43] Analysis of mitochondrial function and assembly

Abstract

This chapter describes the analysis of mitochondrial function and assembly. The first approach for analysis involves in vitro assays employing isolated mitochondria and has illuminated many of the key steps in mitochondrial protein import. A second approach has been the cloning, analysis, and manipulation of genes encoding imported mitochondrial proteins. This type of investigation has revealed targeting signals of many imported polypeptides. A third approach has involved the isolation and characterization of mutant yeast defective in mitochondrial protein import. Such studies have led to the identification of specific components of the import apparatus. The experimental procedures described in the chapter allow the analysis of many aspects of mitochondrial function and assembly. The approaches focus on the analysis of mitochondria in intact cells, the isolation of functional mitochondria, and the assay of mitochondrial function and protein import in cell-free systems. These procedures can be employed to characterize the fate of a specific mitochondrial protein or to analyze the effect of a mutation on mitochondrial function or on protein import.