Factors influencing the in vitro translocation of the Escherichia coli maltose-binding protein.
An in vitro system has been utilized to study the translocation of newly synthesized Escherichia coli maltose-binding protein (MBP) into inverted membrane vesicles. Approximately 40% of precursor MBP (pMBP) synthesized with a wild-type signal peptide was imported into vesicles. However, MBP species with even minor alterations in the signal peptide hydrophobic core were imported into vesicles with an efficiency much lower than predicted from in vivo studies. Posttranslational import of wild-type pMBP into vesicles could be demonstrated if membranes were added after the termination of protein synthesis. However, if vesicles were present throughout the synthesis reaction, most pMBP import occurred either cotranslationally or very soon after completion of synthesis. The wild-type pMBP rapidly became incompetent for posttranslational translocation upon continued incubation in the absence of membranes, whereas pMBP species with altered folding properties remained competent for significantly longer periods. The rate of in vitro pMBP folding was affected by the nature of the signal peptide. The evidence suggests that one or more soluble factors may interact with the newly synthesized pMBP to help maintain it in a translocation-competent state and to promote its entrance into the export pathway.
Weiss
J B
JB
Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27514.
MacGregor
C H
CH
Collier
D N
DN
Fikes
J D
JD
Ray
P H
PH
Bassford
P J
PJ
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics*; D009004:Monosaccharide Transport Proteins; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D008320:Maltose