Preprotein Import into Chloroplasts via the Toc and Tic Complexes Is Regulated by Redox Signals in Pisum sativum

Anna Stengel 0 1 J. Philipp Benz 0 1 Bob B. Buchanan 0 1 Ju rgen Soll 0 1 Bettina B olter 0 1 0 muenchen. de , fax 49-89-2180-74752, tel. 49-89-2180-74759. The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE , SIBS, CAS. doi: 10.1093/mp/ssp043, Advance Access publication 6 July 2009 Received 19 February 2009 ; accepted 29 May 2009 1 a Munich Center for Integrated Protein Science CiPS The import of nuclear-encoded preproteins is necessary to maintain chloroplast function. The recognition and transfer of most precursor proteins across the chloroplast envelopes are facilitated by two membrane-inserted protein complexes, the translocons of the chloroplast outer and inner envelope (Toc and Tic complexes, respectively). Several signals have been invoked to regulate the import of preproteins. In our study, we were interested in redox-based import regulation mediated by two signals: regulation based on thiols and on the metabolic NADP1/NADPH ratio. We sought to identify the proteins participating in the regulation of these transport pathways and to characterize the preprotein subgroups whose import is redox-dependent. Our results provide evidence that the formation and reduction of disulfide bridges in the Toc receptors and Toc translocation channel have a strong influence on import yield of all tested preproteins that depend on the Toc complex for translocation. Furthermore, the metabolic NADP1/NADPH ratio influences not only the composition of the Tic complex, but also the import efficiency of most, but not all, preproteins tested. Thus, several Tic subcomplexes appear to participate in the translocation of different preprotein subgroups, and the redox-active components of these complexes likely play a role in regulating transport. - INTRODUCTION Chloroplasts are photosynthetic organelles characteristic of algae and land plants. They appear to have originated from an endosymbiotic event in which an ancestral photosynthetic cyanobacterium was engulfed by a mitochondriate eukaryotic host cell (Margulis, 1970). During evolution, most of the endosymbionts genes were transferred to the host nucleus (Leister, 2005), so that the majority of chloroplast proteins are translated in the cytosol and post-translationally targeted and imported into the organelle (Jarvis and Soll, 2001; Schleiff and Soll, 2004). The constant adaptation of chloroplasts to developmental or environmental changes requires a flexible organelle proteome that is regulated at several levels, namely transcription, mRNA stability and translation, posttranslational modification, targeting of precursor proteins, translocation across the double membrane, and, finally, protein turnover inside the organelle (Kessler and Schnell, 2006). The import of preproteins into chloroplasts is mediated by two hetero-oligomeric protein complexes located in the inner and outer envelope membranes, respectively, designated Toc (translocon at the outer envelope of chloroplasts) and Tic (translocon at the inner envelope of chloroplasts). The Toc translocon consists of five subunits (Stengel et al., 2007; Jarvis, 2008): Toc159, Toc34, and Toc64, which function as receptor components responsible for binding precursor proteins (Kessler et al., 1994; Qbadou et al., 2006); Toc75, which constitutes the translocation pore (Schnell et al., 1994) and forms the Toc core complex together with Toc159 and Toc34; and Toc12, which is located in the inter-membrane space, where it seems to form a complex with Toc64 and a Tic component, Tic22 (Becker et al., 2004a). The Tic complex in the inner envelope is thought to consist of at least seven proteins (Stengel et al., 2007; Jarvis, 2008; Benz et al., 2008): Tic110, Tic62, Tic55, Tic40, Tic32, Tic22, and Tic20. Tic110, the most abundant protein of the Tic complex, forms the central translocation channel (Heins et al., 2002; Balsera et al., 2008). Three other Tic components were proposed to act as potential regulatory redox-active subunits (the redox regulon): Tic62, Tic32, and Tic55. Tic55 represents a member of CAO/PAO-like oxygenases, containing a Rieske-type [2Fe2S] cluster and an additional mononuclear iron binding site (Caliebe et al., 1997). Tic32 and Tic62 both belong to the family of short-chain dehydrogenases and possess dehydrogenase activity in vitro (K uchler et al., 2002; Ho rmann et al., 2004; Chigri et al., 2006; Stengel et al., 2008). Tic32 was originally identified as an interaction partner of the N-terminal domain of Tic110 (H ormann et al., 2004) and found to be the target of Ca2+/calmodulin (CaM)regulation of protein import (Chigri et al., 2006). Tic62 was found to bind FNR (ferredoxinNADP+oxidoreductase) specifically, and to act as a shuttle between chloroplast membranes and the stroma in a redox-dependent manner (K uchler et al., 2002; Stengel et al., 2008). The import of nuclear-encoded preproteins into chloroplasts using the Toc and Tic machineries is considered to be the general import pathway. A small number of proteins, however, enter the organelle via alternative import pathways: for example, several outer envelope proteins, including most Toc components, lack a cleavable transit peptide and seem to insert into the outer envelope membrane directly and spontaneously from the cytosolic side (Stengel et al., 2007; Jarvis, 2008). There are also examples of inner envelope proteins without cleavable N-terminal targeting signals, such as the chloroplast envelope quinone oxidoreductase homolog (ceQORH) and Tic32. The import of both of these proteins was found to be independent of the Toc/Tic machineries (Miras et al., 2002; Nada and Soll, 2004). An additional chloroplast-targeting pathway has been suggested by which certain proteins (e.g. carbonic anhydrase 1, CAH1) first enter the endoplasmatic reticulum (ER) with an ER signal peptide and are subsequently transported via the Golgi apparatus to the chloroplast as vesicle cargo (Villarejo et al., 2005; Radhamony and Theg, 2006). While only a few examples of alternative import pathways are known, it is conceivable that other proteins also import independently of the Toc/Tic complexes, since many nuclearencoded chloroplast proteins lacking a cleavable transit peptide have been identified in the chloroplast proteome (Kleffmann et al., 2004). The need to adapt to change in developmental and environmental conditions suggests that protein import into chloroplasts is regulated. Moreover, it has been shown that regulation can take place at several levels, notably (1) the cytosol, (2) the Toc complex, and (3) the Tic complex. Phosphorylation of the preprotein transit peptide in the cytosol was shown to influence translocation rate (Waegemann and Soll, 1996; Martin et al., 2006). Regulation of protein import at the level of the Toc complex can be achieved by GTP/GDP binding and by phosphorylation of the receptor components Toc34 and Toc159 (Kessle (...truncated)