FUSION PROTEINS CONTAINING THE CYTOCHROME B(2) PRESEQUENCE ARE SORTEDTO THE MITOCHONDRIAL INTERMEMBRANE SPACE INDEPENDENTLY OF HSP60

hsp60 is a chaperonin located in the mitochondrial matrix. It has been suggested that hsp60 participates in two processes: protein folding i n the matrix, and the sorting of imported proteins to the intermembran e space. We analyzed hsp60 function by allowing isolated mitochondria to import two model precursor proteins and then measuring the binding of these proteins to the chaperonin. Of the methods that we tested for monitoring the association of imported proteins with hsp60, only co-i mmunoprecipitation with specific anti-hsp60 antibodies proved to be re liable. A chimeric matrix-targeted precursor, consisting of a mitochon drial presequence fused to a chloroplast-encoded protein, bound stably to hsp60 after import. In contrast, there was no detectable binding t o hsp60 with a fusion protein that was targeted to the intermembrane s pace by the bipartite cytochrome b(2) presequence. Analysis of a trans location intermediate demonstrated that the cytochrome b(2) presequenc e arrests import through the inner membrane, with the result that the attached passenger protein is never exposed to hsp60.