Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles

Abstract

Mitochondria retain their own genome and translational apparatus that is highly specialized in the synthesis of a handful of proteins, essential components of the oxidative phosphorylation system. During evolution, the players and mechanisms involved in mitochondrial translation have acquired some unique features, which we have only partially disclosed. The study of the mitochondrial translation process has been historically hampered by the lack of an in vitro translational system and has largely relied on the analysis of the incorporation rate of radiolabeled amino acids into mitochondrial proteins in cellulo or in organello. In this chapter, we describe methods to monitor mitochondrial translation by labeling newly synthesized mitochondrial polypeptides with [S³⁵]-methionine in either yeast or mammalian whole cells or isolated mitochondria.

Keywords: Human cells; Mitochondrial translation; Newly synthesized polypeptides; Protein synthesis; Pulse-chase labeling; Yeast; [S35]-methionine.