Tic21 Is an Essential Translocon Component for Protein Translocation across the Chloroplast Inner Envelope Membrane

Abstract

An Arabidopsis thaliana mutant defective in chloroplast protein import was isolated and the mutant locus, cia5, identified by map-based cloning. CIA5 is a 21-kD integral membrane protein in the chloroplast inner envelope membrane with four predicted transmembrane domains, similar to another potential chloroplast inner membrane protein-conducting channel, At Tic20, and the mitochondrial inner membrane counterparts Tim17, Tim22, and Tim23. cia5 null mutants were albino and accumulated unprocessed precursor proteins. cia5 mutant chloroplasts were normal in targeting and binding of precursors to the chloroplast surface but were defective in protein translocation across the inner envelope membrane. Expression levels of CIA5 were comparable to those of major translocon components, such as At Tic110 and At Toc75, except during germination, at which stage At Tic20 was expressed at its highest level. A double mutant of cia5 At tic20-I had the same phenotype as the At tic20-I single mutant, suggesting that CIA5 and At Tic20 function similarly in chloroplast biogenesis, with At Tic20 functioning earlier in development. We renamed CIA5 as Arabidopsis Tic21 (At Tic21) and propose that it functions as part of the inner membrane protein-conducting channel and may be more important for later stages of leaf development.

A 1-Megadalton Translocation Complex Containing Tic20 and Tic21 Mediates Chloroplast Protein Import at the Inner Envelope Membrane

“…By thorough screening of antisera, we found that an antibody against Arabidopsis Tic21 caused a significant shift in this assay when Arabidopsis chloroplasts were used ( Figure 5A, lane 2). Tic21 was recently identified by Arabidopsis genetics (Teng et al, 2006). When antibodies against pea Tic110 and SPA-820 monoclonal antibody (StressGen), which recognizes an as yet unidentified intermembrane space Hsp70 (Schnell et al, 1994;Becker et al, 2004), were added, the mobility of the 1-MD complex on BN-PAGE was not affected ( Figure 5A).…”

“…First, we performed transient expression and targeting of preproteins using Arabidopsis knockout mutants. Because both homozygous tic21/pic1 and tic20 mutants show severe albino phenotypes (Teng et al, 2006), standard import experiments using isolated plastids are difficult. Therefore, we used transient expression system in protoplasts (Jin et al, 2001;Lee et al, 2008).…”

“…Teng et al (2006) identified Tic21 as a preprotein translocon at the inner envelope membrane, whereas Duy et al (2007) independently identified the same protein as an iron transporter (PERMEASE IN CHLOROPLASTS1 [PIC1]). To clarify these controversial findings, we employed two independent experimental approaches.…”

“…At the inner membrane, eight proteins (Tic110, Tic40, Tic20, Tic21, Tic22, Tic55, Tic62, and Tic32) are reported to be involved in the import process Bé dard and Jarvis, 2005;Teng et al, 2006;Jarvis, 2008;Kessler and Schnell, 2009). Tic20 has been proposed to function as the protein-conducting channel of the inner membrane.…”

The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

“…The latter was proposed by Teng and co-workers (2006) as the third potential protein-conducting channel at the inner membrane of chloroplasts. This notion was disputed in another study, where the reported Tic21 (Teng et al , 2006 ) most likely represents an ancient metal permease, which regulates iron uptake and metal homeostasis in chloroplast and not a protein conducting channel (Duy et al , 2007 ). Despite the occurrence of several proteinconducting channel candidates for the inner membrane of the chloroplast, considerable lines of evidence clearly pinpoint Tic110 as the central subunit of the TIC complex, forming a high-conductance cation-selective channel Balsera et al , 2009 ).…”