Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug 15;438(1):143-53.
doi: 10.1042/BJ20110448.

OEP61 is a chaperone receptor at the plastid outer envelope

Ottilie von Loeffelholz  1 , Verena Kriechbaumer, Richard A Ewan, Rafal Jonczyk, Susann Lehmann, Jason C Young, Ben M Abell
Affiliations

OEP61 is a chaperone receptor at the plastid outer envelope

Ottilie von Loeffelholz et al. Biochem J. .

Abstract

Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. In the present paper, we identify an uncharacterized protein in Arabidopsis thaliana OEP61 which shares common features with Toc64, and potentially provides an alternative route to the chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type TPR (tetratricopeptide repeat) domain capable of binding molecular chaperones, and a C-terminal TMD (transmembrane domain). Phylogenetic comparisons show sequence similarities between the TPR domain of OEP61 and those of the Toc64 family. Expression of mRNA and protein was detected in all plant tissues, and localization at the chloroplast outer envelope was demonstrated by a combination of microscopy and in vitro import assays. Binding assays show that OEP61 interacts specifically with Hsp70 (heat-shock protein 70) via its TPR clamp domain. Furthermore, OEP61 selectively recognizes chloroplast precursors via their targeting sequences, and a soluble form of OEP61 inhibits chloroplast targeting. We therefore propose that OEP61 is a novel chaperone receptor at the chloroplast outer envelope, mediating Hsp70-dependent protein targeting to chloroplasts.

