FIGURE 5:

Mrx15 interacts with nascent chains but does not form a stable complex with Mba1. (A) Cross-linking to nascent mitochondrial polypeptide chains. Mitochondrial translation products were radiolabeled in wild-type mitochondria or mitochondria containing a FLAG-tagged variant of Mrx15. After 15 min of labeling, the chemical cross-linker DFDNB was added to covalently link molecules in direct physical contact. The different reactions were stopped, and Mrx15 cross-linking products were purified with a FLAG resin. Mba1 and cross-linked molecules were iummunoprecipitated employing a PA resin and an Mba1 antibody. As a control, the reactions were performed without a FLAG tag or an Mba1 antibody. (B) Purification of FLAG-tagged Mrx15 variants. Mitochondria were lysed in two detergents (Triton X-100, digitonin) and Mrx15 purified by employing a FLAG resin. Western blots of the purification were probed for FLAG, Mba1, Mdm38, and Aco1. (C) Serial-dilution growth test on fermentable (YPD) and nonfermentable (YPG) carbon sources of indicated strains. E, elution; NB, not bound; T, total; WT, wild type.