Determinants of membrane-targeting and transmembrane translocation during bacterial protein export.
We have separately analyzed membrane-targeting and membrane translocation of an exported bacterial protein. The precursor of the outer membrane protein LamB of Escherichia coli was synthesized in vitro and translocated into inverted plasma membrane vesicles under co- and post-translational conditions. The translation/translocation products of LamB were subsequently resolved into soluble and membrane-associated material. Dissipation of the H(+)-motive force, depletion of ATP and treatment of membranes with N-ethylmaleimide each inhibited processing and translocation of preLamB without preventing its binding to the membranes. Hence, all three conditions block transmembrane passage rather than membrane-targeting. The latter was abolished by pretreatment of salt-extracted membrane vesicles with trypsin. It was also drastically reduced when preLamB was synthesized in cell extracts derived from either a secA amber or a secB null mutant. Membrane-targeting of preLamB therefore requires soluble SecA and SecB as well as a protease-sensitive membrane receptor. The finding that SecA is involved in targeting whereas ATP is required for the transmembrane passage suggests that SecA, which harbors an ATPase activity [Lill et al. (1989), EMBO J., 8, 961-966], might have a dual function in bacterial protein export.
Swidersky
U E
UE
Biochemisches Institut der Universität Freiburg, FRG.
Hoffschulte
H K
HK
Müller
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000378:metabolism; D001692:Biological Transport; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism; D018272:Porins; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011991:Receptors, Virus / Q000096:biosynthesis / Q000235:genetics; D014357:Trypsin / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008051:Lipid Bilayers; D018272:Porins; D011498:Protein Precursors; D011991:Receptors, Virus; C016532:maltoporins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D014357:Trypsin; D005033:Ethylmaleimide