Membrane insertion of the chloroplast outer envelope protein, Toc34:constrains for insertion and topology

Qbadou, Soumya; Tien, R.; Soll, Jürgen; Schleiff, Enrico

doi:10.1242/jcs.00291

Cited by 41 publications

(39 citation statements)

References 46 publications

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“…A model was proposed that Toc159 functions as a soluble receptor that shuttles from the cytosol to the chloroplasts (17); however, the experimental support for this idea is currently missing, and other ideas are also feasible (e.g., a soluble pool exists because Toc159 might have a high turnover rate). Import of the soluble form of Toc159 depends on guanosine nucleotide binding (36,37), which might induce a favorable conformation for translocation, similar to the GTP dependence of Toc34 import (26). Furthermore, the interaction between the G domains of Toc34 and Toc159 also stimulates Toc159 insertion into the outer envelope membrane.…”

Section: Discussionmentioning

confidence: 96%

A GTP-driven motor moves proteins across the outer envelope of chloroplasts

Schleiff

Jelic

Soll

2003

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

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110

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The translocation of proteins across cellular membranes is a key mechanistic problem for every cell. The preprotein translocon at the chloroplast outer envelope is responsible for precursor protein recognition and translocation across the outer envelope. We have reconstituted the translocation process into proteoliposomes from single subunits or by using the purified translocon. Precursor proteins are recognized by the Toc34 receptor in an initial GTPdependent process. Translocation across the plane of the membrane then occurs through the Toc75 channel in a GTP-dependent process. Correspondingly, GTP hydrolysis of Toc proteoliposomes is 100-fold enhanced in the presence of preprotein. Complete translocation is demonstrated by processing of the precursor form to the mature form by the stromal processing peptidase and by protease resistance of the imported protein. Molecular chaperones are not involved in this translocation event. We show that Toc159 acts as a GTP-driven motor in a sewing-machine-like mechanism.

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Section: Discussionmentioning

confidence: 96%

A GTP-driven motor moves proteins across the outer envelope of chloroplasts

Schleiff

Jelic

Soll

2003

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

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110

124

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“…In vitro import and intracellular localization of dsRED fusions in chloroplasts. For in vitro import assays, 35 S-labelled proteins were synthesized and used for import experiments and were detected after subfractionation of chloroplasts 29,30 . For intracellular localization of the dsRED fusion, a complementary DNA fragment coding for the first 134 amino-acid residues of STN8 was fused 5 0 to dsRED and inserted into the vector pLEELA (Invitrogen), placing it under the transcriptional control of the cauliflower mosaic virus 35S promoter.…”

Section: Methodsmentioning

confidence: 99%

Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases

Bonardi

Pesaresi

Becker

et al. 2005

Nature

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409

496

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Illumination changes elicit modifications of thylakoid proteins and reorganization of the photosynthetic machinery. This involves, in the short term, phosphorylation of photosystem II (PSII) and light-harvesting (LHCII) proteins. PSII phosphorylation is thought to be relevant for PSII turnover 1,2 , whereas LHCII phosphorylation is associated with the relocation of LHCII and the redistribution of excitation energy (state transitions) between photosystems 3,4 . In the long term, imbalances in energy distribution between photosystems are counteracted by adjusting photosystem stoichiometry 5,6 . In the green alga Chlamydomonas and the plant Arabidopsis, state transitions require the orthologous protein kinases STT7 and STN7, respectively 7,8 . Here we show that in Arabidopsis a second protein kinase, STN8, is required for the quantitative phosphorylation of PSII core proteins. However, PSII activity under high-intensity light is affected only slightly in stn8 mutants, and D1 turnover is indistinguishable from the wild type, implying that reversible protein phosphorylation is not essential for PSII repair. Acclimation to changes in light quality is defective in stn7 but not in stn8 mutants, indicating that short-term and long-term photosynthetic adaptations are coupled. Therefore the phosphorylation of LHCII, or of an unknown substrate of STN7, is also crucial for the control of photosynthetic gene expression.STT7 and STN7 are orthologous protein kinases required for LHCII phosphorylation and for state transitions in Chlamydomonas and Arabidopsis, respectively 7,8 . In Arabidopsis, another STT7/STN7-like protein (STN8) exists that is not required for state transitions 8 . STN8 is located in the chloroplast, as shown by in vivo subcellular localization of its amino-terminal region fused to the dsRED protein and by the import of, and transit peptide removal from, STN8 translated in vitro (Fig. 1a, b). Chloroplast subfractionation after import revealed that the protein is associated, like STT7 and STN7, with thylakoids ( Fig. 1c) (refs 7, 8).Insertion mutants for STN8 and STN7 were obtained from the Salk collection 9 , and for each gene two independent mutant alleles lacking the respective transcript were identified (Supplementary Fig. S1). The stn7 stn8 double mutant was generated by crossing stn7 and stn8 single knockouts and screening the resulting F 2 generation for homozygous double mutants. All mutants were indistinguishable from the wild type with regard to the timing of seed germination and growth rate in the greenhouse ( Supplementary Fig. S1). In stn7 and stn7 stn8 mutants, a slight decrease in the levels of neoxanthin, lutein and total chlorophyll was found (Supplementary Table S1). These subtle changes can be attributed to a minor decrease in LHCII content, not detectable by polyacrylamide-gel electrophoresis (PAGE) analysis ( Supplementary Fig. S2).Photosynthetic electron flow, measured on the basis of chlorophyll fluorescence, was not altered in the mutants (Supplementary Table S2). State transitions w...

