Mitochondrial biogenesis requires translocation of numerous preproteins across both outer and inner membranes into the matrix of the organelle. This translocation process requires a membrane potential (⌬⌿) and ATP. We have recently demonstrated that the efficient import of a urea-denatured preprotein into the matrix requires GTP hydrolysis (Sepuri, N. B. V., Schü lke, N., and Pain, D. (1998) J. Biol. Chem. 273, 1420 -1424). We now demonstrate that GTP is generally required for efficient import of various preproteins, both native and urea-denatured. The GTP participation is localized to a particular stage in the protein import process. In the presence of ⌬⌿ but no added nucleoside triphosphates, the transmembrane movement of preproteins proceeds only to a point early in their translocation across the inner membrane. The completion of translocation into the matrix is independent of ⌬⌿ but is dependent on a GTP-mediated "push." This push is likely mediated by a membrane-bound GTPase on the cis side of the inner membrane. This conclusion is based on two observations: (i) GTP does not readily cross the inner membrane barrier and hence, primarily acts outside the inner membrane to stimulate import, and (ii) the GTP-dependent stage of import does not require soluble constituents of the intermembrane space and can be observed in isolated mitoplasts. Efficient import into the matrix, however, is achieved only through the coordinated action of a cis GTP-dependent push and a trans ATP-dependent "pull."Most mitochondrial proteins are synthesized as precursors in the cytosol and subsequently translocated into the organelle. Proteins targeted to various mitochondrial subcompartments (outer membrane, intermembrane space, inner membrane or matrix) follow a variety of import pathways that differ in their energy requirements. For example, a membrane potential is required for the translocation of a preprotein across the inner membrane, but not into or across the outer membrane (1-3). Translocation of proteins across both membranes into the matrix of the organelle is particularly complex. It requires the coordinated action of two separate translocases, one (Tom) located in the mitochondrial outer membrane and the other (Tim) located in the inner membrane (4 -6). Also required are ATP-dependent interactions with molecular chaperones both outside and inside the organelle (for review, see Refs. 7 and 8).Among the cytosolic chaperones are Hsp70 and mitochondrial import stimulating factor (MSF).1 Hsp70 generally interacts with preproteins to keep them in a translocation-competent conformation. MSF, on the other hand, specifically binds mitochondrial signal sequences, preventing and/or reversing preprotein aggregation, then targeting the precursors to the outer membrane. The MSF-dependent import pathway requires cytosolic ATP, whereas the Hsp70-dependent pathway does not (7). The import of urea-denatured precursors does not require added cytosolic chaperones and may or may not require ATP (9 -12).Matrix-localized Hsp70 (mt-Hsp70) interac...