atTic110 Functions as a Scaffold for Coordinating the Stromal Events of Protein Import into Chloroplasts

Abstract

The translocon of the inner envelope membrane of chloroplasts (Tic) mediates the late events in the translocation of nucleus-encoded preproteins into chloroplasts. Tic110 is a major integral membrane component of active Tic complexes and has been proposed to function as a docking site for translocation-associated stromal factors and as a component of the protein-conducting channel. To investigate the various proposed functions of Tic110, we have investigated the structure, topology, and activities of a 97.5-kDa fragment of Arabidopsis Tic110 (atTic110) lacking only the amino-terminal transmembrane segments. The protein was expressed both in Escherichia coli and Arabidopsis as a stable, soluble protein with a high ␣-helical content. Binding studies demonstrate that a region of the atTic110-soluble domain selectively associates with chloroplast preproteins at the late stages of membrane translocation. These data support the hypothesis that the bulk of Tic110 extends into the chloroplast stroma and suggest that the domain forms a docking site for preproteins as they emerge from the Tic translocon.Chloroplast biogenesis is dependent upon the import of ϳ3000 different nucleus-encoded proteins (1). The majority of these proteins are synthesized as preproteins carrying an amino-terminal transit peptide that serves as the essential signal for targeting to the organelle. The transit peptide is recognized by receptor components of the translocon at the outer envelope membrane of chloroplasts (Toc), and a GTP-regulated switch initiates translocation through the protein-conducting channel of the Toc complex. At this stage, the Toc complex associates with the translocon at the inner envelope membrane (Tic), and this Toc-Tic supercomplex mediates the direct transport of the preproteins from the cytoplasm into the chloroplast stroma (2).Although many mechanistic details remain to be defined, the activities of the Toc components have been extensively investigated. Two membrane-associated GTPases, Toc159 and Toc34/33, mediate transit peptide recognition and regulate the initiation of translocation (3-7). The Toc GTPases form a complex with Toc75, an integral membrane protein that, along with Toc159, constitutes a major component of the protein-conducting channel (1, 8 -10).In contrast to the Toc complex, the activities and functions of the Tic components are less well defined. The biochemical analysis of Tic function has been complicated by the fact that assembly of functional Tic complexes is dynamic and occurs in response to preprotein translocation (11). Therefore, the isolation of a stable Tic complex has thus far been elusive. Tic110 was the first Tic component identified and represents a major component of active Tic complexes (12, 13). It is an integral inner envelope membrane protein, and structural predictions suggest that it consists of two predicted transmembrane helices at its extreme amino terminus and a 97.5-kDa carboxyl-terminal region that is largely hydrophilic. Tic110 transiently associates with at least five o...

AtOSA1, a Member of the Abc1-Like Family, as a New Factor in Cadmium and Oxidative Stress Response

“…The observed localization was identical with that obtained for the Tic110-GFP (Fig. 5B), an integral inner envelope membrane protein of the chloroplast import machinery (Inaba et al, 2003). Our results confirm in silico and proteomic data, suggesting a localization of the AtOSA1 protein in the chloroplast envelope of Arabidopsis (Froehlich et al, 2003).…”

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

“…The protein import complexes located on the outer and inner envelope membranes are termed as TOC (translocon at the outer envelop membrane of chloroplast) and TIC (translocon at the inner envelop membrane of chloroplast) complexes, respectively 1,2,5,6 . The Toc159, Toc75 and Toc34 proteins form the core TOC complex and are involved in protein transport across the outer envelope [6][7][8] . Tic110, Tic20 and Tic21 have been suggested to function as the protein translocation channel of the inner envelope 1,2,7,8 .…”

“…The Toc159, Toc75 and Toc34 proteins form the core TOC complex and are involved in protein transport across the outer envelope [6][7][8] . Tic110, Tic20 and Tic21 have been suggested to function as the protein translocation channel of the inner envelope 1,2,7,8 . Several stromal proteins, including chaperones, proteases and redox regulators, are also involved in the import process 6,9,10 .…”

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).…”