Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits.
The LolCDE complex of Escherichia coli belongs to the ATP-binding cassette transporter superfamily and mediates the detachment of lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane. The complex is composed of one copy each of membrane subunits LolC and LolE, and two copies of ATPase subunit LolD. To establish the conditions for reconstituting the LolCDE complex from separately isolated subunits, the ATPase activities of LolD and LolCDE were examined under various conditions. We found that both LolD and LolCDE were inactivated on incubation at 30 degrees C in a detergent solution. ATP and phospholipids protected LolCDE, but not LolD. Furthermore, phospholipids reactivated LolCDE even after its near complete inactivation. LolD was also protected from inactivation when membrane subunits and phospholipids were present together, suggesting the phospholipid-dependent reassembly of LolCDE subunits. Indeed, the functional lipoprotein-releasing machinery was reconstituted into proteoliposomes with E. coli phospholipids and separately purified LolC, LolD and LolE. Preincubation with phospholipids at 30 degrees C was essential for the reconstitution of the functional machinery from subunits. Strikingly, the lipoprotein-releasing activity was also reconstituted from LolE and LolD without LolC, suggesting the intriguing possibility that the minimum lipoprotein-releasing machinery can be formed from LolD and LolE. We report here the complete reconstitution of a functional ATP-binding cassette transporter from separately purified subunits.
Kanamaru
Kyoko
K
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Taniguchi
Naohiro
N
Miyamoto
Shigehiko
S
Narita
Shin-ichiro
S
Tokuda
Hajime
H
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; C407982:LolC protein, E coli; C407983:LolD protein, E coli; C407984:LolE protein, E coli; D010743:Phospholipids; D021122:Protein Subunits; D000251:Adenosine Triphosphatases