The researchers elucidated the supramolecular architecture of the TOC-TIC supercomplex through cryo-electron microscopy.
Thirteen different protein subunits in this supercomplex were discovered. With the exception of Tic214 encoded by the chloroplast genome, all the other subunits are nuclear encoded. They are assembled into the TOC complex in the outer membrane, the intermembrane space complex (ISC) and the TIC complex in the inner membrane. Remarkably, it was found that the largest membrane protein Tic214 spans the inner membrane, the intermembrane space and the outer membrane, linking the other protein subunits like a bridge and most likely also acting as a scaffold.
The TOC complex in the outer membrane is mainly composed of Toc34, Toc90 and Toc75, flanked on the Toc90 side by the Ctap4-Ctap3 complex. A hybrid barrel-shaped channel is formed by Toc90 and Toc75 on the outer membrane. The channel contains an entrance on the cytosolic side and two exits opening toward the intermembrane space, as well as a lateral gate facing the lipid bilayer. A phytic acid (also known as inositol hexaphosphate/InsP6) molecule intercalates at the interface between Toc90 and Tic214, stabilizing their assembly like a wedge.
The intermembrane-space domain of Tic214, Tic100, Tic56, Ctap3 and Ctap5 intertwine with each other to form a tower-like structure connecting TOC with TIC. In the inner membrane, the membrane-embedded domains of Tic214, Tic20, Ctap5 and three small subunits (named Simp1, Simp2 and Simp3) form the TIC complex. Four lipid molecules serve to stabilize the assembly of a funnel-like channel located at the interface between Tic214 and Tic20 and prevent the channel from leaking.
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