Analysis of mitochondrial protein import pathway in Saccharomyces cerevisiae with translocation intermediates

This chapter focuses on the methods for characterizing translocation intermediates that span the mitochondrial membranes and discusses the utility of their study for elucidating new aspects of the mechanism for protein translocation across membranes. The standard protocols for the in vitro synthesis and import of precursor proteins into isolated mitochondria are described. The future analysis of membrane-spanning translocation intermediates in studies on mitochondrial protein import should help identify the functions of newly identified components of the import apparatus. It will be, especially useful to accumulate translocation intermediates in mitochondria isolated from yeast strains harboring conditional mutations in specific import components. The analysis of translocation intermediates will also help determine the sequential series of interactions that occurs between precursor proteins and import components during protein import into the matrix. Determination of these reactions will provide an estimate of the affinity of different import components for the presequence and mature regions of precursor proteins. This information will provide a starting point for the reconstitution of protein translocation reactions with purified components. © 1995 Elsevier Inc.