Proximity Labelling Reveals Spatial Organisation of Mitochondrial Protein Import Complexes

Mitochondrial protein import and local translation at the mitochondrial outer membrane (MOM) require coordinated interactions between protein translocases and RNA-associated factors, yet the molecular organisation of these interactions remains largely unresolved. Here, we used APEX2-mediated proximity labelling to define the proximal proteomes associated with the cytosolic face of the TOM and SAM complexes by fusing APEX2 to TOMM22 and MTX2, respectively. Quantitative mass spectrometry identified known TOM/SAM-associated proteins together with multiple RNA-binding proteins (RBPs), supporting the emerging role of the MOM in localised translation and other RNA-related processes. Among identified RBPs, the exonuclease EXD2 was consistently enriched in APEX2-TOMM22 and MTX2-APEX2 datasets and remained associated upon puromycin treatment, indicating a translation-independent interaction. Together, our findings provide insights into the molecular organisation of mitochondrial import sites and identify EXD2 as a TOM/SAM-associated factor at the MOM.