Large-scale preparation of the homogeneous LolA lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB-dependent manner.
An ATP-binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA-lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA-lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has been speculated, based on crystallographic and biochemical observations, that LolA undergoes an ATP-dependent conformational change upon lipoprotein binding. Thus, preparation of a large amount of the LolA-lipoprotein complex is difficult. Moreover, lipoproteins bound to LolA are heterogeneous. We report here that the coexpression of LolA and outer membrane-specific lipoprotein Pal from a very efficient plasmid causes the unusual accumulation of the LolA-Pal complex in the periplasm. The complex was purified to homogeneity and shown to be a functional intermediate of the lipoprotein localization pathway. In vitro incorporation of Pal into outer membranes revealed that a single molecule of LolB catalyzes the incorporation of more than 100 molecules of Pal into outer membranes. Moreover, the LolB-dependent incorporation of Pal was not affected by excess-free LolA, indicating that LolB specifically interacts with liganded LolA. Finally, the LolB depletion caused the accumulation of a significant amount of Pal in the periplasm, thereby establishing the conditions for preparation of the homogeneous LolA-lipoprotein complex.
Watanabe
Shoji
S
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
Oguchi
Yuki
Y
Yokota
Naoko
N
Tokuda
Hajime
H
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010457:Peptidoglycan / Q000302:isolation & purification / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C031163:ExcC protein, E coli; D008074:Lipoproteins; C116111:LolA protein, E coli; D010457:Peptidoglycan; D033902:Periplasmic Binding Proteins