Tic22 Is an Essential Chaperone Required for Protein Import into the Apicoplast

Glaser, Stephanie; Dooren, Giel G. van; Agrawal, Swati; Brooks, Carrie F.; McFadden, Geoffrey I.; Striepen, Boris; Higgins, Matthew K.

doi:10.1074/jbc.m112.405100

Cited by 56 publications

(68 citation statements)

References 35 publications

(67 reference statements)

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“…Tic22 from the apicomplexan Toxoplasma gondii has been shown to have chaperone-like activity in vitro (42), suggesting that Tic22 also may act as an IMS chaperone for translocating precursors in chloroplasts. Mutants lacking both Tic22 homologs in Arabidopsis are import-deficient, and it was recently shown that Tic22 protein levels are up-regulated in plants expressing POTRA-deleted versions of Toc75 (15).…”

Section: Discussionmentioning

confidence: 99%

“…In this assay, insulin B aggregation is induced by the reduction of A chain-B chain disulfide bonds with 20 mM DTT, and aggregation is measured over time by light scattering at 360 nm (42). In our system, chaperone activity was assessed by the ability of the added POTRAs to prevent B chain aggregation and the resulting reduction in light scattering.…”

Section: Potra2-3 Mediates An Interaction With the Model Chloroplastmentioning

confidence: 99%

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The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

O'Neil

Richardson

Paila

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a β-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded β-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana, revealing three tandem polypeptide transportassociated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperonelike activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space.protein import | chloroplast | outer membrane | Toc75 | POTRA domain

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Section: Discussionmentioning

confidence: 99%

Section: Potra2-3 Mediates An Interaction With the Model Chloroplastmentioning

confidence: 99%

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

O'Neil

Richardson

Paila

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…To localize TgTom40, we amplified the 3Ј region with primers 1 and 2 (Table 1) and inserted this into the vector pHA 3 .LIC.DHFR (a kind gift from Michael White, University of South Florida) by ligation-independent cloning (LIC), as described previously (50), before linearization with EcoRV, transfection into ⌬ku80 strain parasites, and selection on pyrimethamine as described (52). To localize TgSam50, we amplified the open reading frame with primers 3 and 4, digested the resulting product with BglII and AvrII, ligated into the vector pBTM 3 (53), transfected into parasites, and selected on phleomycin as described (54). To localize TgTim22, we amplified the 3Ј region with primers 5 and 6, digested the resulting product with BglII and AvrII, ligated into pHH vector, a modified version of the pgCM3 vector described previously (55) that has a selectable marker for mycophenolic acid selection.…”

Section: Methodsmentioning

confidence: 99%

“…To generate a strain expressing the mitochondrial targeting sequence of TgHsp60 fused to c-Myc-tagged mouse DHFR, we digested the TgHsp60 leader sequence from the vector Hsp60-RFP in pBTR (58) with BglII and AvrII, ligated this into the equivalent sites of the vector mDHFR in pBTM 3 (53), transfected into the TgTom22 knockdown cell line, and selected on phleomycin.…”

Section: Methodsmentioning

confidence: 99%

The Import of Proteins into the Mitochondrion of Toxoplasma gondii

Dooren

Yeoh

Striepen

et al. 2016

Journal of Biological Chemistry

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146

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Outside of well characterized model eukaryotes, relatively little is known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular parasites that cause major diseases in humans and animals and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbor a mitochondrion that is essential for parasite survival and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbors homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for parasite survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for parasite survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan parasites.

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“…Cytosolic molecular chaperones, Hsc70 and Hsp90, have been proposed to assist in delivery of preproteins to the TOC receptors, Toc34 and Toc159, and the intrinsic GTPase activities of the receptors initiate outer membrane translocation via the β-barrel membrane channel, Toc75 (2,3). Preprotein translocation occurs simultaneously through the TOC and TIC complexes with the assistance of Tic22, a chaperone in the intermembrane space (6,7), and the putative channel components, Tic20 and Tic21 (2,3). The TIC machinery interacts with three ATP-driven molecular chaperones in the stroma (3,5).…”

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confidence: 99%

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

Inoue

Schnell

2013

Proc. Natl. Acad. Sci. U.S.A.

108

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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.

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Tic22 Is an Essential Chaperone Required for Protein Import into the Apicoplast

Cited by 56 publications

References 35 publications

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

The Import of Proteins into the Mitochondrion of Toxoplasma gondii

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

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