Chapter 23 Cross-Linking Reagents as Tools for Identifying Components of the Yeast Mitochondrial Protein Import Machinery

This chapter discusses cross-linking reagents as tools for identifying components of the yeast mitochondrial protein import machinery. Cross-linking is used to characterize a putative mitochondrial receptor for synthetic matrix-targeting peptides (p30). Cross-linking approaches can be very powerful in identifying molecules that are located next to each other. When the cross-link forms, the two molecules may exist as a stable complex or may be trapped in a transient state. Cross-Linking using bifunctional reagents are discussed in the chapter. A wide variety of cross-linkers are presented in the chapter and these reagents can be subdivided into two general classes: homobifunctional cross-linkers, which carry two identical functional groups, and heterobifunctional cross-linkers, which carry two reactive groups with different specificities. These later reagents have the advantage that cross-linking can be performed in a stepwise manner. The reactive groups are also discussed that are most commonly used in protein cross-linking experiments. The ability to cross-link two polypeptides depends on two conditions: (1) there must be two suitably located target sites on two proteins that can be attacked by an activated moiety of the cross-linker and (2) these two target sites must have the right distance from each other.