Binding of a soluble factor of Escherichia coli to preproteins does not require ATP and appears to be the first step in protein export.
We have constructed a mutant form of the maltose binding protein precursor, termed preMBP*, that, unlike its wild-type counterpart preMBP, retains translocation competence after synthesis. In a homologous Escherichia coli translation/translocation system, preMBP* was translocated either co- or posttranslationally with virtually 100% efficiency into inverted vesicles (INV) derived from the E. coli plasma membrane. Translation of preMBP* mRNA in a wheat germ system and subsequent incubation with INV yielded no translocation. However, addition of increasing amounts of an E. coli postribosomal supernatant (PRS) to the wheat germ extract stimulated preMBP* translocation with virtually 100% efficiency occurring at 100 micrograms of PRS per 50 microliters of incubation mixture. The activity in the E. coli PRS appears to be identical to the previously described "export" factor. The soluble activity can bind to preMBP* posttranslationally and in the absence of ATP. Subsequent targeting to INV and/or translocation, however, requires ATP. Binding of the soluble activity to preMBP* thus appears to be the first step in a multistep translocation reaction.
Watanabe
M
M
Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Blobel
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011498:Protein Precursors; C059025:preMBP protein, E coli; D000255:Adenosine Triphosphate