Import of Soluble Proteins into Chloroplasts and Potential Regulatory Mechanisms

Abstract

Chloroplasts originated from an endosymbiotic event in which a free-living cyanobacterium was engulfed by an ancestral eukaryotic host. During evolution the majority of the chloroplast genetic information was transferred to the host cell nucleus. As a consequence, proteins formerly encoded by the chloroplast genome are now translated in the cytosol and must be subsequently imported into the chloroplast. This process involves three steps: (i) cytosolic sorting procedures, (ii) binding to the designated receptor-equipped target organelle and (iii) the consecutive translocation process. During import, proteins have to overcome the two barriers of the chloroplast envelope, namely the outer envelope membrane (OEM) and the inner envelope membrane (IEM). In the majority of cases, this is facilitated by two distinct multiprotein complexes, located in the OEM and IEM, respectively, designated TOC and TIC. Plants are constantly exposed to fluctuating environmental conditions such as temperature and light and must therefore regulate protein composition within the chloroplast to ensure optimal functioning of elementary processes such as photosynthesis. In this review we will discuss the recent models of each individual import stage with regard to short-term strategies that plants might use to potentially acclimate to changes in their environmental conditions and preserve the chloroplast protein homeostasis.

Keywords: TIC; TOC; acclimation; chloroplast; plastid proteostasis; protein import.

FIGURE 1

Chaperone involvement in cytosolic targeting and recognition of preproteins at the outer envelope membrane of chloroplasts. Preproteins could be chaperoned by the guidance complex or by Hsp90 alone. The guidance complex is represented by Hsp70 that binds to both mature region and cTP of the preprotein and 14-3-3 proteins which bind to the phosphorylated cTP. Hsp70-chaperoned preproteins are recognized by the GTP-dependent receptor proteins Toc159 and Toc34, followed by delivery to the import channel Toc75, whereas precursor proteins bound to Hsp90 are docked to the third receptor Toc64 via its TPR domain and are then handed over to Toc34.

FIGURE 2

Crossing the inner envelope membrane of chloroplasts via the TIC complex. The counterpart of the outer channel protein is the IEM protein Tic110 which is a functional dimer. Two hydrophobic domains anchor the protein into the IEM whereas further eight amphipathic helices are involved in the channel formation. Tic40 is supposed to interact with Tic110 with its Sti1 domain and acts further as a scaffold for stromal chaperones. Controversial, the 1MDa-complex depicted on the right side comprises atTic20 as the channel protein, atTic56 embedded in the complex, atTic100 located at the IMS and the plastid encoded Ycf1 (atTic214) with its six transmembrane domains and a large stromal C-terminus.

FIGURE 3

The stromal chaperone system. Two different models have been hypothesized concerning the main import motor of the chaperones. One model (A) involves a secondary function of Hsp93, assuming that this protein acts mainly in the quality control pathway by degrading mistargeted or wrongly folded proteins. In this model the main energy is consumed by Hsp70 and not by Hsp93 (Flores-Pérez et al., 2016). A recent study suggest that Hsp93 interacts subsequently with incoming preprotein at the N-terminal cTP, whereas Hsp70 binds to the mature parts of the protein (Huang et al., 2015). This enable the two chaperone systems to interact at least partially in parallel with the preproteins. After completing of the import by processing the cTP, proteins are folded with the help of various chaperones like Cpn60 and Hsp70 (B).

FIGURE 4

Redox regulation at the outer envelope membrane. Disulfide bridges between conserved cysteine residues of the TOC constituents are involved in the redox modulation of the constituents of the OEM. Under reducing conditions, the TOC receptors are loosely attached, thus forming the open and active TOC complex (A). Upon oxidation due to various external stimuli the generated intra- and intermolecular disulfide bridges lead to a blocked TOC complex which inhibits import of precursor proteins either by blocking the channel or altering the binding capacity of the receptor toward the preproteins (B).

FIGURE 5

Import regulation of the TIC complex from the stromal site. Similar to the redox regulation at the OEM import of precursor proteins is accelatered under reducing conditions, suggestively due to an open conformation of the main channel, Tic110. A second regulation mechanism involves the stromal redox state, which is reflected by the NADPH/NADP⁺ ratio. A low NADPH/NADP⁺ ratio could be shown to enhance the import rate compared to a higher NADPH/NADP⁺ ratio.