Analysis of ubiquitin signaling in the human mitochondrial protein import
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Abstract
Mitochondrial protein import is essential for eukaryotic organisms. Approximately 99% of mitochondrial proteins are imported from the cytoplasm, thereby ensuring proper mitochondrial function. Successful protein import is required for critical mitochondrial functions such as energy production, metabolism and immune signaling. Consequently, cells have developed a broad repertoire of mitochondrial import quality control mechanisms. Import defects are linked to several pathological conditions such as Alzheimer’s and Parkinson’s disease, and cancer, underscoring mitochondrial protein import quality control a topic of high medical and cell-biological relevance.
Ubiquitin, an essential eukaryotic modifier, appears to be central in the mitochondrial quality control in S. cerevisiae, but its role in human cells is poorly characterized. To address this gap of knowledge, a protein-based mitochondrial import clogging model was established in human cells, enabling protein engineering to study mitochondrial quality control. The mitochondrial quality control deals with the mitochondrial clogger by rapid degradation. The mitochondrial clogger is highly ubiquitylated, most likely leading to its degradation in a proteasome-dependent manner. In addition, proximity labeling was employed to characterize the interactome of both the mitochondrial clogger and the clogged mitochondrial surface. This provides an in-depth view on mitochondrial protein-protein interaction networks upon import stress in human cells. A cluster of proteins that regulate OXPHOS and mitochondrial gene expression was enriched in the proximity of the clogged mitochondrial surface. The interactome was unexpectedly enriched in nuclear speckle splicing factors whereas a cluster of proteins centered around the tumor suppressor BRCA1 decreased. Collectively these observations suggest that the mitochondrial surface under mitochondrial import stress defines a mitochondrial-nuclear signaling axis.
The analysis further reveals the potential role of atypical ubiquitylation of the mitochondrial clogger. However, tools to study atypical ubiquitylation in vivo remain limited. Therefore, the recently developed Ubiquiton system, which enables inducible ubiquitylation in vivo, was extended to include atypical linkages K6 and K11. This provides a perspective for future studies on ubiquitylation during mitochondrial import clogging.
In conclusion, this work connects two essential eukaryotic pathways, mitochondrial protein import and ubiquitin signaling and thereby contributes to a better understanding of the mitochondrial quality control which is crucial for cellular and organismic health.