Proximity labeling reveals differential interaction partners of the human mitochondrial import receptor proteins TOMM20 and TOMM70

Import of most mitochondrial proteins requires that their precursor proteins are bound by the (peripheral) receptor proteins TOM20, TOM22, and TOM70. For budding yeast TOM20 and TOM70, there is evidence of specific yet overlapping substrate recognition, but no such data is available for metazoan cells. Using APEX2-based proximity labeling, we thus created association profiles for human TOMM20 and TOMM70 in HeLa cells. We particularly focused on their interaction with RNA-binding proteins (RBPs) since there is evidence for RNA association with the mitochondrial outer membrane (MOM) and local translation at the mitochondrial surface, but these processes are poorly understood. Our results show a preferred association of several RBPs and translation factors with TOMM20 over TOMM70. These include SYNJBP2, a previously identified membrane-bound RBP that binds and protects mRNAs encoding mitochondrial proteins. Translational inhibition by puromycin resulted in an even increased association of these RBPs with TOMM20 compared to TOMM70, suggesting that TOMM20 but not TOMM70 might play a role in preserving cellular hemostasis during translation stress by retaining protective RBPs and translation-related proteins at the MOM.