CIS AND TRANS SITES OF THE TOM COMPLEX OF MITOCHONDRIA IN UNFOLDING AND INITIAL TRANSLOCATION OF PREPROTEINS

Translocation of preproteins across the mitochondrial outer membrane i s mediated by the TOM: complex, Our previous studies led to the concep t of two preprotein binding sites acting in series, the surface expose d cis site and the trans site exposed to the intermembrane space, We r eport here that preproteins are bound to the cis site in a labile fash ion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration, The sta bility of the trans site intermediate results from interactions of bot h the presequence and unfolded parts of the mature part of the preprot ein with the TOM complex, Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction, Efficient binding to the trans site and unfolding were observed with both outer membran e vesicles and intact mitochondria whose membrane potential, Delta Psi , was dissipated, Upon re establishing Delta Psi, trans site-bound pre protein resumed translocation into the matrix, The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria, We conclude that preprotein unfol ding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.