Coupling of protein synthesis and mitochondrial import in a homologous yeast in vitro system.

M Fujiki,K Verner

doi:10.1016/s0021-9258(20)89577-5

Abstract

We made use of a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae to investigate the coupling of protein synthesis and import. Mitochondrial precursor proteins were synthesized in a yeast lysate either in the presence or absence of isolated yeast mitochondria. We were, therefore, able to analyze protein import into mitochondria either in a strictly posttranslational reaction (when isolated mitochondria were added only after protein synthesis has been arrested by the addition of cycloheximide) or in a reaction in which synthesis and import were permitted to occur simultaneously. We found that the import of a precursor protein consisting of the amino-terminal mitochondrial targeting sequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase is very inefficient in a strictly posttranslational reaction, whereas efficient import is observed if precursor synthesis and import are coupled. The same result was obtained when we analyzed the import of bulk endogenous yeast mitochondrial proteins in this system. Finally, we found that the insertion of the yeast outer membrane protein porin is also several times more efficient when synthesis and insertion are coupled.

Highlights

Inthisstudy, we utilized the homologous yeastimport system to examine the import of a fusion protein consisting
We examined the insertion of the yeast outer advance that has contributed to our understanding of the mitochondrial membrane protein porin and demonstrate that import process is thedevelopment of i n vitro import systems. it is inserted into the mitochondrial membrane much more
In this report we have demonstrated that the synthesisof mitochondrial precursor proteins and their import into isolated mitochondria can be very tightly coupled inayeast homologous in vitro system

Summary

Introduction

Inthisstudy, we utilized the homologous yeastimport system to examine the import of a fusion protein consisting. Precursor proteins were efficiently imported when their synthesis in a yeast cell-free translation system occurred in the Biotechnology Inc.) were equilibrated with25 ml of elution buffer (20 presence of isolated yeast mitochondria(12). Effect of Adding Inhibitors of Protein Synthesis on Precursor Import into Mitochondria-The experiment presented in Fig. 20 rnin 30min 60

Results

Conclusion