HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Watanabe, S. Articles by Tokuda, H. Search for Related Content PubMed PubMed Citation Articles by Watanabe, S. Articles by Tokuda, H. Social Bookmarking                   What's this?

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

Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan

(RECEIVED July 2, 2007; FINAL REVISION September 3, 2007; ACCEPTED September 25, 2007)

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.

Keywords: bacterial lipoprotein; membrane sorting; LolA; Pal; coexpression; LolB

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Y. Oguchi, K. Takeda, S. Watanabe, N. Yokota, K. Miki, and H. Tokuda Opening and Closing of the Hydrophobic Cavity of LolA Coupled to Lipoprotein Binding and Release J. Biol. Chem., September 12, 2008; 283(37): 25414 - 25420. [Abstract] [Full Text] [PDF]

				S. Watanabe, Y. Oguchi, K. Takeda, K. Miki, and H. Tokuda Introduction of a Lethal Redox Switch That Controls the Opening and Closing of the Hydrophobic Cavity in LolA J. Biol. Chem., September 12, 2008; 283(37): 25421 - 25427. [Abstract] [Full Text] [PDF]