Assessing Mitochondrial Unfolded Protein Response in Mammalian Cells.

Abstract

Mitochondria serve a key role in the supply of energy to cells in the form of ATP, the supply of essential cellular components such as phospholipids and heme, in apoptosis and as a mediator of cellular signaling pathways. Mitochondria have their own DNA, consisting of a small number of genes, but the majority of the total protein complement is encoded in the nucleus, synthesized in the cytosol, and is imported into the mitochondria in a largely, if not completely unfolded form. These proteins need to be folded into their functional form within the organelle with the concomitant requirement that the organelle has its own suite of molecular chaperones and complexes to degrade damaged proteins to avoid stress arising from accumulation of unfolded proteins. This mitochondrial unfolded protein response can also be induced in cells and protein regulation can be determined using western blot, luciferase reporter assay, and sensitive mass spectrometry techniques. In this chapter, we describe a method to induce mtUPR in mammalian cells and the three methods to analyze components involved in it.