title: Proximity labeling approaches to mitochondrial import receptors TOMM20 and TOMM70
Creators: name: Akram, Saira
Publisher: name: Universität Tübingen
Year: 2025
Subjects: subject: 500
subject: TOMM20
subject: TOMM70
subject: Proximity labeling
subject: TOM complex
subject: APEX2

description: In yeast, import of most mitochondrial proteins requires that their precursor proteins are bound by the peripheral receptor proteins Tom20, Tom22, and Tom70, a process conserved from yeast to mammals. For budding yeast Tom20 and Tom70, there is evidence of specific yet overlapping substrate recognition, however, no such data is available for the metazoan cells. Although mitochondrial protein import has been extensively studied in past years, the cytosolic stage of this process remains enigmatic. The cytosolic ribosomes interacting with the TOM complex have been found at the mitochondrial outer membrane (MOM). mRNA localization to specific subcellular compartments is an essential process in the eukaryotic cells, allowing precise and targeted distribution of mRNAs to particular site, where proteins can be synthesized locally, exactly where they are needed. Numerous nuclear-encoded mitochondrial mRNAs are increasingly recognized to be localized to MOM. mRNA localization to the mitochondria is well documented in polarized cells like neurons but global occurrence of mRNA localization in other mammalian cells remains to be analyzed. The key players involved in this process mainly include RNA binding proteins (RBPs), which presumably regulate the targeting and local translation of their putative targets at MOM. In my project, I aimed to identify the association profiles for TOMM20 and TOMM70 in mammals. I particularly focused on identifying novel RBPs, associating with these receptors and probably being involved in localized translation at the TOM complex. For that, I established an APEX2-based proximity labeling methodology, and targeted it to human TOMM20 and TOMM70 receptors in HeLa cells. This approach allowed me to capture the distinct interactomes of TOMM20 and TOMM70 receptors within their nano-environments. Each receptor showed enrichment of a unique set of proteins, though several RBPs and translation factors were preferentially more enriched in TOMM20 interactome over TOMM70 interactome. These include SYNJ2BP, a previously identified MOM-localized 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 homeostasis during translation stress by retaining protective RBPs and translation-related proteins at the MOM. Further validation of identified interactors by orthogonal experiments could potentially identify their complex interaction and function at TOM complex. Then, nearby mRNAs biotinylated by TOMM20 bait protein were identified in a preliminary RNA-seq analysis. Not any particular enrichment of nuclear-encoded mitochondrial transcripts over non-mitochondrial ones was identified. It appears like RNA-Seq pipeline requires further optimization to identify local transcriptome at the TOM complex. However, I established an APEX2 based methodology for studying the local proteome of the human TOMM20 and TOMM70 receptors at MOM in HeLa cells. In future, this methodology could be further applied in other cells, such as neurons, to identify receptor specific interactomes. The identified interactomes could be further characterized and compared with analyses of other TOM receptors to reveal complex interactions at MOM, and provide deeper insights into localized translation processes and their contribution to mitochondrial import.