Journal of Molecular Biology
Volume 367, Issue 5, 13 April 2007, Pages 1330-1346
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Toc64 - A Preprotein-receptor at the Outer Membrane with Bipartide Function

https://doi.org/10.1016/j.jmb.2007.01.047Get rights and content

Abstract

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.

Introduction

The requirement of protein translocation is a consequence of the subdivision of the eukaryotic cell. Hence, molecular machines facilitating this translocation can be found in all membranes.1 Here, the general mechanism can be divided into two major pathways promoting either co-translational or post-translational translocation. Chloroplasts import most of their protein complement post-translationally from the cytosol. The majority of these preproteins contain an N-terminal transit peptide, which is cleaved off upon translocation.2., 3., 4., 5. The machinery facilitating recognition and translocation of preproteins across the outer envelope membrane of the plastid is constituted of Toc75, Toc159 and Toc34 assembling the Toc core-complex6 and the dynamically associated Toc64 and Toc12.7 Toc159 and Toc34 expose a GTPases domain into the cytosol, which is involved in preprotein recognition and translocation.8., 9., 10., 11. Toc34 is discussed to act as a receptor within the Toc core complex11., 12. and Toc159 is crucial for the import process.9., 11., 13. The mode of recognition is proposed to be precursor dependent.14 One class of preproteins can be phosphorylated at the transit peptide, which initiates the assembly of a cytosolic “guidance complex” with a 14-3-3 protein as the central component.15 The phosphorylation is not essential for targeting and translocation of preproteins,16 but enhances the rate of translocation15 and the affinity of recognition by Toc34.17 The cytosolic complex formed by 14-3-3, the phosphorylated preprotein and further unknown components are directly targeted to Toc34.14 Dephosphorylation of the preprotein has to occur before passage through the import channel of the outer envelope membrane.12., 18.

A second class of preproteins is associated with Hsp90.14 The cytosolic exposed C-terminal tetratrico-peptide repeat (TPR) motif-containing domain of Toc64 is the initial recognition site on the surface of the chloroplast for Hsp90 associated preproteins. However, the clamp type TPR domain19 of Toc64 does not directly interact with the preprotein but with Hsp90. The dissociation of the preprotein from Hsp90 and therefore from Toc64 is ATP-dependent. The preprotein is subsequently transferred to and recognized by Toc34.14 Additionally, a third class of preproteins might exist occurring in a monomeric form in vitro and in vivo, which can also be recognized by Toc34.17 Therefore, at present it can be assumed that these three pathways merge at the receptor site Toc34 and that these preproteins will be processed by the translocation channel Toc75.

The subsequent transport across the intermembrane space is not as explored as recognition on the cytosolic site. Recently, a so-called intermembrane space complex composed of Toc64, Toc12, Tic22 and an intermembrane space localized Hsp70 was identified.7 Toc12 contains a J-like domain found to recruit the intermembrane space localized Hsp70 (isHsp70) in an ATP-dependent fashion to the translocon and to stimulate the ATPase activity of the chaperone.7 It is proposed that Toc12 modulates the function of the isHsp70, which together with Tic2220 and Toc64 might facilitate the translocation across the intermembrane space.7 Therefore, the participation on the intermembrane space complex indicates a role of Toc64 on the trans site of the outer envelope membrane. The specificity and the function of this complex, however, remains to be further explored.

Toc64 seems to be involved in the steps of protein translocation at both sides of the membrane.7., 14. The protein is dynamically associated with the Toc core complex6., 14. and contains three distinct regions annotated as amidase, charged and TPR-containing domain.21., 22. The amidase region at the N terminus is silenced in its enzymatic function by an amino acid exchange of the central and essential serine to glycine.21 As mentioned, the C-terminal TPR domain of Toc64 is involved in the recognition of preproteins associated with Hsp90 chaperones.14 Toc64 contains an N-terminal transmembrane region, which is essential and sufficient for targeting to chloroplasts.23 Initial experiments on isoforms of Toc64 from Physcomitrella patens suggested a largely cytosolic exposed protein conformation.24 This proposed topology, however, cannot explain the observed tight association of Toc64 with Toc12, Tic22 and the imsHsp70.7

For an understanding of Toc64 function and the translocation processes in the intermembrane space a detailed analysis of the second binding site of Toc64 for incoming preproteins was conducted. Further, the domain-dependent association of Toc64 with the core complex was probed. This analysis is complemented by a detailed study of the insertion and topology.

Section snippets

Toc64 acts in the intermembrane space

The C-terminal TPR domain of Toc64 acts as a docking site for Hsp90 delivered preproteins.14 However, a chaperone-independent interaction of the receptor with preproteins like the small subunit of RubisCO (pSSU) targeted to a binding site distinct from the TPR was reported as well.14 Hence, we aimed to identify this association site within Toc64. pSSU was incubated with pea chloroplasts in the absence (Figure 1(a), lanes 7–12) or presence of ATP (lanes 1–6) followed by wash steps of the

The topology of Toc64 revisited

Protein translocation across the chloroplast envelopes starts with the recognition of the preprotein by a receptor complex.1., 2., 3., 4., 5. The subsequent transfer across the intermembrane space is thought to be assisted for instance by Toc647 (Figure 1). To understand the function of Toc64 at both sides of the outer envelope we first investigated the topology of the protein. The N-terminal transmembrane domain of Toc64 acts as a targeting signal19 and inserts with a Nin-Cout orientation23 as

General

RNase free DNase, RNase I, proteinase K, trypsin, chymotrypsin, thermolysin, reticulocyte lysate, were purchased from Roche (Mannheim, Germany). GE-Healthcare (Munich, Germany) supplied the [35S]methionine and Percoll. The data are presented using Adobe Photoshop 6.0 (Adobe Systems Inc., Munich, Germany), Corel Draw 10 (Eastman Kodak Company, USA) and Sigma Plot 7.0 (SPSS Inc., Chicago, USA). The generation, in vitro translation or expression of pSSU or Toc64 was described.7., 12., 14., 17.

Acknowledgements

We thank P. Jarvis and H. Aronsson for helpful discussions for experimental designs and O. Mirus for discussing the topology. The work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie (to E.S. and J.S.), from the Volkswagenstiftung (to E.S.) and the Herbert Quandt Foundation (to S.Q.).

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