From nucleus to mitochondria : a ubiquitination study

Ubiquitination is a post-translational modification achieved by an enzymatic cascade. This post-translational modification is involved in many crucial cellular processes across different cellular compartments such as protein turnover via the ubiquitin proteasome system and various signaling pathways from the DNA damage response to the immune response. Ubiquitin has seven lysine residues (Lys 6, 11, 27, 29, 33, 48 and 63) onto which other ubiquitin moieties can be conjugated, forming ubiquitin chains of different types. These different ubiquitin chains can have different functions and the relationship between both is often referred to as “the ubiquitin code”. Although the enzymatic cascade leading to ubiquitination of proteins is well described, the ubiquitin code remains largely unresolved. Accumulation of insoluble ubiquitinated proteins is a hallmark of neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease, making the study of these cellular processes relevant to human health. Specific proteins can also impair the function of the proteasome such as progerin in the Hutchinson-Gilford Progeria Syndrome (HGPS). The importance of ubiquitination in many cellular processes and its involvement in many human pathologies inspired us to develop an inducible ubiquitination system that could be used as a tool to better understand the ubiquitin code and its role in different cellular compartments. In paper I, we have engineered a ubiquitin ligase, ProxE3, which assembles specific ubiquitin chain (lysine 63) onto a fluorescent substrate. We have used this tool to generate ubiquitin chains on the surface of mitochondria and investigate mitophagy, more precisely if lysine 63 (K63) ubiquitin chains are sufficient to trigger aggregation of mitochondria or mitophagy. Upon successful ubiquitination of the surface of mitochondria by ProxE3 and depolarization of mitochondria by carbonyl cyanide mchlorophenyl hydrazone (CCCP), peri-nuclear clustering of mitochondria was observed but not mitophagy. The lack of ...