Tài liệu Báo cáo khoa học: Complete reconstitution of an ATP-binding cassette transporter LolCDE

Complete reconstitution of an ATP-binding cassette

transporter LolCDE complex from separately isolated

subunits

Kyoko Kanamaru*,

, Naohiro Taniguchi, Shigehiko Miyamoto, Shin-ichiro Narita

and Hajime Tokuda

Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan

Escherichia coli has at least 90 species of lipoproteins

[1], which have the N-terminal Cys modified with thio￾ether-linked diacylglycerol and an amino-linked acyl

chain [2]. Most lipoproteins are present in the outer

membrane, but there are some in the inner membrane.

Sorting of lipoproteins depends on the species of

the residue at position 2 [3–5], and is catalyzed by the

Lol system, composed of five Lol proteins [6]. The

LolCDE complex in the inner membrane belongs to

the ATP-binding cassette (ABC) transporter super￾family, and mediates detachment of lipoproteins from

the inner membrane [7]. This results in the formation

of a complex between lipoprotein and LolA [8], a peri￾plasmic molecular chaperone for lipoproteins. LolB in

the outer membrane then accepts lipoproteins from

LolA and incorporates them into the outer membrane

[9]. Inner membrane-specific lipoproteins, which have

Asp at position 2, avoid the action of LolCDE,

thereby remaining in the inner membrane [10]. Such

a LolCDE avoidance function of Asp depends on

Keywords

ABC transporter; lipoprotein; LolCDE;

reconstitution

Correspondence

H. Tokuda, Institute of Molecular and

Cellular Biosciences, University of Tokyo,

1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032,

Japan

Fax: +81 3 5841 8464

Tel: +81 3 5841 7830

E-mail: [email protected]

*Present address

Department of Biological Mechanisms and

Functions, Graduate School of Bioagricultu￾ral Sciences, Nagoya University, Nagoya,

Japan

†

These authors contributed equally to this

work

(Received 6 November 2006, revised

11 April 2007, accepted 17 April 2007)

doi:10.1111/j.1742-4658.2007.05832.x

The LolCDE complex of Escherichia coli belongs to the ATP-binding cas￾sette 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 sepa￾rately 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 C in a detergent solution.

ATP and phospholipids protected LolCDE, but not LolD. Furthermore,

phospholipids reactivated LolCDE even after its near complete inactiva￾tion. 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 lipo￾protein-releasing machinery was reconstituted into proteoliposomes with

E. coli phospholipids and separately purified LolC, LolD and LolE. Prein￾cubation with phospholipids at 30 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 transpor￾ter from separately purified subunits.

Abbreviations

ABC, ATP-binding cassette; BN, blue native; DDM, n-dodecyl-b-D-maltopyranoside; His-tag, hexahistidine tag.

3034 FEBS Journal 274 (2007) 3034–3043 ª 2007 The Authors Journal compilation ª 2007 FEBS