Live cell Imaging of yeast cells expressing human PINK1-GFP and the TOM complex subunits

The PINK1-Parkin axis plays a major role in mitochondrial quality control and mutations have been closely associated with familial cases of Parkinson's disease. To assess the correct functioning of mitochondria, PINK1 acts as a sensor by monitoring their import capabilities. While proper import leads to degradation of PINK1, failed import causes PINK1 to form a complex with the translocon of the outer mitochondrial membrane, allowing it to be stabilized and activated by cross- phosphorylation. Here we describe a protocol for live-cell imaging of Saccharomyces cerevisiae cells expressing human PINK1 with GFP tag at the C-terminus as well as all the human TOM complex subunits (TOMs 5, 6, 7, 20, 22, 40 and 70) immobilized using Concanavalin A coating. The aim of this experiment is to investigate if human PINK1 expressed in yeast cells is localized to the mitochondria. In this case, galactose was used to induce expression of the fluorescence-tagged protein-of interest and colocalization was tested using a respective mitochondrial tracker.