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“…Fusion of Toc34 at the N terminus of EGFP (i.e., BsToc34-EGFP), however, produced punctate structures of variable size ( Figure 2E). Since the C-terminal transmembrane domain of Toc34 and its flanking sequences contain important sorting information and the protein is integrated into the outer chloroplast membrane with an N out -C in topology (Qbadou et al, 2003;Dhanoa et al, 2010), the addition of a bulky tag (i.e., 27 kD) to the C terminus might result in mistargeting of the fusion protein and formation of insoluble aggregates. Fusion constructs of Toc159 and Toc132 with a C-terminal EGFP (i.e., BsToc159-EGFP and BsToc132-EGFP) did not produce any fluorescent signal in B. sinuspersici protoplasts in repeated experiments using different sequencing-verified clones.…”

Section: Toc159 and Toc34 Demonstrated Distinctive Subcellular Localimentioning

confidence: 99%

“…Similarly to other tail-anchored proteins, the targeting of Toc34 to the chloroplast envelope is dependent on its transmembrane domain plus the flanking C terminus (Chen and Schnell, 1997;Li and Chen, 1997;Qbadou et al, 2003;Dhanoa et al, 2010). Li and Chen (1997) demonstrated that this C-terminal region is sufficient for targeting and efficient insertion of a passenger protein, regardless of the location of fusion at the N or C terminus.…”

Section: The Toc159 Targeting Pathway Awaited Discovery Of a Sorting mentioning

confidence: 99%

“…Depending on the location of the transmembrane helix at the N or C terminus, these proteins are commonly referred to as signal-anchored proteins (e.g., OEP7/14 and OEP64) and tailanchored proteins (e.g., OEP9 and Toc34), respectively. The targeting of these proteins and their efficient insertion with the correct topology depend on the information embedded in the transmembrane domains and the flanking charged regions, whereas the cytosolic GTPase domain of Toc34 also plays an essential role (Chen and Schnell, 1997;Schleiff et al, 2001;Qbadou et al, 2003;Hofmann and Theg, 2005;Dhanoa et al, 2010). Although two previous studies documented that the GTPase domain mediates the targeting and insertion of Toc159 (Bauer et al, 2002;Smith et al, 2002a), this domain per se presumably does not contain any sorting signal, and the targeting specificity of this receptor to the chloroplast envelope has not been elucidated.…”

Section: Introductionmentioning

confidence: 99%

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A Transit Peptide–Like Sorting Signal at the C Terminus Directs the Bienertia sinuspersici Preprotein Receptor Toc159 to the Chloroplast Outer Membrane

Lung

Chuong

2012

Plant Cell

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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.

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Membrane insertion of the chloroplast outer envelope protein, Toc34:constrains for insertion and topology

Cited by 41 publications

References 46 publications

A GTP-driven motor moves proteins across the outer envelope of chloroplasts

A GTP-driven motor moves proteins across the outer envelope of chloroplasts

Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases

A Transit Peptide–Like Sorting Signal at the C Terminus Directs the Bienertia sinuspersici Preprotein Receptor Toc159 to the Chloroplast Outer Membrane

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