Precursor proteins made in the cytoplasm must be in an unfolded confor
mation during import into mitochondria, Some precursor proteins have t
ightly folded domains but are imported faster than they unfold spontan
eously, implying that mitochondria can unfold proteins, We measured th
e import rates of artificial precursors containing presequences of var
ying length fused to either mouse dihydrofolate reductase or bacterial
barnase, and found that unfolding of a precursor at the mitochondrial
surface is dramatically accelerated when its presequence is long enou
gh to span both membranes and to interact with mhsp70 in the mitochond
rial matrix, If the presequence is too short, import is slow but can b
e strongly accelerated by urea-induced unfolding, suggesting that impo
rt of these 'short' precursors is limited by spontaneous unfolding at
the mitochondrial surface, With precursors that have sufficiently long
presequences, unfolding by the inner membrane import machinery can be
orders of magnitude faster than spontaneous unfolding, suggesting tha
t mhsp70 can act as an ATP-driven force-generating motor during protei
n import.