multitude of soluble and membrane components. In this review we wil1 focus on early events of the translocation pathway of nuclear coded plastidic precursor proteins and compare import routes for polypeptide of the outer chloroplast envelope to that of internal chloroplast compartments. A number of proteins housed in the chloroplast envelopes have been implied to be involved in the translocation process, but so far a certain function has not been assigned to any of these proteins. The only exception could be an envelope localized hsc 70 homologue which could retain the import competence of a precursor protein in transit into the organelle.     

The role of lipids in plastid protein transport

The elaborate compartmentalization of plant cells requires multiple mechanisms of protein targeting and trafficking. In addition to the organelles found in all eukaryotes, the plant cell contains a semi-autonomous organelle, the plastid. The plastid is not only the most active site of protein transport in the cell, but with its three membranes and three aqueous compartments, it also represents the most topologically complex organelle in the cell. The chloroplast contains both a protein import system in the envelope and multiple protein export systems in the thylakoid. Although significant advances have identified several proteinaceous components of the protein import and export apparatuses, the lipids found within plastid membranes are also emerging as important players in the targeting, insertion, and assembly of proteins in plastid membranes. The apparent affinity of chloroplast transit peptides for chloroplast lipids and the tendency for unsaturated MGDG to adopt a hexagonal II phase organization are discussed as possible mechanisms for initiating the binding and/or translocation of precursors to plastid membranes. Other important roles for lipids in plastid biogenesis are addressed, including the spontaneous insertion of proteins into the outer envelope and thylakoid, the role of cubic lipid structures in targeting and assembly of proteins to the prolamellar body, and the repair process of D1 after photoinhibition. The current progress in the identification of the genes and their associated mutations in galactolipid biosynthesis is discussed. Finally, the potential role of plastid-derived tubules in facilitating macromolecular transport between plastids and other cellular organelles is discussed.     

Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast

In contrast to animal and fungal cells, green plant cells contain one or multiple chloroplasts, the organelle(s) in which photosynthetic reactions take place. Chloroplasts are believed to have originated from an endosymbiotic event and contain DNA that codes for some of their proteins. Most chloroplast proteins are encoded by the nuclear genome and imported with the help of sorting signals that are intrinsic parts of the polypeptides. Here, we show that a chloroplast-located protein in higher plants takes an alternative route through the secretory pathway, and becomes N-glycosylated before entering the chloroplast.     

Emerging roles of the chloroplast outer envelope membrane

The chloroplast is essential for the viability of plants. It is enclosed by a double-membrane envelope that originated from the outer and plasma membranes of a cyanobacterial endosymbiont. Chloroplast biogenesis depends on binary fission and import of nuclear-encoded proteins. Our understanding of the mechanisms and evolutionary origins of these processes has been greatly advanced by recent genetic and biochemical studies on envelope-localized multiprotein machines. Furthermore, the latest studies on outer envelope proteins have provided molecular insights into organelle movement and membrane lipid remodeling, activities that are vital for plant survival under diverse environmental conditions. Ongoing and future research on the chloroplast outer envelope should add to our knowledge of organelle biology and the evolution of eukaryotic cells.     

The Chloroplast Outer Envelope Membrane： The Edge of Liaht and Excitement

The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites including nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous phases, the cytoplasm and the chloroplast stroma. The chloroplast envelope is also the location where the biosynthesis and accumulation of various lipids take place. By contrast to the inner membrane, which contains a number of specific transporters and acts as the permeability barrier, the chloroplast outer membrane has often been considered a passive compartment derived from the phagosomal membrane. However, the presence of galactoglycerolipids and β-barrel membrane proteins support the common origin of the outer membranes of the chloroplast envelope and extant cyanobacteria. Furthermore, recent progress in the field underlines that the chloroplast outer envelope plays important roles not only for translocation of various molecules, but also for regulation of metabolic activities and signaling processes. The chloroplast outer envelope membrane offers various interesting and challenging questions that are relevant to the understanding of organelle biogenesis, plant growth and development, and also membrane biology in general.     

Chloroplast ribosomal proteins of Chlamydomonas synthesized in the cytoplasm are made as precursors

Polyadenylated RNA from Chlamydomonas was translated in a cell-free rabbit reticulocyte system that employed [35S]methionine. Antibodies made to four chloroplast ribosomal proteins synthesized in the cytoplasm and imported into the organelle were used for indirect immunoprecipitation of the labeled translation products, which were subsequently visualized on fluorographs of SDS gels. The cytoplasmically synthesized chloroplast ribosomal proteins were first seen as precursors with apparent molecular weights of 1,000 to 6,000 greater than their respective mature forms. Processing of the ribosomal protein precursors to mature proteins was affected by adding a postribosomal supernatant that had been extracted from cells of Chlamydomonas. In contrast to the chloroplast ribosomal proteins synthesized in the cytoplasm, two such proteins made within the chloroplast were found to be synthesized in mature form in cell-free wheat germ translation systems programmed with nonpolyadenylated RNA.     

