A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle

Lübeck, Jens; Heins, Lisa; Soll, Jürgen

doi:10.1083/jcb.137.6.1279

Cited by 78 publications

(89 citation statements)

References 47 publications

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“…As a model substrate we used the integral translocon subunit Tic110, which had been shown before to form such a stromal intermediate [13]. Tic110 is anchored to the inner envelope by two hydrophobic a-helices located at the N-terminus of the protein.…”

Section: Resultsmentioning

confidence: 99%

“…In chloroplasts very little is known about the molecular characteristics of those pathways, though both seem to exist. While some inner envelope preproteins seem to import without a soluble stromal intermediate, like the phosphate-triose-phosphate-translocator [11], others have been shown to accumulate in the stroma, like Tic110 or Tic40, before being inserted into the inner envelope [12,13].…”

Section: Introductionmentioning

confidence: 99%

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Requirements for a conservative protein translocation pathway in chloroplasts

Vojta¹,

Soll²,

Bölter³

2007

FEBS Letters

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The chloroplast inner envelope translocon subunit Tic110 is imported via a soluble stromal translocation intermediate. In this study an in-organellar import system is established which allows for an accumulation of this intermediate in order to analyze its requirements for reexport. All results demonstrate that the re-export of Tic110 from the soluble intermediate stage into the inner envelope requires ATP hydrolysis, which cannot be replaced by other NTPs. Furthermore, the molecular chaperone Hsp93 seems prominently involved in the reexport pathway of Tic110, because other stromal intermediates like that of the oxygen evolving complex subunit OE33 (iOE33) en route to the thylakoid lumen interacts preferentially with Hsp70.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Requirements for a conservative protein translocation pathway in chloroplasts

Vojta¹,

Soll²,

Bölter³

2007

FEBS Letters

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“…Our goal was to examine the hypotheses that Tic110 functions primarily as a docking site for stromal import factors (12,20), an inner membrane-anchored protein of the intermembrane space that mediates Toc and Tic translocon interactions (13,19), or a ␤-barrel membrane channel for the Tic translocon (23). Our analyses demonstrate that the carboxyl-terminal 97.5 kDa of atTic110 folds into a soluble domain when expressed both in E. coli (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In the first model, the carboxyl-terminal region of pea Tic110, psTic110, was predicted to extend into the intermembrane space between the outer and inner envelope membranes and thereby mediate the interactions between the Toc and Tic complexes during the translocation reaction (13,19). However, this model was discounted by detailed topology mapping studies (20).…”

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confidence: 99%

atTic110 Functions as a Scaffold for Coordinating the Stromal Events of Protein Import into Chloroplasts

Inaba

Alvarez-Huerta

et al. 2003

Journal of Biological Chemistry

117

148

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The translocon of the inner envelope membrane of chloroplasts (Tic) mediates the late events in the translocation of nucleus-encoded preproteins into chloroplasts. Tic110 is a major integral membrane component of active Tic complexes and has been proposed to function as a docking site for translocation-associated stromal factors and as a component of the protein-conducting channel. To investigate the various proposed functions of Tic110, we have investigated the structure, topology, and activities of a 97.5-kDa fragment of Arabidopsis Tic110 (atTic110) lacking only the amino-terminal transmembrane segments. The protein was expressed both in Escherichia coli and Arabidopsis as a stable, soluble protein with a high ␣-helical content. Binding studies demonstrate that a region of the atTic110-soluble domain selectively associates with chloroplast preproteins at the late stages of membrane translocation. These data support the hypothesis that the bulk of Tic110 extends into the chloroplast stroma and suggest that the domain forms a docking site for preproteins as they emerge from the Tic translocon.Chloroplast biogenesis is dependent upon the import of ϳ3000 different nucleus-encoded proteins (1). The majority of these proteins are synthesized as preproteins carrying an amino-terminal transit peptide that serves as the essential signal for targeting to the organelle. The transit peptide is recognized by receptor components of the translocon at the outer envelope membrane of chloroplasts (Toc), and a GTP-regulated switch initiates translocation through the protein-conducting channel of the Toc complex. At this stage, the Toc complex associates with the translocon at the inner envelope membrane (Tic), and this Toc-Tic supercomplex mediates the direct transport of the preproteins from the cytoplasm into the chloroplast stroma (2).Although many mechanistic details remain to be defined, the activities of the Toc components have been extensively investigated. Two membrane-associated GTPases, Toc159 and Toc34/33, mediate transit peptide recognition and regulate the initiation of translocation (3-7). The Toc GTPases form a complex with Toc75, an integral membrane protein that, along with Toc159, constitutes a major component of the protein-conducting channel (1, 8 -10).In contrast to the Toc complex, the activities and functions of the Tic components are less well defined. The biochemical analysis of Tic function has been complicated by the fact that assembly of functional Tic complexes is dynamic and occurs in response to preprotein translocation (11). Therefore, the isolation of a stable Tic complex has thus far been elusive. Tic110 was the first Tic component identified and represents a major component of active Tic complexes (12, 13). It is an integral inner envelope membrane protein, and structural predictions suggest that it consists of two predicted transmembrane helices at its extreme amino terminus and a 97.5-kDa carboxyl-terminal region that is largely hydrophilic. Tic110 transiently associates with at least five o...

