Abstract
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.
Highlights
From the Departmentof Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
We found that the import of bulk mitochondrial proteins was inhibiteind both the homologous in vitroreaction andin vivoupon arrest of cytosolic protein synthesis with the addition of cycloheximide
DHFR bypreventing theDHFR moiety from unfolding. ing the events that occur at the mitochondrial surface and,wefound thatantifolateadditionhadno inside the organelle, there are a number of unanswered inhibitory effect on the import of COXIV-DHFR in questions involving events that occur before precursor provivo, suggesting that its import into mitochondria in teins engage the importreceptors
Summary
From the Departmentof Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033. Considerable cytmnrheieudoaalesnattmdeticdro-eicnaylhlleslmoaofsroetomcepcxwbhutirerrtaanshnmyecltihohptoyteorcdtaehrpnanorsztonlioatdaontlrineaioantnolidpaocilrnonleoyldclp.auuhpcFrosseuoertrehrtheceptarhor, beowolsmanh,cyeicwtlnuoe--cwyae6faf-aam)cc, totaoounrrnssNtmi-noeafctylwhubydoelermikanavlhcoeyailtsvomesriodedlseiiunc-lstemehndeisatiiotntcsihhtvohoecnecosdkumr-gilaipgkloeensptpeironroonttte(eti7hin)na,,tiamhncsydppto7oar0sto.2(l8i5Sc-,uch found that the ability to be imported by a strictly kDa protein isolated from both rabbit reticulocyte and rat posttranslational mechanism upon reestablishing the liver lysates [8,9] This latter component cabne isolated from membrane potential varied from one precursor to an- high salt washes of isolated rat liver mitochondria aswell as other, suggesting that cotranslational import may be from cytosolic fractions which has led the authors tosuggest mandatory for the importof some proteins in vivo.In that the role of this factor in mitochondrial protein import summary, our findingasre entirely consistent with the may resemble that of the signal recognition particle in the notion that importof proteins into yeast mitochondria transport of secretory proteins into tehnedoplasmic reticulum occurs cotranslationally under normal conditions vivo. A coupled cell-free translation/import reaction with a Miscellaneous-Published procedures wereused for SDS-polyfinal volume of 625 p1 contained 237.6 p1 of non-nuclease-treated acrylamide gel electrophoresis and fluorography [18]with the modiyeast lysate, 62.5 p1 of 10 mg/ml mitochondria, and was adjusted to fications described previously [12]. Protein was measured by the Biothe followingconcentrations: 48 mM HEPES-KOH, pH 7.4, 170 mM Rad protein assay according to themanufacturer's instructions. potassium acetate, 17 mM creatinephosphate, 70 pg/ml creatine phosphokinase, 25 p~ of each amino acid except methionine, 1 mM
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