Characterization of Tic110, a Channel-forming Protein at the Inner Envelope Membrane of Chloroplasts, Unveils a Response to Ca2+ and a Stromal Regulatory Disulfide Bridge

Abstract

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca 2؉ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.After the proposed endosymbiotic event that gave rise to chloroplasts and, as a consequence, to the massive transfer of genes from the endosymbiont to the host cell nucleus, two new machineries were developed at the outer and inner envelope of chloroplasts, the TOC and TIC complexes, respectively. The machineries transport plastid proteins, whose synthesis was moved to the cytosol, back to the plastid (for a review, see Refs. 1 and 2). Whereas the channel of the TOC complex is homologous to the Omp85 family of transporters in bacteria (3), the TIC complex shows no homology to known transport systems, since the plasma membrane-bacterial transport machineries were relocated to the thylakoid membrane. Moreover, there was an essential requirement for establishing new lines of communication between the organelle and other parts of the cell (4). In chloroplasts of land plants, redox regulation is linked to oxygen and/or light. The regulation of chloroplast-specific processes by redox in accord with the status of the organelle applies not only for transcription and translation but also for translocation. Redox regulation at the translocation level has been proposed for the TIC machinery on the basis of protein composition and dynamics (5-8) as well as specific in vitro import experiments (9). However, despite advances in our biochemical understanding of the import process, little is presently known regarding details of the role of redox or of the structure of the TIC complex, particularly with respect to its channel subunits.Tic110 is essential for protein import into plastids (10 -12). It has been proposed to be a pore-forming protein and one of the main components of the TIC complex (13). Previous studies have provided information about certain aspects of Tic110 structure-function. The region from residu...

Structural characterizations of chloroplast translocon protein Tic110

“…As shown in Figure 2, CmTic110 is predicted to have a chloroplast-targeting transit peptide at its N terminus, followed by the two transmembrane helices agreed upon by both models (TM1 and TM2, Figure 2; shaded in yellow), and a large C-terminal region, similar to other Tic110s. As previously reported (Balsera et al, 2009), CmTic110 has similar secondary structure features to Tic110s from green plants except that it contains several insertions in the regions between TM2 and TM3, and between TM3 and TM4 of model 1. The C-terminal half Figure 2) conserved in all Tic110s (Kalanon and McFadden, 2008).…”

“…The first model describes Tic110 with six transmembrane helices (herein referred to as TM1 to TM6; Figure 1a, model 1) located throughout the polypeptide. The first two transmembrane helices (TM1 and TM2) function as a signal-anchor sequence to target the protein to the inner membrane (L€ ubeck et al, 1997), and the rest of the polypeptide traverses the inner membrane four more times (TM3 to TM6) to form a Ca 2+ -sensitive and cation-selective channel (Heins et al, 2002;Balsera et al, 2009;Kovacs-Bogdan et al, 2011). Tic110 is therefore proposed to be the major channel for translocation of protein across the inner membrane.…”

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

“…These three proteins (Tic110, Tic40, and Hsp93) are thought to drive protein import into the stroma through repeated cycles of binding and release. Although an alternative model for the topology and function of Tic110 has also been proposed, in which Tic110 is a polytopic membrane protein that functions as a protein-conducting channel Balsera et al, 2009), a truncated version of Tic110 lacking the N-terminal transmembrane helices was shown to exist as a soluble protein when expressed in Escherichia coli or in the stroma of transgenic Arabidopsis (Inaba et al, 2003).…”

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

“…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 ). Electrophysiology measurements indicate a pore size of 1.7 nm, similar to that of Toc75.…”

“…After tranlocating through the outer envelope (OE), preproteins are imported either via Tic110 or Tic20 through the IE. Tic110 is thought to form a homodimer with a total of eight amphipathic transmembrane helices forming the translocation channel and four hydrophobic α -helices involved in the insertion into the membrane (Lubeck et al , 1996 ;Balsera et al , 2009 ). The proposed Tic20 channel is depicted as a homo-oligomer with a proposed molecular mass of > 700 kD but only three molecules are drawn for simplicity.…”