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.     

A gateway to chloroplasts - protein translocation and beyond

The biogenesis of chloroplasts requires the coordinated interplay with the nucleus and the cytoplasm. The majority of chloroplast proteins are encoded by the nuclear genome and must be faithfully and efficiently delivered to the organelle upon completion of translation in the cytosol. This high-fidelity targeting is accomplished by specific chloroplast targeting signal peptides. Several cytoplasmic factors recognise, modify, and bind this targeting sequence, and deliver the preproteins to the chloroplast translocation machinery. The multisubunit translocation complex at the outer envelope contains receptor proteins, a translocation channel, and accessory subunits. Complete import into the stroma utilizes both outer and inner envelope translocons and molecular chaperones in the intermembrane space and in the stroma. The entire import process appears to be regulated by phosphorylation, nucleotide binding, and hydrolysis. Recent evidence indicates that several subunits of the chloroplast import machinery may have evolved from cyanobacterial ancestors.     

Protein import into chloroplasts: new aspects of a well-known topic

Protein import into plant chloroplasts is a fascinating topic that is being investigated by many research groups. Since the majority of chloroplast proteins are synthesised as precursor proteins in the cytosol, they have to be posttranslationally imported into the organelle. For this purpose, most preproteins are synthesised with an N-terminal presequence, which is both necessary and sufficient for organelle recognition and translocation initiation. The import of preproteins is facilitated by two translocation machineries in the outer and inner envelope of chloroplasts, the Toc and Tic complexes, respectively. Translocation of precursor proteins across the envelope membrane has to be highly regulated to react to the metabolic requirements of the organelle. The aim of this review is to summarise the events that take place at the translocation machineries that are known so far. In addition, we focus in particular on alternative import pathways and the aspect of regulation of protein transport at the outer and inner envelope membrane.     

A Survey of Chloroplast Protein Kinases and Phosphatases in Arabidopsis thaliana

Protein phosphorylation is a major mode of regulation of metabolism, gene expression and cell architecture. In chloroplasts, reversible phosphorylation of proteins is known to regulate a number of prominent processes, for instance photosynthesis, gene expression and starch metabolism. The complements of the involved chloroplast protein kinases (cpPKs) and phosphatases (cpPPs) are largely unknown, except 6 proteins (4 cpPKs and 2 cpPPs) which have been experimentally identified so far. We employed combinations of programs predicting N-terminal chloroplast transit peptides (cTPs) to identify 45 tentative cpPKs and 21 tentative cpPPs. However, test sets of 9 tentative cpPKs and 13 tentative cpPPs contain only 2 and 7 genuine cpPKs and cpPPs, respectively, based on experimental subcellular localization of their N-termini fused to the reporter protein RFP. Taken together, the set of enzymes known to be involved in the reversible phosphorylation of chloroplast proteins in A. thaliana comprises altogether now 6 cpPKs and 9 cpPPs, the function of which needs to be determined in future by functional genomics approaches. This includes the calcium-regulated PK CIPK13 which we found to be located in the chloroplast, indicating that calcium-dependent signal transduction pathways also operate in this organelle.Key Words: Arabidopsis thaliana, chloroplast, chloroplast transit peptide, protein kinase, protein phosphatase, protein phosphorylation, proteomics.     

Calcium regulation of chloroplast protein import

The majority of chloroplast proteins is nuclear-encoded and therefore synthesized on cytosolic ribosomes. In order to enter the chloroplast, these proteins have to cross the double-membrane surrounding the organelle. This is achieved by means of two hetero-oligomeric protein complexes in the outer and inner envelope, the Toc and Tic translocon. The process of chloroplast import is highly regulated on both sides of the envelope membranes. Our studies indicate the existence of an undescribed mode of control for this process so far, at the same time providing further evidence that the chloroplast is integrated into the calcium-signalling network of the cell. In pea chloroplasts, the calmodulin inhibitor Ophiobolin A as well as the calcium ionophores A23187 and Ionomycin affect the translocation of those chloroplast proteins that are imported with an N-terminal cleavable presequence. Import of these proteins is inhibited in a concentration-dependent manner. Addition of external calmodulin or calcium can counter the effect of these inhibitors. Translocation of chloroplast proteins that do not possess a cleavable transit peptide, that is outer envelope proteins or the inner envelope protein Tic32, is not affected. These results suggest that the import of a certain subset of chloroplast proteins is regulated by calcium. Our studies furthermore indicate that this regulation occurs downstream of the Toc translocon either within the intermembrane space or at the inner envelope translocon. A potential promoter of the calcium regulation is calmodulin, a protein well known as part of the plant's calcium signalling system.