Reconstitution of human PINK1 and outer mitochondria TOM complex in yeast v1

PTEN induced kinase 1(PINK1) is a mitochondria kinase that phosphorylates ubiquitin and Ubl domain of parkin coincidentally at structurally obscured S65 in both proteins and initiate mitophagy. Dysregulation of this process has been implicated in cancer, obesity, cardiac disease, and neurodegenerative diseases (Youle and Narendra, 2011). PINK1 and TOM complex are key regulators in the mitochondrial quality control pathway. Physiologically under ideal condition PINK1 via its MTS forms a complex with the tom complex comprising of seven individual toms (TOMs 5, 6, 7, 20, 22, 40 and 70) going through the pores of TOM40 into the inner mitochondria (IMM) (Rasool et al., 2022). In the IMM PINK1 is cleaved within the MTS region precisely between residues 103 and 104 by two IMM proteases, MPP and PARL. The processed PINK1 is then released back into the cytosol where it is channelled towards proteasomal degradation (Rasool et al., 2022). In a pathological condition however, that’s upon mitochondrial damage, PINK1 import is blocked, preventing its processing. This results in PINK1 accumulation at the outer mitochondria membrane (OMM) and the activation of PINK1 through autophosphorylation, which subsequently initiates the mitophagy pathway to prevent the accumulation of reactive oxygen species from damaged mitochondria which could lead to cell apoptosis (Rasool et al., 2022). Here we describe in detail the protocol for making yeast cells stably expressing PINK1 and Human TOMs which has allowed us to characterise PINK1 when co-express with the TOMs.