Mitochondrial protein import: Nucleoside triphosphates are involved in conferring import-competence to precursors

Abstract

The role of nucleoside triphosphates (NTPs) in mitochondrial protein import was investigated with the precursors of N. crassa ADPlATP carrier, F1-ATPase subunit 9, F,-ATPase subunit 9, and fusion proteins between subunit 9 and mouse dihydrofolate reductase. NTPs were necessary for the initial interaction of precursors with the mitochondria and for the completion of translocation of precursors from the mitochondrial surface into the mitochondria. Higher levels of NTPs were required for the latter reactions as compared with the early stages of import. Import of precursors having identical presequences but different mature protein parts required different levels of NTPs. The sensitivity of precursors in reticulocyte lysate to proteases was decreased by removal of NTPs and increased by their readdition. We suggest that the hydrolysis of NTPs is involved in modulating the folding state of precursors in the cytosol, thereby conferring import competence.

Protein transport into mitochondria

“…However, when the dissipation of A^is combined with a lowering of the temperature (to 2°C) or of the levels of ATP, the precursor remains on the surface of the mitochondria accessible to externally added proteases. Raising the temperature or the levels of ATP leads to insertion of the precursor into the second binding sites described above (3,8). These studies show the existence of two subsequent binding sites on the import pathway of ADP/ATP carrier.…”

“…Major portions of the precursors, however, were still outside the outer membrane as they were accessible to proteases or to antibodies added to isolated mitochondria. Thus, the precursor proteins were reaching from the outside of the outer membrane into the matrix thereby spanning sites of close contact between both membranes (translocation contact sites) (3,8,(10)(11)(12). The translocation intermediates with bound antibodies were decorated with protein-A-gold particles.…”

“…Nucleoside triphosphates are required to keep cytosolic precursors in an unfolded conformation (3,4). The precursors specifically interact with proteinaceous binding sites on the mitochondrial surface (5-9).…”

Import of a mutant mitochondrial precursor fails to respond to stimulation by a cytosolic factor

“…Recently, it has been reported that an enzymatic unfolding activity is necessary for the denaturation of native proteins that are to be translocated across microsomal [11,12] and mitochondrial [11,19] membranes. This activity has been attributed to a member(s) of the 70 kDa heat shock protein family (hsp 70).…”

Contact sites between inner and outer membranes

“…Various methods were developed for the accumulation of precursor proteins in mitochondrial contact sites: import of precursor proteins at low temperature [9,15,20]; lowering the levels of ATP in the import reaction [21]; prebinding of specific antibodies to portions of the precursor proteins [9,20]; and, induction of a stable tertiary structure in a domain of a precursor protein [18,19,22]. The underlying principle of these methods is that a (usually carboxyl-terminal) portion of a precursor protein is either not or only partially unfolded and thus cannot be inserted into the mitochondrial membranes.…”