Chapter 11 Assaying protein import into mitochondria

Abstract

Publisher Summary Most proteins found in the mitochondria originate in the cytosol and therefore must be imported into this organelle following their synthesis. Most of the translocase components of the outer and inner mitochondrial membranes (TOM and TIM, respectively) have been identified in Saccharomyces cerevisiae and Neurospora crassa . A preprotein is directed to the mitochondria by virtue of its targeting signal, which is most often situated at the N terminus. Preproteins are first bound to receptors of the mitochondrial membrane—Tom70, Tom22, and Tom20—before being translocated across the outer membrane channel, which is formed by Tom40. Many preproteins destined to reside in the outer membrane transverse laterally out of the channel upon contact of their hydrophobic anchors with the TOM machinery. Further, another group of inner membrane proteins contain internal targeting signals that direct them from the inner face of the TOM machinery to a different TIM complex (the TIM22 complex) via their association with small intermembrane space TIM proteins. The characterization of these different import pathways has been achieved mainly through the use of mitochondrial in vitro import studies. In such cases, isolated mitochondria are incubated with in vitro -translated 35 S-labeled preproteins under varying conditions, and their import characteristics and requirements are analyzed. This chapter describes various methods employed to characterize both the mitochondrial import of preproteins and the protein import machinery in vitro .