Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli.
The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined. IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1-ATPase was also observed in the presence of ATP. An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation.
Tokuda
H
H
Institute of Applied Microbiology, University of Tokyo, Japan.
Shiozuka
K
K
Mizushima
S
S
D000255:Adenosine Triphosphate; D000906:Antibodies / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001666:Binding Sites, Antibody / Q000187:drug effects; D001692:Biological Transport; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005960:Glucosides; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins; D010743:Phospholipids; D011510:Proteolipids / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D000069816:SEC Translocation Channels
D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D008565:Membrane Proteins; D010743:Phospholipids; D011510:Proteolipids; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; C020485:proteoliposomes; C018619:octyl-beta-D-glucoside; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases