Abstract
The association between ribosomes and the pore proteins at the endoplasmic reticulum membrane is important to co-translational translocation. To determine if a similar association occurs between the ribosome and mitochondrial membrane protein(s) during protein import in higher eukaryotes, we examined ribosome-mitochondria binding. By using spectral measurements, analysis of mitochondrial associated RNA, and electron microscopy, we demonstrated that ribosomes stably bind to purified rat liver mitochondria in vitro. Binding of ribosomes to mitochondria was markedly reduced by GTP and nearly abolished by the non-hydrolyzable GTP analogue, guanosine-5'-[thio]-triphosphate (GTPgammaS), but was only modestly reduced by GDP or ATP and unaffected by CTP. The initial rate of GTP hydrolysis by mitochondria was increased by ribosomes, whereas the rate of ATP hydrolysis by mitochondria was not affected. Ribosomes programmed with mRNA for 92 amino acids of the N terminus of mitochondrial malate dehydrogenase bound to mitochondria, but unlike unprogrammed rat liver ribosomes, neither GTP nor GDP disrupted binding; however, GTPgammaS did. These data show that receptors specific for ribosomes are present on the mitochondrial membrane, and a GTP-dependent process mediates this binding. The presence of a nascent chain alters these binding characteristics. These findings support the hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria.
Highlights
Proteins can cross membranes by either post-translational or co-translational translocation
Because recent studies have shown that GTP-binding proteins are involved in initiating protein translocation at the membrane of the endoplasmic reticulum (ER),1 but are involved in protein import into chloroplast [19], we determined the effect of ATP and GTP on ribosomes binding to mitochondria and the early events in protein translocation
Ribosomes Bind to Mitochondria—Rat liver mitochondria prepared by differential centrifugation have been shown to contain other organelles including a significant amount of rough endoplasmic reticulum-derived microsomes [26]
Summary
Proteins can cross membranes by either post-translational or co-translational translocation. Methotrexate, which inhibits post-translational import of cytochrome oxidase subunit IV-dihydrofolate reductase by preventing its unfolding, does not inhibit cytochrome oxidase subunit IV-dihydrofolate reductase import in vivo [9] This indicates that co-translational translocation may eliminate the need for precursor proteins to be maintained in a translocation-competent state by chaperones in the cytoplasm. “targeting factor” or mitochondrial stimulating factor, and the precursor is recognized and imported by a multisubunit translocation complex in the mitochondrial membranes [13,14,15,16,17,18] Since these early events in targeting and translocation clearly and involve the N-terminal presequence, it is entirely possible that mitochondrial protein import can be initiated long before translation is complete. Because recent studies have shown that GTP-binding proteins are involved in initiating protein translocation at the membrane of the endoplasmic reticulum (ER), but are involved in protein import into chloroplast [19], we determined the effect of ATP and GTP on ribosomes binding to mitochondria and the early events in protein translocation
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