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“…Interaction Partners of Tic110 -To find interaction partners of the N terminus of Tic110, a hybrid protein of mature Tic110 (amino acids 39 -269) and a C-terminal fusion with mSSU-His 6 (35) or with a His 6 tag alone were overexpressed in E. coli as inclusion bodies, dissolved in 20 mM Tris/HCl, pH 8.0, 250 mM NaCl, and 8 M urea, immobilized onto nickel-nitrilotriacetic acid matrix, and washed with the above buffer. After washing, the refolding of the protein on the matrix was achieved by creating refolding conditions on the matrix with a linear gradient of 8-0 M urea in 20 mM Tris/HCL, pH 8.0 (36).…”

Section: Tic32 Is Essential In Chloroplast Biogenesis 34757mentioning

confidence: 99%

Tic32, an Essential Component in Chloroplast Biogenesis

Hörmann¹,

Küchler²,

Sveshnikov³

et al. 2004

Journal of Biological Chemistry

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Chloroplast protein import across the inner envelope is facilitated by the translocon of the inner envelope of chloroplasts (Tic). Here we have identified Tic32 as a novel subunit of the Tic complex. Tic32 can be purified from solubilized inner envelope membranes by chromatography on Tic110 containing affinity matrix. Co-immunoprecipitation experiments using either Tic32 or Tic110 antisera indicated a tight association between these polypeptides as well as with other Tic subunits, e.g. Tic40, Tic22, or Tic62, whereas the outer envelope protein Toc75 was not found in this complex. Chemical cross-linking suggests that Tic32 is involved late in the overall translocation process, because both the precursor form as well as the mature form of Rubisco small subunit can be detected. We were unable to isolate Arabidopsis null mutants of the attic32 gene, indicating that Tic32 is essential for viability. Deletion of the attic32 gene resulted in early seed abortion because the embryo was unable to differentiate from the heart stage to the torpedo stage. The homology of Tic32 to short-chain dehydrogenases suggests a dual role of Tic32 in import, one as a regulatory component and one as an important subunit in the assembly of the entire complex.Choroplasts must import most of their protein constituents from the cytosol in a posttranslational process (1-4). Cytosolically synthesized precursor proteins are generally made with an N-terminal transit peptide that is both necessary and sufficient for chloroplast recognition and translocation initiation. Translocation across the outer and inner envelopes of chloroplasts is achieved by the Toc 1 and Tic complexes, respectively (translocon at the outer/inner envelope of chloroplasts). Upon import, the targeting peptide is removed by a stromal processing peptidase, and the mature protein is further sorted and assembled into active structures (5).Preproteins are recognized in a GTP-dependent manner by the Toc34 receptor at the chloroplast surface (6 -8). The GTPase activity of Toc34 is greatly stimulated by precursor binding, and upon GTP hydrolysis the preprotein is released from Toc34 GDP to Toc159 (9). Toc159, another GTP-dependent protein, interacts with the preprotein in a manner that also involves GTP hydrolysis (6, 10, 11). Toc159 facilitates the translocation of the preprotein through the import channel Toc75 (12, 13). The preprotein then engages the Tic complex to move across the inner envelope in an ATP-requiring fashion (14 -16). Several subunits of the Tic complex have been identified. Tic110, which is the second most abundant protein in the inner envelope (17-20), can form an aqueous ion channel in vitro and is therefore thought to form the translocation pore (21). However, because of different proposals about the topology and structure of Tic110 (21, 22), the exact function of Tic110 is not resolved. In addition, Tic110 could interact with the molecular chaperones cpn60 and Hsp93 on the stromal face of the membrane. It could also act as a chaperone recruitment factor (17,...

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A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle

Cited by 78 publications

References 47 publications

Requirements for a conservative protein translocation pathway in chloroplasts

Requirements for a conservative protein translocation pathway in chloroplasts

atTic110 Functions as a Scaffold for Coordinating the Stromal Events of Protein Import into Chloroplasts

Tic32, an Essential Component in Chloroplast Biogenesis

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