Presentation description

Mitochondrial dysfunction is a hallmark of age-related cellular decline, and to maintain mitochondrial homeostasis, cells utilize numerous mitochondrial degradation pathways. The Hughes lab identified Mitochondrial-Derived Compartments (MDCs) as a unique structure in a quality control pathway that removes a subset of inner mitochondrial membrane (IMM) and outer mitochondrial membrane proteins (OMM) in yeast and mammalian cells. OMM protein, Tom70, is both an MDC cargo and required for MDC formation, but the mechanisms by which it contributes remain unclear. Given ubiquitination's significance in membrane remodeling and protein sorting, we investigated its possible role in MDC formation by inhibiting ubiquitination of Tom70. Deubiquitinase (DUB), a ubiquitin-removing protein, was tagged to Tom70 in yeast cells, followed by four MDC induction experiments by metabolic stress or protein overload: 1) Amino acid buildup by cycloheximide (CHX) treatment. 2) SRP knockdown by auxin treatment leading to protein overload at the mitochondrial surface. 3) OMM protein overload through SCM4 overexpression (OE) by genetic modification. 4) Amino acid buildup by rapamycin (rap) treatment, a known MDC inducer. Fluorescent marker mCherry on IMM protein Tim50, which is excluded from MDCs, was used to visualize the IMM. Experiments 1-3 tagged GFP to MDC cargo protein, Tcd2, while experiment 4 tagged GFP to Tom70-DUB. Experiments 1 and 3, alongside prior data, showed that the DUB on or near Tom70 blocked Tcd2-positive MDCs under tested stress. In experiment 2, we saw no blockage of Tcd2-positive MDCs. Experiment 4 found that Tom70-positive MDCs were blocked by the DUB, suggesting that the DUB directly inhibited MDC formation rather than just preventing cargo entry into MDCs. These findings demonstrate ubiquitin's involvement in Tcd2 or Tom70-positive MDC formation under specific stress. Future studies should explore whether the observed ubiquitination requirement is specific to Tom70 or if it is ubiquitin’s presence on the mitochondrial surface that is necessary for MDC formation.