Preprotein recognition by the Toc complex (original) (raw)
Related papers
The Roles of Toc34 and Toc75 in Targeting the Toc159 Preprotein Receptor to Chloroplasts
Journal of Biological Chemistry, 2003
The Toc complex at the outer envelope of chloroplasts initiates the import of nuclear-encoded preproteins from the cytosol into the organelle. The core of the Toc complex is composed of two receptor GTPases, Toc159 and Toc34, as well as Toc75, a -barrel membrane channel. Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, suggesting that assembly of the Toc complex is dynamic. In the present study, we used the Arabidopsis thaliana orthologs of Toc159 and Toc34, atToc159 and atToc33, respectively, to investigate the requirements for assembly of the trimeric Toc complex. In addition to its intrinsic GTPase activity, we demonstrate that integration of at-Toc159 into the Toc complex requires atToc33 GTPase activity. Additionally, we show that the interaction of the two GTPase domains stimulates association of the membrane anchor of atToc159 with the translocon. Finally, we employ reconstituted proteoliposomes to demonstrate that proper insertion of the receptor requires both Toc75 and Toc34. Collectively these data suggest that Toc34 and Toc75 act sequentially to mediate docking and insertion of Toc159 resulting in assembly of the functional translocon.
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.
The Plant Cell, 2012
Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C 4 species with dimorphic chloroplasts in individual chlorenchyma cells. Fluorescent protein tagging and immunogold studies revealed that the localization patterns of Toc159 were distinctive from those of Toc34, suggesting different targeting pathways. Bioinformatics analyses indicated that the C-terminal tails (CTs) of Toc159 possess physicochemical and structural properties of chloroplast transit peptides (cTPs). These results were further confirmed by fluorescent protein tagging, which showed the targeting of CT fusion proteins to the chloroplast surface. The CT of Bs Toc159 in reverse orientation functioned as a cleavable cTP that guided the fluorescent protein to the stroma. Moreover, a Bs Toc34 mutant protein was retargeted to the chloroplast envelope using the CTs of Toc159 or reverse sequences of other cTPs, suggesting their conserved functions. Together, our data show that the C terminus and the central GTPase domain represent a novel dual domain-mediated sorting mechanism that might account for the partitioning of Toc159 between the cytosol and the chloroplast envelope for preprotein recognition.
Phospholipids Can Switch the GTPase Substrate Preference of a GTPase-activating Protein
Journal of Biological Chemistry, 2003
The major cellular inhibitors of the small GTPases of the Ras superfamily are the GTPase-activating proteins (GAPs), which stimulate the intrinsic GTP hydrolyzing activity of GTPases, thereby inactivating them. The catalytic activity of several GAPs is reportedly inhibited or stimulated by various phospholipids and fatty acids in vitro, indicating a likely physiological role for lipids in regulating small GTPases. We find that the p190 RhoGAP, a potent GAP for the Rho and Rac GTPases, is similarly sensitive to phospholipids. Interestingly, however, several of the tested phospholipids were found to effectively inhibit the RhoGAP activity of p190 but stimulate its RacGAP activity. Thus, phospholipids have the ability to "switch" the GTPase substrate preference of a GAP, thereby providing a novel regulatory mechanism for the small GTPases.