PubMed Disclaimer

Figures

Figure 1
Figure 1. OEP61 relationships with other TPR clamp proteins
Schematic structure of OEP61 (Swiss-Prot accession number Q8GWM6). The N-terminal TPR domain is defined by PROSITE and a C-terminal TMD is predicted by SMART between amino acids 531 and 553. The alignment file shows the TPR domain of OEP61 with the known chaperone binding TPR clamp domains from Hop (hHop1 and hHop2A; P31948), FKBP5 (hFKBP5; Q13451), FKBP4 (hFKBP4; Q02790), cyclophilin-40 (hCyc40; Q08752), serine/threonine phosphatase 5 (hSTP5; P53041), cyclophilin seven suppressor (scCyc7s; A6ZL97), Tom70 (hTom70; O94826), Tom34 (hTom34C; Q15785) and Unc-45 (hUNC45A; Q9H3U1).
Figure 2
Figure 2. OEP61 is expressed throughout the mature plant
(A) RNA levels of OEP61 relative to actin were measured in rosette leaves, cauline leaves, stems, roots, buds, flowers and siliques from adult plants by qRT-PCR. Results are means ± S.E. (n = 3). (B) Immunoblot against immunoprecipitated OEP61 from young leaves (2 weeks after germination), and rosette leaves, cauline leaves, stems, roots, buds, flowers and siliques from adult plants. Recombinant OEP61 lacking its TMD (OEP61-TM) is used as a comparison.
Figure 3
Figure 3. OEP61 is localized to the outer envelope of chloroplasts
(A) Confocal microscopy image of the YFP-tagged OEP61 construct transiently expressed in tobacco leaves. Its subcellular localization is overlaid with chlorophyll autofluorescence. Areas of significant co-localization (by ImageJ) are shown in white. (B) Competitive targeting assay. OEP61 was synthesized by in vitro translation in WGE (lane 1, 10% input) and incubated with purified chloroplasts and mitochondria. The chloroplasts (Cp, lane 2) and mitochondria (Mito, lane 3) were repurified and washed with sodium carbonate before analysis by SDS/PAGE. (C) Topology of OEP61 at chloroplasts. Radiolabelled in vitro translated protein was incubated with purified pea chloroplasts, which were pelleted and washed with sodium carbonate, followed by thermolysin proteolysis in lanes 2, 4 and 6. Tic22 resides in the intermembrane space and Lhcb1 resides in the thylakoids. Molecular mass markers are shown in kDa on the left-hand side. (D) Topology of native OEP61 at chloroplasts. A. thaliana chloroplasts were isolated and washed with sodium carbonate prior to treatment of equal quantities with ( + Th) or without thermolysin, and then immunoblotted alongside supernatant (AtSN) and recombinant OEP61-TM.
Figure 4
Figure 4. OEP61 interacts specifically with Hsp70 via its TPR clamp domain
(A) Variants of OEP61 constructs: OEP61FL (amino acids 1–554; full length), OEP61TPR (amino acids 103–213), OEP61L (amino acids 214–534), OEP61-TM (amino acids 1–534) and OEP61R185A (point mutation of amino acid 185 from arginine to alanine in OEP61-TM). (B) OEP61-TM and OEP61R185A were incubated with WGE and isolated by their His-tag. The resulting binding partners were analysed by SDS/PAGE and Coomassie staining. Recombinant OEP61 is indicated by a star; the additional band at 70 kDa is indicated by a circle. (C) His–OEP61-TM was incubated with Hsp70 and Hsp81, and a pull down with equal amounts of each recombinant protein was performed. The resulting binding partners were analysed by SDS/PAGE and Coomassie staining. Hsp70 and Hsp81 input are shown as − Ni, and the removal of their His-tags was tested by pull down without His–OEP61 ( + Ni). (D) OEP61 variants and the HopTPR1 domain were incubated with WGE and isolated by their His-tag. Binding to Hsp70 was assessed by immunobloting with anti-Hsp70 IgG, and the signals were calculated in relation to the strongest signal, which was set as 100% (lane 3). The effect of binding competition with the peptide GAGPKIEEVD was also tested (lanes 7–12). The molecular mass in kDa is indicated on the left-hand side.
Figure 5
Figure 5. OEP61 associates specifically with precursors destined for the chloroplast via its TPR clamp domain
(A) Radiolabelled precursors of Toc33, Toc34, Lhcb1, Tic22, At2g17, ANT homologue (ANT-h), Tom22-I, Tim9, Tim10 and At5g013 were synthesized in WGE, treated with apyrase and then incubated with His-tagged OEP61-TM or the mutated form OEP61R185A. Products bound to His-tagged protein and pulled down with Ni-NTA beads were analysed by SDS/PAGE. Loading was adjusted to correct for the translation efficiency of each protein. Full-length products are indicated by a black square, specific pull downs are indicated by a star and non-specific pull downs are indicated with a circle. Molecular masses are given in kDa on the left-hand side. (B) Pull down by OEP61-TM was also performed for Toc33 and pSSU with and without their targeting sequences (TS).
Figure 6
Figure 6. Soluble OEP61 inhibits chloroplast targeting
(A) Radiolabelled precursors were synthesized by in vitro translation and incubated with chloroplasts or ER membranes (Toc33 and Sec61β), or chloroplasts and mitochondria in a competitive assay (mitoAt5g58), in the presence of OEP61-TM or PEX19. The organelles were pelleted and analysed by SDS/PAGE. (B) Chloroplast targeting assays were performed for Toc33, Lhcb1 and pSSU in the presence of OEP61-TM, the Hsp70-binding domain of Hop (HopTPR1) or PEX19. Molecular mass markers are shown in kDa on the left-hand side.
Figure 7
Figure 7. Proposed scheme of OEP61 and Toc64 action
OEP61 may be involved in the early recognition of Hsp70-bound precursors, in parallel with the recognition of Hsp90-bound precursors by Toc64, and it is also possible that precursors may engage both Toc64 and OEP61. Binding of the chaperone by the chaperone receptor would deliver the targeting sequence of the precursor to Toc34 (or Toc33) and promote release of the molecular chaperones. The precursor would then become translocated across the outer envelope using established mechanisms involving Toc159 and the pore formed by Toc75. OEP61 and Toc64 would then dislocate from the core complex.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wickner W, Schekman R. Protein translocation across biological membranes. Science. 2005;310:1452–1456. - PubMed
    1. May T, Soll J. 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants. Plant Cell. 2000;12:53–64. - PMC - PubMed
    1. Qbadou S, Becker T, Mirus O, Tews I, Soll J, Schleiff E. The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64. EMBO J. 2006;25:1836–1847. - PMC - PubMed
    1. Bae W, Lee YJ, Kim DH, Lee J, Kim S, Sohn EJ, Hwang I. AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis. Nat Cell Biol. 2008;10:220–227. - PubMed
    1. Dhanoa PK, Richardson LG, Smith MD, Gidda SK, Henderson MP, Andrews DW, Mullen RT. Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope. PLoS ONE. 2010;5:e10098. - PMC - PubMed

Publication types

MeSH terms