THE REQUIREMENT OF MATRIX ATP FOR THE IMPORT OF PRECURSOR PROTEINS INTO THE MITOCHONDRIAL MATRIX AND INTERMEMBRANE SPACE

The role of ATP in the matrix for the import of precursor proteins int o the various mitochondrial subcompartments was investigated by studyi ng protein translocation at experimentally defined ATP levels. Protein s targeted to the matrix were neither imported or processed when matri x ATP was depleted. Import and processing of precytochrome b(2) (pb(2) ), a precursor carrying a bipartite presequence, into the intermembran e space was also strongly dependent on matrix ATP. Preproteins, consis ting of 220 or more residues of pb(2) fused to dihydrofolate reductase , showed the same requirement for matrix ATP, whereas the import of sh orter fusion proteins (up to 167 residues of pb(2)) was largely indepe ndent of matrix ATP. For those intermembrane-space-targeted proteins t hat did need matrix ATP, the dependence could be relieved either by un folding these proteins prior to import or by introducing a deletion in to the mature portion of the protein thereby impairing the tight foldi ng of the cytochrome b(5) domain. These results suggest the following: (a) The import of matrix-targeted preproteins, in addition to a membr ane potential Delta psi, requires matrix ATP [most likely to facilitat e reversible binding of mitochondrial heat-shock protein 70 (mt-Hsp70) to incoming precursors], for two steps, securing the presequence on t he matrix side of the inner membrane and for the completion of translo cation; (b) in the case of intermembrane-space-targeted precursors wit h bipartite signals, the function of ATP/mt-Hsp70 is not obligatory, a s components of the intermembrane-space-sorting pathway may substitute for ATP/mt-Hsp70 function (however, if a tightly folded domain is pre sent in the precursor, ATP/mt-Hsp70 is indispensable); (c) unfolding o n the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix-ATP/mt-Hsp70.