Protein Transport to the Inner Chloroplast Membrane

Proteins targeted to the inner chloroplast membrane follow two distinct routes: the stop-transfer pathway and the re-insertion pathway. The stop-transfer pathway uses an N-terminal hydrophobic region that prevents complete translocation into the stroma, anchoring the protein directly to the inner membrane. The re-insertion pathway first translocates soluble precursors through the general TOC/TIC import pathway into the stroma, then reinserts them after signal peptidase cleavage.

In the stop-transfer pathway, an N-terminal hydrophobic region of the precursor acts as a transmembrane anchor that halts translocation through the TIC complex. This hydrophobic segment prevents the polypeptide from fully entering the stroma, causing the stalled precursor to be spontaneously inserted laterally into the inner membrane where it remains anchored.

After the transit signal is cleaved by signal peptidases in the stroma, the TIC stromal chaperone components—Hsp93, TIC40, and TIC110—bind the processed precursor. TIC40 coordinates with Hsp93 and TIC110 to facilitate reinsertion of the precursor into the inner membrane, ensuring proper protein folding and membrane integration.

A third pathway translocates plastid proteins that lack N-terminal transit sequences using the HP30-HP30-2 heteromer, which interacts with HP20 protein to facilitate translocation and insertion. This pathway targets proteins including chloroplast envelope quinone oxidoreductase homologs and other specialized plastid proteins that cannot use standard TOC/TIC routes.

Most plastid proteins carry N-terminal transit sequences and internal import sequences that target them to specific chloroplast subcompartments. In the stop-transfer pathway, internal hydrophobic sequences inhibit translocation into the stroma. In the re-insertion pathway, an N-terminal re-insertion signal exposed after transit signal cleavage guides the precursor to the Hsp93-TIC40-TIC110 complex for membrane insertion.

When a precursor with an N-terminal hydrophobic region is arrested across the TIC complex, it undergoes lateral release directly into the inner membrane. The hydrophobic segment serves as a membrane anchor, securing the protein in place without requiring complete translocation into the stroma or subsequent reinsertion steps.

After the transit signal is cleaved by stromal signal peptidases, the re-insertion signal becomes exposed on the processed precursor. This signal directs the precursor to the Hsp93-TIC40-TIC110 complex, where it is folded and inserted into the inner membrane with assistance from TIC40 and TIC110 proteins.