New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development

Abstract

The translocons at the outer (TOC) and the inner (TIC) envelope membranes of chloroplasts mediate the targeting and import of several thousand nucleus-encoded preproteins that are required for organelle biogenesis and homeostasis. The cytosolic events in preprotein targeting remain largely unknown, although cytoplasmic chaperones have been proposed to facilitate delivery to the TOC complex. Preprotein recognition is mediated by the TOC GTPase receptors Toc159 and Toc34. The receptors constitute a GTP-regulated switch, which initiates membrane translocation via Toc75, a member of the Omp85 (outer membrane protein 85)/TpsB (two-partner secretion system B) family of bacterial, plastid and mitochondrial β-barrel outer membrane proteins. The TOC receptor systems have diversified to recognize distinct sets of preproteins, thereby maximizing the efficiency of targeting in response to changes in gene expression during developmental and physiological events that impact organelle function. The TOC complex interacts with the TIC translocon to allow simultaneous translocation of preproteins across the envelope. Both the two inner membrane complexes, the Tic110 and 1 MDa complexes, have been implicated as constituents of the TIC translocon, and it remains to be determined how they interact to form the TIC channel and assemble the import-associated chaperone network in the stroma that drives import across the envelope membranes. This review will focus on recent developments in our understanding of the mechanisms and diversity of the TOC-TIC systems. Our goal is to incorporate these recent studies with previous work and present updated or revised models for the function of TOC-TIC in protein import.

Keywords: TIC complex; TOC complex; chaperones; plastid biogenesis.

Figure 1

Role of cytoplasmic chaperone systems in targeting preproteins to the core TOC complex. Three pathways have been proposed for the targeting of newly synthesized preproteins to the TOC system. A. Cytoplasmic Hsp90 in conjunction with the HOP and immunophilin FKB73 co-chaperones bind preproteins and dock at the outer envelope membrane via an interaction with the cytoplasmically exposed tetratricopeptide repeat (TPR) domain of Toc64, an integral outer membrane protein [26,27]. Toc64 is proposed to deliver preproteins to the core TOC complex by interacting with Toc34 and transferring the preprotein to the Toc34 and Toc159 receptors. B. Cytoplasmic Hsp70 and a 14-3-3 protein of unknown identity form a guidance complex, which binds to the transit peptide and mature regions of newly synthesized preproteins and facilitates targeting to the TOC receptors [25,37]. Binding of the guidance complex is proposed to be regulated by reversible phosphorylation of the transit peptide although in vivo evidence in support of a phosphorylation cycle is lacking. The mechanism of preprotein transfer from the guidance complex to the TOC receptors is unknown, but might be facilitated by OEP61, an Hsp70 receptor at the outer envelope membrane. C. Cytoplasmic preproteins also can bind directly to the Toc34 and Toc159 receptors at the core TOC complex. Genetic studies suggest that the Hsp90 and Hsp70 chaperone systems are not essential for protein import, but could increase the efficiency of targeting by preventing mistargeting or misfolding of preproteins prior to binding at the TOC complex (for review see [24]).

Figure 2

A GDP/GTP regulated switch model for the initiation of import at the TOC complex. A. The core TOC complex consists of two membrane bound GTPase receptors, Toc159 and Toc34, and the β-barrel channel protein, Toc75. The receptors are proposed to exist in their GDP-bound states in the absence of protein import. B. Toc34 and Toc159 can simultaneously recognize the transit peptides of cytoplasmic preproteins at the chloroplast surface via binding sites within their GTPase domains (G-domains). C. Transit peptide binding dissociates receptor homodimers, triggering the exchange of GDP for GTP, and promotes Toc34-Toc159 heterodimerization. This step corresponds to an ‘activated’ complex, which is primed to initiate transfer of the preprotein into the TOC channel. D. GTP hydrolysis at the receptors initiates preprotein insertion across the outer membrane via Toc75, and across the inner membrane via the TIC complex. The binding of molecular chaperones to the preprotein in the intermembrane space (IMS) and stroma would drive unidirectional translocation across the envelope. E. The core TOC complex would reset to it's GDP-bound ‘resting’ state following the completion of translocation.

