Chapter 18 Pulse Labeling of Yeast Cells as a Tool to Study Mitochondrial Protein Import

Abstract

This chapter presents a simple labeling protocol that measures protein import into mitochondria of intact yeast cells. The method exploits the fact that most nuclear-encoded mitochondrial proteins are synthesized in the cytoplasm as larger precursors. The amino-terminal presequence is cleaved off by the matrix protease during import into the mitochondria, thereby resulting in a smaller protein inside the mitochondria. Thus, by pulse labeling yeast cells with a radioactive amino acid, the newly synthesized precursor and the newly imported mature form can be detected as labeled polypeptides after immunoprecipitation, SDS-gel electrophoresis, and fluorography. This assay depends on the availability of monospecific antibodies that recognize both the precursor and the mature form of the protein. Most polyclonal antibodies raised against mature proteins fulfill this criterion. In vivo labeling proved to be a valuable part of the methodology to analyze mitochondrial protein import. As classic methods to study mitochondrial protein import, pulse-labeling experiments with intact cells have contributed significantly to the identification of important features and components of the mitochondrial protein import machinery. Thus, they showed that most nuclear-encoded mitochondrial proteins contain presequences, which upon import are removed by the matrix-localized protease and which some nuclear encoded mitochondrial polypeptides contain complex presequences that are cleaved twice, first by the matrix protease and then by other proteases.