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However, the expression level of is lower at the earliest stage during germination. of leaf development. INTRODUCTION Most proteins in chloroplasts are encoded by the nuclear genome and are synthesized in the cytosol as precursors with N-terminal targeting signals called transit peptides. Import of precursor proteins into chloroplasts is mediated by a protein translocon complex, which is composed of the Toc (for translocon at the outer envelope membrane of chloroplasts) and the Tic (for translocon at the inner envelope membrane of chloroplasts) proteins and stromal chaperones (for reviews, see Soll and Schleiff, 2004; Kessler and Schnell, 2006). Three Toc proteins, Toc159, Toc75, and Toc34, form the core Toc complex. Toc159 and Toc34 are homologous GTPases and function as the initial receptors for incoming precursors. Toc75 is the protein-conducting channel across the outer membrane. Toc75 is predicted to form a -barrelCtype channel similar to transporters found in the outer membrane of gram-negative bacteria and the Tom40 protein-conducting channel of the mitochondrial outer membrane. The identity and composition of the protein-conducting channel across the chloroplast inner envelope membrane are much less clear. Two proteins, Tic20 and Tic110, have been suggested to function as the inner membrane channel. Tic20 was identified by direct cross-linking to importing precursors in pea (has two genes encoding Tic20: At and At gene, compared with At in generated a range of phenotypes from seedling lethal, pale, and stunted growth to pale cotyledons but fully recovered mature leaves (Chen et al., 2002). At is expressed CP 945598 HCl (Otenabant HCl) at its highest level during germination and at an equal level in etiolated and light-grown seedlings. These data indicate that At Tic20 is important for early plastid development (Chen et al., CP 945598 HCl (Otenabant HCl) 2002). However, it has been reported that pea Tic20 is present at a 10-fold lower abundance compared with Toc75, arguing against a function for Tic20 as the general protein-conducting channel across the inner membrane (Vojta et al., 2004; Becker et al., 2005). Tic110 is the major Tic component identified. It is predicted to have a small N-terminal hydrophobic domain and a large C-terminal hydrophilic domain. Incorporation of denatured recombinant Tic110 into proteoliposomes gives rise to cation-selective channels (Heins et al., 2002). However, topology analyses in intact chloroplasts and expression in transgenic plants have indicated that Tic110 is anchored to the inner Rabbit Polyclonal to MOV10L1 membrane with the N-terminal hydrophobic domain, and the large C-terminal domain is soluble and is located in the stroma (Jackson et al., 1998; Inaba et al., 2003, 2005). Part of the Tic110 stromal hydrophilic domain binds transit peptides directly and therefore may function as the stromal docking site for precursors when they are first translocated across the inner membrane into the stroma (Inaba et al., 2003). It is still likely that the membrane anchor of Tic110 CP 945598 HCl (Otenabant HCl) participates in channel formation. Tic20 is detected in Tic/Toc supercomplexes in association with Tic110. It is therefore possible that the Tic channel is formed by interaction of Tic20 and Tic110 (Kessler and Schnell, 2006). However, the low-abundance Tic20 in leaves needs to be accounted for, possibly by the presence other channel components. To further understand the mechanism of protein import into chloroplasts, we have designed a transgene-based screening strategy to isolate mutants defective in CP 945598 HCl (Otenabant HCl) chloroplast protein import (Sun et al., 2001). We have named these mutants (for chloroplast import apparatus). Characterization of the mutants and the CIA5 protein indicates that CIA5 is an essential translocon component for protein translocation across the inner membrane. Expression data and single and double mutant phenotypes of and At suggest that CIA5 and Tic20 may perform similar functions and that CIA5 is more important for later and Tic20 is more important for earlier stages of leaf development. RESULTS Molecular Cloning of the Locus Using a transgene-based screening strategy, possible chloroplast protein import mutants were selected based on hygromycin resistance.