Figure 3

Three-dimensional model of the Toc75 β-barrel channel of the TOC complex. A. The structural model of Toc75 illustrates the C-terminal domain consisting of a predicted 16-stranded β-barrel membrane channel and an N-terminal domain consisting of three repeats of POTRA (POlypeptide-TRansport Associated) domains [89]. The composite structure was generated by homology modeling to the crystal structure of the β-barrel membrane channel of the BamA component (PDB ID: 4K3B) of the β-barrel translocase of Neisseria gonorrhoeae [86] and the crystal structure of the three POTRA domains from the closely related OMP85 protein from the cyanobacterium, Anabaena sp. PCC 7120 (PDB ID: 3MC8) [88]. B. and C. Crystal structures of BamA (PDB ID: 4K3B) of Neisseria gonorrhoeae [86] and the TpsB transporter, FhaC, of Bordetella pertussis (PDB ID: 2QDZ) [84], respectively. The first and last β-strands of the β-barrel domains of the predicted structure of Toc75 and the known structures of BamA and FhaC are illustrated in green. The individual POTRA domains of each structure are color-coded and labeled P1-P5. Toc75 contains three POTRA domains. BamA and FhaC contain five and two POTRA domains, respectively. Crystal structures of the NgBamA (Barrel assembly machinery A; PDBID: 4K3B) and POTRA1-3 of Anabaena Omp85 (PDBID: 3MC8) were obtained from RCSB (http://www.rcsb.org/pdb) and used as templates for modeling the β-barrel and POTRAs of Toc75, respectively. The final alignment was used to construct the model using the software Modeller (version 9.12) [201]. A set of 200 models was generated, from which the lowest energy structure was used. The RMSD values of the β-barrel and POTRA domains are 1.48°A and 1.07°A, respectively. The initial low energy model obtained from Modeller was validated by using PROCHECK [202] and VERIFY_3D [203] servers.

Figure 4

Model for the coordinate function of proposed TIC components in protein import across the inner envelope membranes. Two inner membrane protein complexes have been implicated as core components of the TIC complex. A. A complex composed of the integral membrane proteins, Tic110, Tic40, and Tic20, associate with molecular chaperones in the intermembrane space (Tic22) and the stroma (cpHsp70, Hsp90C and Hsp93/ClpC). Tic22 is proposed to facilitate transit of the preprotein between the TOC and TIC complexes and prevent mistargeting to the IMS. Tic40 contains TPR and Sti1 domains characteristic of chaperone organizing proteins and is proposed to coordinate the assembly of the stromal chaperones to form the import motor [127,158]. The C-terminal stromal domain of Tic110 serves as the scaffold for assembly of the stromal chaperones [122] and interacts with Tic20 and Tic21, two constituents of the TIC membrane channel [120,130,134]. B. A 1 MDa complex consisting of Tic20/21, Tic56, Tic100 and Tic214 also has been shown to associate with preprotein import intermediates and is proposed to constitute the TIC translocon [92,145]. C. An alternative model is proposed in which the Tic110 (A) and 1 MDa (B) complexes would associate dynamically to form the functional TIC complex at the inner envelope membrane. In this model, the two complexes would associate to form the translocation channel via the common components, Tic20/21. Interaction of the preprotein with either individual complex as it emerges across the TOC complex into the intermembrane space could trigger assembly of the functional TIC complex. The stromal chaperones docked at Tic110 would form the ATP-dependent translocation motor to drive import of the preprotein into the stroma.

Figure 5

Alternative functions of the import associated chaperone network in the stroma. A. Tic110 and Tic40 play key roles in assembling the complex of stromal chaperones that constitute the import motor of the TIC complex. Binding of the stromal domain of Tic110 to the transit peptide of a preprotein as it emerges from the TIC membrane channel triggers binding of the Hsp93/ClpC chaperone to Tic110 with the aid of the TPR and Sti1 chaperone organizing domains of the Tic40 co-chaperone [122,158]. Tic40 also is proposed to facilitate the assembly of the cpHsp70 and Hsp90C chaperones at Tic110. At least two models have been proposed to account for the individual contributions of cpHsp70, Hsp90C and Hsp93/ClpC to protein import. B. In the first model, ATP-driven binding and release of the preprotein from cpHsp70 would constitute the rate limiting step in the import motor [169]. CpHsp70, Hsp90C and Hsp93/ClpC would act coordinately or in sequence to facilitate membrane translocation. C. In the second model, the cpHsp70-Hsp90C complex and Hsp93/ClpC would constitute separate motors with preferences for preproteins with different physical properties [164].

Figure 6

Models for quality control systems operating in the cytoplasm and stroma to monitor preprotein import and folding. A. The capacity of the TOC-TIC system is sufficient to ensure efficient import of newly synthesized preproteins under normal conditions. When the expression of preproteins exceeds the capacity of the import system, preproteins misfold prior to engaging the TOC receptors, or the TOC-TIC system is damaged, preproteins can accumulate in the cytoplasm and potentially form harmful aggregates. The cytoplasmic Hsp70-4 chaperone recognizes the transit peptides of these excess preproteins and facilitates their degradation by the ubiquitin-proteasome system (UPS) via the activities of the E3 ubiquitin ligase, CHIP, and the 26S proteasome [185]. B. The ClpP protease associates with the inner envelope membrane in stoichiometric amounts relative to the Hsp93/ClpC chaperone [187]. This raises the possibility that Hsp93/ClpC and ClpP function to degrade newly imported proteins that do not fold or assemble properly during or shortly after import. In this model the Hsp93/ClpC docked at the TIC complex would have dual functions as a component of the import motor and in the quality control of newly imported proteins.