A GTP-dependent 'push' is generally required for efficient protein translocation across the mitochondrial inner membrane into the matrix

Abstract

Mitochondrial biogenesis requires translocation of numerous preproteins across both outer and inner membranes into the matrix of the organelle. This trans, location process requires a membrane potential (ΔΨ) 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., Schulke, N., and Pain, D. (1998) J. Biol. Chem. 273, 14201424). 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 ΔΨ 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 ΔΨ 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'.