Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.
We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was the case not only with the OmpA protein, which is synthesized by free polysomes and hence is presumably exported posttranslationally in the cell, but also with alkaline phosphatase, which is synthesized only by membrane-bound polysomes and has been shown to be secreted cotranslationally in the cells. Prolonged incubation rendered the precursors inactive for subsequent translocation. Posttranslational translocation was impaired, like cotranslational translocation, by inhibitors of the proton motive force and by treatment of the vesicles with protease. Since it appears that E. coli can translocate the same proteins either cotranslationally or posttranslationally, the cotranslational mode may perhaps be more efficient, but not obligatory, for the secretion of bacterial proteins.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001692:Biological Transport; D002451:Cell Compartmentation; D002474:Cell-Free System; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational
D001425:Bacterial Outer Membrane Proteins; D008565:Membrane Proteins; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D019286:Endopeptidase K