Functional analysis of the protein-interacting domains of chloroplast SRP43.
(9/207)
The chloroplast signal recognition particle (cpSRP) consists of an evolutionarily conserved 54-kDa subunit (cpSRP54) and a dimer of a unique 43-kDa subunit (cpSRP43). cpSRP binds light-harvesting chlorophyll proteins (LHCPs) to form a cpSRP/LHCP transit complex, which targets LHCP to the thylakoid membrane. Previous studies showed that transit complex formation is mediated through the binding of the L18 domain of LHCP to cpSRP43. cpSRP43 is characterized by a four-ankyrin repeat domain at the N terminus and two chromodomains at the C terminus. In the present study we used the yeast two-hybrid system and in vitro binding assays to analyze the function of different domains of cpSRP43 in protein complex formation. We report here that the first ankyrin repeat binds to the 18-amino acid domain on LHCP that binds to cpSRP43, whereas the third and fourth ankyrin repeats are involved in the dimerization of cpSRP43. We show further that the interaction of cpSRP43 with cpSRP54 is mediated via binding of the methionine-rich domain of cpSRP54 to the C-terminally located chromodomains of cpSRP43. Both chromodomains contain essential elements for binding cpSRP54, indicating that the closely spaced chromodomains together create a single binding site for cpSRP54. In addition, our data demonstrate that the interaction of cpSRP54 with the chromodomains of cpSRP43 is enhanced indirectly by the dimerization motif of cpSRP43. (+info)
Functional characterization of recombinant chloroplast signal recognition particle.
(10/207)
The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting. We have expressed and purified the two components of the Arabidopsis thaliana chloroplast signal recognition particle (cpSRP) involved in post-translational transport: cpSRP54 and the chloroplast-specific protein, cpSRP43. Recombinant cpSRP supports the efficient in vitro insertion of pea preLhcb1 into isolated thylakoid membranes. Recombinant cpSRP is a stable heterodimer with a molecular mass of approximately 100 kDa as determined by analytical ultracentrifugation, gel filtration analysis, and dynamic light scattering. The interactions of the components of the recombinant heterodimer and pea preLhcb1 were probed using an immobilized peptide library (pepscan) approach. These data confirm two previously reported interactions with the L18 region and the third transmembrane helix of Lhcb1 and suggest that the interface of the cpSRP43 and cpSRP54 proteins is involved in substrate binding. Additionally, cpSRP components are shown to recognize peptides from the cleavable, N-terminal chloroplast transit peptide of preLhcb1. The interaction of cpSRP43 with cpSRP54 was probed in a similar experiment with a peptide library representing cpSPR54. The C terminus of cpSRP54 is essential for the formation of the stable cpSRP complex and cpSPR43 interacts with distinct regions of the M domain of cpSRP54. (+info)
Compartmentation of metabolism within mitochondria and plastids.
(11/207)
An important component of metabolic regulation is compartmentation and specialization. Subcellular compartmentation and the role of individual organelles is well studied, though less consideration has been given to the extent to which organelles differ between cells. Organelles extracted from whole tissue homogenates will have generally originated from a range of cell types. This review describes and assesses the regulation of metabolic activity in plants at both the cellular and subcellular level by considering specialization of mitochondria and plastids. (+info)
The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts?
(12/207)
The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b(6)/f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the DeltapH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH(2)-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of DeltapH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways. (+info)
Leaf-specific upregulation of chloroplast translocon genes by a CCT motif-containing protein, CIA 2.
(13/207)
Chloroplasts are a major destination of protein traffic within leaf cells. Protein import into chloroplasts is mediated by a set of translocon complexes at the chloroplast envelope. Current data indicate that the expression of translocon genes is regulated in a tissue-specific manner, possibly to accommodate the higher import demand of chloroplasts in leaves and the lower demand of plastids in other tissues. We have designed a transgene-based positive screen to isolate mutants disrupted in protein import into plastids. The first locus we isolated, CIA2, encodes a protein containing a motif conserved within the CCT family of transcription factors. Biochemical analysis indicates that CIA2 is responsible for specific upregulation of the translocon genes atToc33 and atToc75 in leaves. Identification of CIA2 provides new insights into the tissue-specific regulation of translocon gene expression. (+info)
The paradox of plastid transit peptides: conservation of function despite divergence in primary structure.
(14/207)
Transit peptides are N-terminal extensions that facilitate the targeting and translocation of cytosolically synthesized precursors into plastids via a post-translational mechanism. With the complete Arabidopsis genome in hand, it is now evident that transit peptides direct more than 3500 different proteins into the plastid during the life of a typical plant. Deciphering a common mechanism for how this multitude of targeting sequences function has been hampered by the realization that at a primary sequence level, transit peptides are highly divergent in length, composition, and organization. This review addresses recent findings on several of the diverse functions that transit peptides must perform, including direct interaction with envelope lipids, association with a cis-acting guidance complex, recognition by envelope receptors, insertion into the Toc/Tic translocon, interaction with molecular motors, and finally, recognition/cleavage by the stromal processing peptidase. In addition to higher plants, transit peptides also direct the import of proteins into complex plastids derived from secondary endosymbiosis. An emerging concept suggests that transit peptides contain multiple domains that provide either distinct or possibly overlapping functions. Although still poorly characterized, evolutionary processes could yield transit peptides with alternative domain organizations. (+info)
Post-translational protein translocation into thylakoids by the Sec and DeltapH-dependent pathways.
(15/207)
Two distinct protein translocation pathways that employ hydrophobic signal peptides function in the plant thylakoid membrane. These two systems are precursor specific and distinguished by their energy and component requirements. Recent studies have shown that one pathway is homologous to the bacterial general export system called Sec. The other one, called the DeltapH-dependent pathway, was originally considered to be unique to plant thylakoids. However, it is now known that homologous transport systems are widely present in prokaryotes and even present in archaea. Here we review these protein transport pathways and discuss their capabilities and mechanisms of operation. (+info)
Molecular chaperones involved in chloroplast protein import.
(16/207)
Transport of cytoplasmically synthesized precursor proteins into chloroplasts, like the protein transport systems of mitochondria and the endoplasmic reticulum, appears to require the action of molecular chaperones. These molecules are likely to be the sites of the ATP hydrolysis required for precursor proteins to bind to and be translocated across the two membranes of the chloroplast envelope. Over the past decade, several different chaperones have been identified, based mainly on their association with precursor proteins and/or components of the chloroplast import complex, as putative factors mediating chloroplast protein import. These factors include cytoplasmic, chloroplast envelope-associated and stromal members of the Hsp70 family of chaperones, as well as stromal Hsp100 and Hsp60 chaperones and a cytoplasmic 14-3-3 protein. While many of the findings regarding the action of chaperones during chloroplast protein import parallel those seen for mitochondrial and endoplasmic reticulum protein transport, the chloroplast import system also has unique aspects, including its hypothesized use of an Hsp100 chaperone to drive translocation into the organelle interior. Many questions concerning the specific functions of chaperones during protein import into chloroplasts still remain that future studies, both biochemical and genetic, will need to address. (+info)