Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Abstract

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

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

“…The Tic20 family consists of four genes in Arabidopsis: Tic20-I, Tic20-IV, Tic20-II, and Tic20-V (Jarvis, 2008). Tic20-I characterized in this study is the most abundant isoform among four proteins and is the closest homolog to the biochemically identified pea Tic20 (Kouranov et al, 1998). The other three Tic20 isoforms in Arabidopsis may be responsible for the import of nonphotosynthetic and housekeeping proteins.…”

“…Toc75, an integral membrane protein, was largely resistant to thermolysin and trypsin. Tic22, which is known to reside in the intermembrane space (Kouranov et al, 1998), was resistant to thermolysin but sensitive to trypsin. Stromal Hsp70 and Tic110, an inner envelope membrane protein that is oriented toward the stroma (Jackson et al, 1998;Inaba et al, 2003), were completely resistant to both thermolysin and trypsin ( Figure 4B).…”

“…Thermolysin is an outer envelope-impermeable protease that selectively digests chloroplast surface-exposed proteins, whereas a certain concentration of trypsin is able to destroy the membrane integrity of the outer envelope and partially digest proteins within the intermembrane space while leaving the stromal proteins undigested (Kouranov et al, 1998;Hirohashi et al, 2001). Toc159, which has a large cytosolically exposed domain, was easily degraded by thermolysin and trypsin, as demonstrated by the resultant 52-kD protease-resistant fragment ( Figure 4B).…”

“…Tic20 has been proposed to function as the protein-conducting channel of the inner membrane. Tic20 was identified by chemical cross-linking to translocating preproteins in pea (Pisum sativum) chloroplasts (Kouranov et al, 1998). Tic20 is an integral membrane protein and is predicted to have three or four transmembrane helices.…”

The Amino-Terminal Domain of Chloroplast Hsp93 Is Important for Its Membrane Association and Functions in Vivo

“…Most Hsp93 proteins are found in the stroma. However, a significant portion of Hsp93 molecules are associated with the inner envelope membrane and can be coisolated with other Tic components (Moore and Keegstra, 1993;Akita et al, 1997;Nielsen et al, 1997;Kouranov et al, 1998;Su and Li, 2010). In Arabidopsis (Arabidopsis thaliana), Hsp93 is encoded by two genes, atHSP93III (At3g48870; ClpC2) and atHSP93V (At5g50920; ClpC1), and atHsp93V is the major functional form.…”