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

Abstract

Chloroplast protein import is mediated by two hetero-oligomeric protein complexes, the Tic and Toc translocons, which are located in the inner and outer envelope membranes. At the inner membrane, many Tic components have been identified and characterized, but it remains unclear how these Tic proteins are organized to form a protein-conducting channel or whether a stable Tic core complex that binds translocating preproteins exists. Here, we report the identification of a 1-megadalton (MD) translocation complex as an intermediate during protein translocation across the inner membrane in Arabidopsis thaliana and pea (Pisum sativum). This complex can be detected by blue native PAGE using the mild detergent digitonin without any chemical cross-linkers. The preprotein arrested in the 1-MD complex can be chased into its fully translocated form after a subsequent incubation. While Tic20 and Tic21 appear to be involved in the 1-MD complex, Tic110, a wellcharacterized Tic component, exists as a distinct entity from the complex. Several lines of evidence suggest that the 1-MD complex functions in between the Toc and Tic110-containing complexes, most likely as a protein-conducting channel at the inner envelope.

YCF1: A Green TIC: Response to the de Vries et al. Commentary

“…We found this TIC complex to be absolutely required for photosynthetic protein import in green tissues and, thus, essential for plant viability in Arabidopsis (Hirabayashi et al, 2011;Kikuchi et al, 2013). Experimental evidence for the presence of a similar TIC complex in pea (Pisum sativum) (Kikuchi et al, 2009) supports the idea that this complex is a general feature of the TIC apparatus. The TIC complex and Ycf1 appear to have evolved dramatically along with the evolution of the green lineage, including green algae and land plants; thus, they can be regarded as a "general green TIC.…”

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

“…1A). Unlike atTic40, mature SSU is a soluble stromal protein, and previous studies have defined specific intermediates at several stages of import through the TOC-TIC complexes in in vitro import reactions (13,(21)(22)(23)(24)(25). Import intermediates for both preproteins coprecipitated Hsp90C as well as Tic110 and Toc75 (Fig.…”

Structural characterizations of chloroplast translocon protein Tic110

“…We cannot exclude the possibility that the region not covered by our analyses, the TM3 and TM4 regions of model 1, provided the channel activity observed in the reconstituted liposome. Of note is the fact that the six-transmembrane-domain topology of Tic110 was deduced using isolated inner membrane vesicles (Balsera et al, 2009), while experiments using intact chloroplasts have shown that the region after TM2 is entirely inside the stroma (Kessler and Blobel, 1996;Jackson et al, 1998;Chou et al, 2003;Inaba et al, 2003Inaba et al, , 2005Kikuchi et al, 2009). Isolated inner membrane vesicles may have mixed orientations and may complicate the experimental results.…”

“…It was suggested that the higher than calculated molecular mass was due to a possible non-globular shape of Tic110 (Balsera et al, 2009). Pea and Arabidopsis Tic110 also migrated as dimers on blue native (BN)-PAGE when solubilized from chloroplast membranes (Kikuchi et al, 2009(Kikuchi et al, , 2013. The calculated molecular masses of CmTic110 B and CmTic110 C are 59.9 and 42.8 kDa, respectively.…”

“…Tic21 has been shown genetically to function in the same pathway as Tic20 (Teng et al, 2006). Furthermore, a complex containing Tic20, Tic21 and translocating precursors, but not Tic110, has been identified in chloroplasts (Kikuchi et al, 2009(Kikuchi et al, , 2013.…”