Functional Interaction Between Toc75 POTRA1 Domain and Tic22‐III in the Intermembrane Space During Chloroplast Protein Import

Chaperones are essential for facilitating the import of nuclear-encoded precursor proteins into chloroplasts. In the intermembrane space (IMS) of the chloroplasts, this process is mediated by the transport-associated domains (POTRA) of the translocon at the outer envelope membrane (Toc75) and translocon at the inner envelope membrane (Tic22) proteins. The present work aims to understand the interaction between the Toc75 POTRA domain and Tic22 in the IMS and determine their relationship in facilitating protein import. Expression of the POTRA1 domain deleted <i>TOC75</i> (<i>TOC75ΔP1</i>) in the <i>tic22-III</i> mutant background resulted in a more severe phenotype than the individual mutants, indicating that the two proteins functionally interact in the IMS. Using an insulin aggregation assay, we have demonstrated that Tic22-III also possesses chaperone-like activity. In vitro import experiments suggest that <i>TOC75ΔP1/tic22-III</i> plants are compromised in importing stromal and thylakoid membrane proteins. Therefore, we propose that the Toc75 POTRA domains and Tic22-III both provide chaperone activity necessary to prevent the misfolding of incoming pre-proteins, acting as chaperones and facilitating the protein import process through the IMS of the chloroplast.