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

## TL;DR

The study reveals how Toc75 POTRA1 and Tic22-III work together in chloroplasts to help import proteins and prevent misfolding.

## Contribution

The study identifies a functional interaction between Toc75 POTRA1 and Tic22-III in chloroplast protein import.

## Key findings

- Deleting the POTRA1 domain of Toc75 in tic22-III mutants causes a more severe phenotype, showing functional interaction.
- Tic22-III has chaperone-like activity, preventing pre-protein misfolding during import.
- TOC75ΔP1/tic22-III plants are impaired in importing stromal and thylakoid membrane proteins.

## Abstract

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 TOC75 (TOC75ΔP1) in the tic22‐III 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 TOC75ΔP1/tic22‐III 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.