Dataset Information

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.

ABSTRACT: 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.

SUBMITTER: Watanabe S

PROVIDER: S-EPMC2222812 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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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.

Watanabe Shoji S Oguchi Yuki Y Yokota Naoko N Tokuda Hajime H

Protein science : a publication of the Protein Society 20071201 12

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 ...[more]

PMID: 18029423

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Project description:1. Human total HDL (high-density lipoprotein), HDL2 and HDL3 were labelled in vitro by incubation with lipoprotein-deficient serum (LPDS) which contained either [3H]cholesteryl oleate or [14C]cholesterol under different conditions. The lipoproteins were then subfractionated by heparin-Sepharose column chromatography, and three subfractions (A, B and C) were successively eluted from each preparation of HDL, HDL2 and HDL3. When the labelling was done at 37 degrees C for 17 h, the subfractions were homogeneously labelled with [3H]cholesteryl oleate. However, when it was performed for only 30 min at 4 degrees C, the subfractions showed marked differences in the 3H specific radioactivity, which was much higher in the C fractions than in the others. 2. 3H-labelled HDL2 and HDL3 subfractions behaved differently under the precipitant action of heparin-Mn2+; fraction C (the richest in apolipoprotein E) produced the largest amount of radioactive and chemical precipitate. More 3H radioactivity, but not the cholesterol, was precipitated from HDL2 or HDL3 by the reagent, demonstrating that 3H-labelled HDL2 and HDL3 behave like their fraction C, which becomes labelled to the highest specific radioactivity despite having the smallest mass. 3. The incubation of 3H-labelled HDL subfractions with human LPDS and very-low-density lipoprotein (VLDL) at 37 degrees C increased the quantity of 3H radioactivity that was precipitated, in proportion to the amount of VLDL present in the media. These changes were attributable to the action of cholesterol ester transfer protein, since they did not occur at 4 degrees C or when human LPDS was replaced with rat LPDS. 4. Kinetics of the transfer of HDL [3H]cholesteryl oleate to VLDL showed a greater apparent Vmax for fractions A than for fractions B from either HDL2 or HDL3, whereas the apparent Km values were very similar, which suggest that this transfer process is influenced by the apoprotein composition of the donor lipoprotein.

Dataset Information

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.

Publications

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.

Similar Datasets

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