A Novel Human Tocopherol-associated Protein: Cloning, in-Vitro Expression and Characterization
Biochemical Society Transactions, 2000
Vitamin E (␣-tocopherol) is an essential dietary nutrient for humans and animals. The mechanisms involved in cellular regulation as well as in the preferential cellular and tissue accumulation of ␣-tocopherol are not yet well established. We previously reported (Stocker, A., Zimmer, S., Spycher, S. E., and Azzi, A. (1999) IUBMB Life 48, 49-55) the identification of a novel 46-kDa tocopherol-associated protein (TAP) in the cytosol of bovine liver. Here, we describe the identification, the molecular cloning into Escherichia coli, and the in vitro expression of the human homologue of bovine TAP, hTAP. This protein appears to belong to a family of hydrophobic ligand binding proteins, which have the CRAL (cis-retinal binding motif) sequence in common. By using a biotinylated ␣-tocopherol derivative and the IASys resonant mirror biosensor, the purified recombinant protein was shown to bind tocopherol at a specific binding site with K d 4.6 ؋ 10 ؊7 M. Northern analyses showed that hTAP mRNA has a size of approximately 2800 base pairs and is ubiquitously expressed. The highest amounts of hTAP message are found in liver, brain, and prostate. In conclusion, hTAP has sequence homology to proteins containing the CRAL TRIO structural motif. TAP binds to ␣-tocopherol and biotinylated tocopherol, suggesting the existence of a hydrophobic pocket, possibly analogous to that of SEC14.
Phospholipase Cγ1 controls surface expression of TRPC3 through an intermolecular PH domain
Nature, 2005
Many ion channels are regulated by lipids 1-3 , but prominent motifs for lipid binding have not been identified in most ion channels. Recently, we reported that phospholipase Cg1 (PLC-g1) binds to and regulates TRPC3 channels 4 , components of agonist-induced Ca 21 entry into cells. This interaction requires a domain in PLC-g1 that includes a partial pleckstrin homology (PH) domain-a consensus lipid-binding and proteinbinding sequence 5,6 . We have developed a gestalt algorithm to detect hitherto 'invisible' PH and PH-like domains, and now report that the partial PH domain of PLC-g1 interacts with a complementary partial PH-like domain in TRPC3 to elicit lipid binding and cell-surface expression of TRPC3. Our findings imply a far greater abundance of PH domains than previously appreciated, and suggest that intermolecular PH-like domains represent a widespread signalling mode.
European Journal of Biochemistry, 1996
To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of E.scherichia coli outer-membrane protein PhoE. It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole,GroPGro) and dioleoylglycerophosphoethanolamine (Ole,GroPEtn), this insertion was mainly driven by hydrophobic forces. Compared with previous results obtained with PhoE signal peptide, the full-length precursor protein does not show the specific interaction with acidic lipids. PrePhoE inserted into a Ole,GroPGro monolayer occupies an area of 28 ? 30 nm2/molecule, which is approximately 10fold larger than the area occupied by the PhoE signal peptide. The purified mature PhoE protein has a lower capacity to insert into Ole,GroPGro and OlezGroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinasc K after interacting with a Ole,GroPGro monolayer. The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers.
The Journal of Cell Biology, 2002
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 bi...
Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane
Journal of …, 1993
The TolA protein is involved in maintaining the integrity of the outer membrane of Escherichia coli, as mutations in toL4 cause the bacteria to become hypersensitive to detergents and certain antibiotics and to leak periplasmic proteins into the medium. This protein also is required for the group A colicins to exert their effects and for many of the filamentous single-stranded bacteriophage to infect the bacterial cell. ToIA is a three-domain protein, with the amino-terminal domain anchoring it to the inner membrane. The helical second domain is proposed to span the periplasmic space to allow the carboxyl-terminal third domain to interact with the outer membrane. A plasmid that allowed the synthesis and transport of the carboxyl-terminal third domain into the periplasmic space was constructed. The presence of an excess of this domain in the periplasm of a wild-type cell resulted in an increased sensitivity to deoxycholate, the release of periplasmic alkaline phosphatase and RNase into the medium, and an increased tolerance to colicins El, E2, E3, and A. There was no effect on the cells' response to colicin D, which depends on TonB instead of ToIA for its action. The presence of the free carboxyl-terminal domain of ToIA in the periplasm in a toUt null mutation did not restore the wild-type phenotype, suggesting that this domain must be part of the intact TolA molecule to perform its function. Our results are consistent with a model in which the carboxyl-terminal domain of ToIA interacts with components in the periplasm or on the inner surface of the outer membrane to function in maintaining the * Corresponding author.