Mapping of the local proteome of the Arabidopsis chloroplast intermembrane space by proximity labelling

The chloroplast intermembrane space, located between the outer and inner envelope membranes, is important for protein import and lipid trafficking. Currently, the proteome of the intermembrane space is largely uncharacterized, with only a relatively few proteins identified, limiting our understanding of the functional roles of this compartment. Here, we used TurboID-based proximity labelling to define the chloroplast intermembrane space proteome in Arabidopsis thaliana. TurboID was targeted to the intermembrane space by fusion to the chloroplast transit peptide (cTP) of Tic22, a 22-kDa component of the inner envelope translocon. Localization of Tic22cTP fusion proteins was confirmed by confocal microscopy and chloroplast protease protection assays. Following optimization of biotinylation, mass spectrometry identified a distinct set of candidate intermembrane space-localized or proximal proteins, including known components of protein import and lipid biosynthesis/trafficking pathways, as well as several novel proteins. Comparative analyses of plants exposed to high light and in the rhomboid-like protein10 lipid mutant revealed changes in the intermembrane space proximal proteome. Selected candidate proteins were further validated for their localization using fluorescent protein fusions and bimolecular fluorescence complementation assays. Together, these findings provide a valuable proteomics-based resource for the chloroplast intermembrane space and offer new insights to the contribution of this compartment to chloroplast function.