Abstract
Protein import into the innermost compartment of mitochondria (the matrix) requires a membrane potential (delta psi) across the inner membrane, as well as ATP-dependent interactions with chaperones in the matrix and cytosol. The role of nucleoside triphosphates other than ATP during import into the matrix, however, remains to be determined. Import of urea-denatured precursors does not require cytosolic chaperones. We have therefore used a purified and urea-denatured preprotein in our import assays to bypass the requirement of external ATP. Using this modified system, we demonstrate that GTP stimulates protein import into the matrix; the stimulatory effect is directly mediated by GTP hydrolysis and does not result from conversion of GTP to ATP. Both external GTP and matrix ATP are necessary; neither one can substitute for the other if efficient import is to be achieved. These results suggest a "push-pull" mechanism of import, which may be common to other post-translational translocation pathways.
Highlights
Most (Ͼ95%) mitochondrial proteins are synthesized on cytoplasmic ribosomes
GTP plays an important role in protein trafficking across various membranes, far there is no evidence that GTP plays a direct role in mitochondrial protein import
Our data demonstrate that GTP hydrolysis does play a direct, essential role in translocation of proteins into the mitochondrial matrix, and that mitochondria can be added to the list of trafficking systems where GTP hydrolysis plays a critical role
Summary
(Received for publication, August 27, 1997, and in revised form, October 30, 1997). From the Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085. Using this modified system, we demonstrate that GTP stimulates protein import into the matrix; the stimulatory effect is directly mediated by GTP hydrolysis and does not result from conversion of GTP to ATP. We demonstrate that GTP stimulates protein import into the matrix; the stimulatory effect is directly mediated by GTP hydrolysis and does not result from conversion of GTP to ATP Both external GTP and matrix ATP are necessary; neither one can substitute for the other if efficient import is to be achieved. To investigate the GTP requirements for protein import into the mitochondrial matrix, we used a purified, urea-denatured precursor (delta-1-pyrroline-5-carboxylate dehydrogenase, pPut) in our assay. Both are necessary and neither one can substitute for the other
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