Spatial proteomics provides new insight into proteostasis processes during acute mitochondrial stress

Objectives

Mitochondria are crucial for metabolism in eukaryotic cells which primarily produce ATP through oxidative phosphorylation. To fulfill their various functions, mitochondria need a healthy proteome that is maintained through the import of mitochondrial precursor proteins, a process highly scrutinized by the ubiquitin-proteasome system (UPS). Mitochondrial dysfunction and defects in the UPS are linked to human diseases. Problems with mitochondrial protein import trigger a cellular stress response, with UPS quickly trying to remove the mislocalized precursor proteins. However, knowledge about the different factors and mechanisms involved in mitochondria-associated degradation (MAD) pathways is incomplete. This study aims to explore the UPS-dependent factors and processes that uphold mitochondrial and cellular proteostasis during acute mitochondrial dysfunction using spatial and functional proteomics approaches.

Methods

We induced mitochondrial protein import stress in human HEK293T cells by inhibiting the presequence protein import pathway with a chemical probe. To study changes in protein abundance and localization, we perform global and spatial proteomics using quantitative MS technology. Subcellular fractions are generated by differential and density gradient centrifugation. Protein correlation profiling analysis with organellar marker proteins is used to reveal relocalization events. Selected candidates from proteomics data are further examined by high-resolution microscopy. Validated UPS factors are characterized for their role in MAD using functional proteomics, including loss-of-function analysis, protein proximity labelling, and ubiquitinome profiling.

Results

Through spatial proteomics, we profiled different subcellular niches and analyzed the subcellular localization of proteins during mitochondrial dysfunction. Our spatial data indicate the involvement of novel components of the UPS in MAD processes. Notably, the dynamic relocalization of central subunits of the proteasome machinery appears to represent a direct mechanism for the efficient clearance of arrested and/or non-imported mitochondrial precursor proteins accumulating at the mitochondrial surface. Our initial screen has also identified several E3 ligases associated with dysfunctional mitochondria and first interactome data indicate a specific recruitment of UPS factors. We are currently validating these findings using multiple experimental approaches.

Conclusion

Our spatial proteomics and microscopic analyses proved to be effective for identifying new UPS factors and processes involved in MAD pathways in human cells. Our data indicate that the specific relocalization of UPS factors to a subcellular niche where proteostasis is disturbed provides a direct, fast mechanism for maintaining a functional cellular proteome during acute mitochondrial stress conditions.