Limits...
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.


His-tagged Mla proteins are able to rescue SDS/EDTA sensitivity in the respective mla mutant strains.Serial dilutions of cultures of wild-type (WT), ∆mlaF, ∆mlaE, ∆mlaD and ∆mlaB strains harboring pET23/42 empty vector, pET23/42mlaF-His (pmlaF-His), pET23/42His-mlaE (pHis-mlaE), pET23/42mlaD-His (pmlaD-His) or pET23/42His-mlaB (pHis-mlaB), respectively, were spotted on LB agar plates containing 200 μg/mL ampicillin, supplemented with or without 0.50% SDS and 0.60 mM EDTA as indicated, and incubated overnight at 37°C.DOI:http://dx.doi.org/10.7554/eLife.19042.004
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016091&req=5

fig1s1: His-tagged Mla proteins are able to rescue SDS/EDTA sensitivity in the respective mla mutant strains.Serial dilutions of cultures of wild-type (WT), ∆mlaF, ∆mlaE, ∆mlaD and ∆mlaB strains harboring pET23/42 empty vector, pET23/42mlaF-His (pmlaF-His), pET23/42His-mlaE (pHis-mlaE), pET23/42mlaD-His (pmlaD-His) or pET23/42His-mlaB (pHis-mlaB), respectively, were spotted on LB agar plates containing 200 μg/mL ampicillin, supplemented with or without 0.50% SDS and 0.60 mM EDTA as indicated, and incubated overnight at 37°C.DOI:http://dx.doi.org/10.7554/eLife.19042.004

Mentions: To determine whether MlaF, MlaE, MlaD and MlaB interact with each other, we performed affinity purification experiments using wild-type (WT) cells exogenously expressing N-terminally His-tagged MlaE protein (His-MlaE). His-MlaE is expressed from a “leaky” expression plasmid that has been shown to yield low cellular levels of other proteins (Wu et al., 2006; Chong et al., 2015). This construct is fully functional, as it is able to restore SDS/EDTA resistance in the ΔmlaE mutant strain (Figure 1—figure supplement 1). Two unique protein bands at ~29 kDa and ~19 kDa co-purified with His-MlaE (Figure 1A). These bands represent MlaF and MlaD, respectively, as they are no longer co-purified in the corresponding ΔmlaF and ΔmlaD mutant strains. Even though His-MlaE enabled enrichment of MlaF and MlaD, we are unable to detect the His-tagged protein in these samples, which have been heated prior to analysis. As there is a tendency for hydrophobic membrane proteins to aggregate when heated, we wondered if this was true for His-MlaE. Indeed, we are able to detect His-MlaE on immunoblots when the samples are not heated (Figure 1A, right panel). His-MlaE migrates anomalously as a diffuse band around 24 kDa (expected 28 kDa), likely because it is still partially folded in the presence of SDS, a phenomenon commonly observed for multi-pass membrane proteins (Rath et al., 2009). Remarkably, MlaD also migrates differently, now as a high molecular weight species, when the samples are not heated, suggesting that it may be forming oligomers. To examine if the interactions between MlaF, MlaE and MlaD were specific, we performed reciprocal affinity purification experiments using WT cells expressing C-terminally His-tagged MlaF (MlaF-His) or MlaD (MlaD-His) proteins, which we show are functional (Figure 1—figure supplement 1). Even though we could not probe for MlaE due to the lack of appropriate antibodies, we demonstrate that MlaF-His and MlaD-His are able to pull down MlaD and MlaF, respectively, likely via interactions with MlaE (Figure 1—figure supplement 2A). Taken together, these results establish that MlaF, MlaE and MlaD interact specifically in a complex.10.7554/eLife.19042.003Figure 1.MlaF, MlaE, MlaD and MlaB form a stable complex.


Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
His-tagged Mla proteins are able to rescue SDS/EDTA sensitivity in the respective mla mutant strains.Serial dilutions of cultures of wild-type (WT), ∆mlaF, ∆mlaE, ∆mlaD and ∆mlaB strains harboring pET23/42 empty vector, pET23/42mlaF-His (pmlaF-His), pET23/42His-mlaE (pHis-mlaE), pET23/42mlaD-His (pmlaD-His) or pET23/42His-mlaB (pHis-mlaB), respectively, were spotted on LB agar plates containing 200 μg/mL ampicillin, supplemented with or without 0.50% SDS and 0.60 mM EDTA as indicated, and incubated overnight at 37°C.DOI:http://dx.doi.org/10.7554/eLife.19042.004
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: His-tagged Mla proteins are able to rescue SDS/EDTA sensitivity in the respective mla mutant strains.Serial dilutions of cultures of wild-type (WT), ∆mlaF, ∆mlaE, ∆mlaD and ∆mlaB strains harboring pET23/42 empty vector, pET23/42mlaF-His (pmlaF-His), pET23/42His-mlaE (pHis-mlaE), pET23/42mlaD-His (pmlaD-His) or pET23/42His-mlaB (pHis-mlaB), respectively, were spotted on LB agar plates containing 200 μg/mL ampicillin, supplemented with or without 0.50% SDS and 0.60 mM EDTA as indicated, and incubated overnight at 37°C.DOI:http://dx.doi.org/10.7554/eLife.19042.004
Mentions: To determine whether MlaF, MlaE, MlaD and MlaB interact with each other, we performed affinity purification experiments using wild-type (WT) cells exogenously expressing N-terminally His-tagged MlaE protein (His-MlaE). His-MlaE is expressed from a “leaky” expression plasmid that has been shown to yield low cellular levels of other proteins (Wu et al., 2006; Chong et al., 2015). This construct is fully functional, as it is able to restore SDS/EDTA resistance in the ΔmlaE mutant strain (Figure 1—figure supplement 1). Two unique protein bands at ~29 kDa and ~19 kDa co-purified with His-MlaE (Figure 1A). These bands represent MlaF and MlaD, respectively, as they are no longer co-purified in the corresponding ΔmlaF and ΔmlaD mutant strains. Even though His-MlaE enabled enrichment of MlaF and MlaD, we are unable to detect the His-tagged protein in these samples, which have been heated prior to analysis. As there is a tendency for hydrophobic membrane proteins to aggregate when heated, we wondered if this was true for His-MlaE. Indeed, we are able to detect His-MlaE on immunoblots when the samples are not heated (Figure 1A, right panel). His-MlaE migrates anomalously as a diffuse band around 24 kDa (expected 28 kDa), likely because it is still partially folded in the presence of SDS, a phenomenon commonly observed for multi-pass membrane proteins (Rath et al., 2009). Remarkably, MlaD also migrates differently, now as a high molecular weight species, when the samples are not heated, suggesting that it may be forming oligomers. To examine if the interactions between MlaF, MlaE and MlaD were specific, we performed reciprocal affinity purification experiments using WT cells expressing C-terminally His-tagged MlaF (MlaF-His) or MlaD (MlaD-His) proteins, which we show are functional (Figure 1—figure supplement 1). Even though we could not probe for MlaE due to the lack of appropriate antibodies, we demonstrate that MlaF-His and MlaD-His are able to pull down MlaD and MlaF, respectively, likely via interactions with MlaE (Figure 1—figure supplement 2A). Taken together, these results establish that MlaF, MlaE and MlaD interact specifically in a complex.10.7554/eLife.19042.003Figure 1.MlaF, MlaE, MlaD and MlaB form a stable complex.

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.