Detergent disruption of bacterial inner membranes and recovery of protein translocation activity.
Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. We treated inner-membrane vesicles of Escherichia coli with mixtures of octyl beta-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure [35S]pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components.
Cunningham
K
K
Molecular Biology Institute, University of California, Los Angeles 90024.
Wickner
W T
WT
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D003902:Detergents / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D029968:Escherichia coli Proteins; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D013501:Surface-Active Agents / Q000494:pharmacology*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008081:Liposomes; D008563:Membrane Lipids; D010743:Phospholipids; D011498:Protein Precursors; D011510:Proteolipids; D013501:Surface-Active Agents; C057184:outer membrane protein A precursor (E coli); C020485:proteoliposomes