Tic32, an Essential Component in Chloroplast Biogenesis

Abstract

Chloroplast protein import across the inner envelope is facilitated by the translocon of the inner envelope of chloroplasts (Tic). Here we have identified Tic32 as a novel subunit of the Tic complex. Tic32 can be purified from solubilized inner envelope membranes by chromatography on Tic110 containing affinity matrix. Co-immunoprecipitation experiments using either Tic32 or Tic110 antisera indicated a tight association between these polypeptides as well as with other Tic subunits, e.g. Tic40, Tic22, or Tic62, whereas the outer envelope protein Toc75 was not found in this complex. Chemical cross-linking suggests that Tic32 is involved late in the overall translocation process, because both the precursor form as well as the mature form of Rubisco small subunit can be detected. We were unable to isolate Arabidopsis null mutants of the attic32 gene, indicating that Tic32 is essential for viability. Deletion of the attic32 gene resulted in early seed abortion because the embryo was unable to differentiate from the heart stage to the torpedo stage. The homology of Tic32 to short-chain dehydrogenases suggests a dual role of Tic32 in import, one as a regulatory component and one as an important subunit in the assembly of the entire complex.Choroplasts must import most of their protein constituents from the cytosol in a posttranslational process (1-4). Cytosolically synthesized precursor proteins are generally made with an N-terminal transit peptide that is both necessary and sufficient for chloroplast recognition and translocation initiation. Translocation across the outer and inner envelopes of chloroplasts is achieved by the Toc 1 and Tic complexes, respectively (translocon at the outer/inner envelope of chloroplasts). Upon import, the targeting peptide is removed by a stromal processing peptidase, and the mature protein is further sorted and assembled into active structures (5).Preproteins are recognized in a GTP-dependent manner by the Toc34 receptor at the chloroplast surface (6 -8). The GTPase activity of Toc34 is greatly stimulated by precursor binding, and upon GTP hydrolysis the preprotein is released from Toc34 GDP to Toc159 (9). Toc159, another GTP-dependent protein, interacts with the preprotein in a manner that also involves GTP hydrolysis (6, 10, 11). Toc159 facilitates the translocation of the preprotein through the import channel Toc75 (12, 13). The preprotein then engages the Tic complex to move across the inner envelope in an ATP-requiring fashion (14 -16). Several subunits of the Tic complex have been identified. Tic110, which is the second most abundant protein in the inner envelope (17-20), can form an aqueous ion channel in vitro and is therefore thought to form the translocation pore (21). However, because of different proposals about the topology and structure of Tic110 (21, 22), the exact function of Tic110 is not resolved. In addition, Tic110 could interact with the molecular chaperones cpn60 and Hsp93 on the stromal face of the membrane. It could also act as a chaperone recruitment factor (17,...

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

“…The identification of LTD in LHCP import and subsequent delivery to the cpSRP pathway was unexpected, as both the envelope and cpSRP transport pathways have been extensively investigated and this component has not been detected before 11,15,20,21,49,50 . One possibility is that the inability of LTD to form the stable complexes with cpSRP43 or with Tic40 and Tic110, making it difficult to be detected using the biochemical assays currently applied in the identification of the components of the envelope and cpSRP transport pathways 11,15,20,21,49,50 .…”

“…One possibility is that the inability of LTD to form the stable complexes with cpSRP43 or with Tic40 and Tic110, making it difficult to be detected using the biochemical assays currently applied in the identification of the components of the envelope and cpSRP transport pathways 11,15,20,21,49,50 . Another possibility is that an intermediate stage of such process cannot be trapped or reconstituted in vitro for further identification of the components involved in because the transport from the inner envelop to the SRP pathway is so rapid.…”

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence

“…TIC55 had been considered to exhibit a redox regulatory role in protein import together with TIC32 and TIC62, two additional proposed components of the protein import machinery at the inner chloroplast envelope (Küchler et al, 2002;Hörmann et al, 2004). Both proteins were shown to possess NADPH binding domains and, in addition, TIC62 is able to interact with FNR (Bölter et al, 2015).…”

Arabidopsis Tic110 Is Essential for the Assembly and Function of the Protein Import Machinery of Plastids

“…Covalent cross-linking studies with isolated chloroplasts demonstrate direct roles for Tic110, Tic40, Tic22, and Tic20 in preprotein import Chou et al, 2003;Inaba et al, 2003). Tic62, Tic55, and Tic32 are redox proteins that are proposed to play a regulatory role in protein import (Caliebe et al, 1997;Kuchler et al, 2002;Hormann et al, 2004).…”