DIRECTIONALITY OF POLYPEPTIDE TRANSFER IN THE MITOCHONDRIAL PATHWAY OF CHAPERONE-MEDIATED PROTEIN-FOLDING

Protein folding in mitochondria depends on the functional cooperation of the Hsp70 and Hsp60 chaperone systems, at least for a subset of mit ochondrial polypeptides. As suggested previously, Hsp70 and Hsp60 act sequentially. However, recent proposals that the chaperonin Hsp60 func tions by releasing substrate protein in an unfolded state would predic t a lateral partitioning of folding intermediates between chaperone sy stems. Firefly luciferase, carrying a mitochondrial targeting signal, was used as a model protein to analyze the degree of coupling and the directionality of substrate transfer between the Hsp70 and Hsp60 chape rones. In vitro, Hsp60 binds unfolded luciferase with high affinity bu t is unable to promote its folding, whereas the Hsp70 system assists t he folding of luciferase efficiently. Upon import into yeast mitochond ria, luciferase interacted first with Hsp70. Surprisingly, most of the protein subsequently accumulated in a complex with Hsp60 and never re ached the native state. Import into mitochondria that lack a functiona l Hsp60 did not result in increased folding, but in the aggregation of luciferase. Thus, in intact organelles the two chaperone systems do n ot function independently in de novo folding of aggregation-sensitive proteins but rather act in an ordered pathway with substrate transfer predominantly in the direction from Hsp70 to Hsp60.