Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo.
We have utilized a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae, in addition to performing a series of in vivo experiments in yeast, to investigate the coupling between cytosolic protein synthesis and protein transport into mitochondria. We found that the import of bulk mitochondrial proteins was inhibited in both the homologous in vitro reaction and in vivo upon arrest of cytosolic protein synthesis with the addition of cycloheximide. Tight coupling of synthesis and import was also demonstrated in vivo for the beta subunit of the mitochondrial F1-ATPase. We also investigated the effect of the antifolate methotrexate on the import of a fusion protein consisting of the mitochondrial targeting signal of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (the COXIV-DHFR fusion protein). Methotrexate has previously been shown to inhibit posttranslational import of COXIV-DHFR by preventing the DHFR moiety from unfolding. However, we found that antifolate addition had no inhibitory effect on the import of COXIV-DHFR in vivo, suggesting that its import into mitochondria in yeast cells occurs cotranslationally. Further, when we treated yeast with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone to collapse the mitochondrial membrane potential and induce the accumulation of extramitochondrial precursor pools, we found that the ability to be imported by a strictly posttranslational mechanism upon reestablishing the membrane potential varied from one precursor to another, suggesting that cotranslational import may be mandatory for the import of some proteins in vivo. In summary, our findings are entirely consistent with the notion that import of proteins into yeast mitochondria occurs cotranslationally under normal conditions in vivo.
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey 17033.
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M
Fujiki
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K
Verner
D000818:Animals; D001692:Biological Transport; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis*; D007163:Immunosorbent Techniques; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003513:Cycloheximide; D013762:Tetrahydrofolate Dehydrogenase; D003576:Electron Transport Complex IV; D006180:Proton-Translocating ATPases; D008727:Methotrexate