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Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry

View Article: PubMed Central - PubMed

ABSTRACT

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.

Doi:: http://dx.doi.org/10.7554/eLife.19042.001

No MeSH data available.


Related in: MedlinePlus

MlaD is not co-purified with MlaF-His in the absence of MlaB.Co-TALON affinity purification using wild-type (WT), ∆mlaB and ∆mlaE strains harboring an empty pET23/42 vector or pmlaF-His. Samples were heated and subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. Positions of relevant molecular weight markers are indicated in kDa.DOI:http://dx.doi.org/10.7554/eLife.19042.014
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fig4s2: MlaD is not co-purified with MlaF-His in the absence of MlaB.Co-TALON affinity purification using wild-type (WT), ∆mlaB and ∆mlaE strains harboring an empty pET23/42 vector or pmlaF-His. Samples were heated and subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. Positions of relevant molecular weight markers are indicated in kDa.DOI:http://dx.doi.org/10.7554/eLife.19042.014

Mentions: MlaE and MlaF constitute TMDs and NBDs of a canonical ABC transporter, respectively (Malinverni and Silhavy, 2009). MlaD binds PLs, consistent with its proposed role as a periplasmic binding protein; however, it is not clear if MlaD has other roles in the activity of the ABC transporter. The function of MlaB is also unknown. To characterize the importance of MlaD and MlaB in the complex, we attempted to over-express and purify sub-complexes containing His-MlaE, including MlaFE, MlaFED and MlaFEB (Figure 4). We were successful in obtaining purified MlaFEB (Figure 4C), but not MlaFE and MlaFED (Figure 4A and B). Despite reasonable expression of MlaF (Figure 4—figure supplement 1), it is not co-purified with His-MlaE in preparations of MlaFE and MlaFED (Figure 4A and B). Therefore, MlaF does not interact with MlaE unless MlaB is present. To exclude artifacts due to over-expression, we also performed affinity purification using His-MlaE expressed at low levels in cells lacking MlaB. Unlike in WT cells, MlaF is not co-purified with His-MlaE in the absence of MlaB (Figure 4D). Similarly, we show that MlaF-His could not pull down MlaD (through MlaE) without MlaB (Figure 4—figure supplement 2). In fact, the affinity-enriched levels of MlaF-His appear to be reduced in the absence of MlaB, suggesting that MlaB modulates the stability of MlaF. Our results demonstrate that MlaB plays a key role in the assembly of the complex.10.7554/eLife.19042.012Figure 4.MlaB is required for the stability and/or assembly of the canonical ABC transporter.


Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
MlaD is not co-purified with MlaF-His in the absence of MlaB.Co-TALON affinity purification using wild-type (WT), ∆mlaB and ∆mlaE strains harboring an empty pET23/42 vector or pmlaF-His. Samples were heated and subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. Positions of relevant molecular weight markers are indicated in kDa.DOI:http://dx.doi.org/10.7554/eLife.19042.014
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5016091&req=5

fig4s2: MlaD is not co-purified with MlaF-His in the absence of MlaB.Co-TALON affinity purification using wild-type (WT), ∆mlaB and ∆mlaE strains harboring an empty pET23/42 vector or pmlaF-His. Samples were heated and subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. Positions of relevant molecular weight markers are indicated in kDa.DOI:http://dx.doi.org/10.7554/eLife.19042.014
Mentions: MlaE and MlaF constitute TMDs and NBDs of a canonical ABC transporter, respectively (Malinverni and Silhavy, 2009). MlaD binds PLs, consistent with its proposed role as a periplasmic binding protein; however, it is not clear if MlaD has other roles in the activity of the ABC transporter. The function of MlaB is also unknown. To characterize the importance of MlaD and MlaB in the complex, we attempted to over-express and purify sub-complexes containing His-MlaE, including MlaFE, MlaFED and MlaFEB (Figure 4). We were successful in obtaining purified MlaFEB (Figure 4C), but not MlaFE and MlaFED (Figure 4A and B). Despite reasonable expression of MlaF (Figure 4—figure supplement 1), it is not co-purified with His-MlaE in preparations of MlaFE and MlaFED (Figure 4A and B). Therefore, MlaF does not interact with MlaE unless MlaB is present. To exclude artifacts due to over-expression, we also performed affinity purification using His-MlaE expressed at low levels in cells lacking MlaB. Unlike in WT cells, MlaF is not co-purified with His-MlaE in the absence of MlaB (Figure 4D). Similarly, we show that MlaF-His could not pull down MlaD (through MlaE) without MlaB (Figure 4—figure supplement 2). In fact, the affinity-enriched levels of MlaF-His appear to be reduced in the absence of MlaB, suggesting that MlaB modulates the stability of MlaF. Our results demonstrate that MlaB plays a key role in the assembly of the complex.10.7554/eLife.19042.012Figure 4.MlaB is required for the stability and/or assembly of the canonical ABC transporter.

View Article: PubMed Central - PubMed

ABSTRACT

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.

Doi:: http://dx.doi.org/10.7554/eLife.19042.001

No MeSH data available.


Related in: MedlinePlus