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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.


mlaFK47R and mlaBT52A are non-functional alleles.Serial dilutions of cultures of wild-type (WT) and ∆mlaF strains harboring pET22/42 empty vector, pET22/42mlaF-His or pET22/42mlaFK47R-His, or WT and ∆mlaB strains harboring pCDF empty vector, pCDFmlaB or pCDFmlaBT52A were spotted on LB agar plates containing appropriate antibiotics, supplemented with or without 0.50% SDS, 0.65/0.70 mM EDTA as indicated, and incubated overnight at 37°C. The strains used here do not express T7 polymerase. IPTG (1 mM) was added to allow de-repression and thus low-level transcription (by endogenous polymerases) from the T7-lacO promoter in these plasmids.DOI:http://dx.doi.org/10.7554/eLife.19042.017
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fig5s1: mlaFK47R and mlaBT52A are non-functional alleles.Serial dilutions of cultures of wild-type (WT) and ∆mlaF strains harboring pET22/42 empty vector, pET22/42mlaF-His or pET22/42mlaFK47R-His, or WT and ∆mlaB strains harboring pCDF empty vector, pCDFmlaB or pCDFmlaBT52A were spotted on LB agar plates containing appropriate antibiotics, supplemented with or without 0.50% SDS, 0.65/0.70 mM EDTA as indicated, and incubated overnight at 37°C. The strains used here do not express T7 polymerase. IPTG (1 mM) was added to allow de-repression and thus low-level transcription (by endogenous polymerases) from the T7-lacO promoter in these plasmids.DOI:http://dx.doi.org/10.7554/eLife.19042.017

Mentions: We next examined whether MlaB plays additional roles in the activity of the ABC transporter. To test this, we constructed a variant with a mutation in the STAS domain of MlaB at position 52, which in two other STAS domain proteins have been shown to be important for function (Aravind and Koonin, 2000; Diederich et al., 1994; Rouached et al., 2005). This mutation does not affect the assembly of the MlaFEB complex (Figure 5A), allowing us to compare the steady-state ATP hydrolysis rates of purified MlaFEBWT and MlaFEBT52A complexes. We demonstrate that MlaFEBWT exhibits high intrinsic ATPase activity in detergent micelles (kcat = 1.8 ± 0.5 μmol ATP s-1/μmol complex) (Figure 5A), comparable to known ABC importers (Reich-Slotky et al., 2000; Tal et al., 2013). In addition, ATP binding is cooperative (Hill coefficient = 1.5 ± 0.5). Remarkably, no activity is detected with the MlaFEBT52A complex, similar to a variant of the complex containing a predicted non-functional mutation in the MlaF Walker A motif (MlaFK47REB) (Walker et al., 1982). Accordingly, the mlaBT52A or mlaFK47R alleles do not complement SDS-EDTA sensitivity observed in ΔmlaB and ΔmlaF strains, respectively (Figure 5—figure supplement 1). These results imply that MlaB assumes critical role(s) during catalysis, and validate this STAS domain protein as an essential and functional component of the IM ABC transporter.10.7554/eLife.19042.015Figure 5.MlaD and MlaB modulate ATP hydrolytic activity of the IM ABC transporter.


Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
mlaFK47R and mlaBT52A are non-functional alleles.Serial dilutions of cultures of wild-type (WT) and ∆mlaF strains harboring pET22/42 empty vector, pET22/42mlaF-His or pET22/42mlaFK47R-His, or WT and ∆mlaB strains harboring pCDF empty vector, pCDFmlaB or pCDFmlaBT52A were spotted on LB agar plates containing appropriate antibiotics, supplemented with or without 0.50% SDS, 0.65/0.70 mM EDTA as indicated, and incubated overnight at 37°C. The strains used here do not express T7 polymerase. IPTG (1 mM) was added to allow de-repression and thus low-level transcription (by endogenous polymerases) from the T7-lacO promoter in these plasmids.DOI:http://dx.doi.org/10.7554/eLife.19042.017
© Copyright Policy
Related In: Results  -  Collection

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

fig5s1: mlaFK47R and mlaBT52A are non-functional alleles.Serial dilutions of cultures of wild-type (WT) and ∆mlaF strains harboring pET22/42 empty vector, pET22/42mlaF-His or pET22/42mlaFK47R-His, or WT and ∆mlaB strains harboring pCDF empty vector, pCDFmlaB or pCDFmlaBT52A were spotted on LB agar plates containing appropriate antibiotics, supplemented with or without 0.50% SDS, 0.65/0.70 mM EDTA as indicated, and incubated overnight at 37°C. The strains used here do not express T7 polymerase. IPTG (1 mM) was added to allow de-repression and thus low-level transcription (by endogenous polymerases) from the T7-lacO promoter in these plasmids.DOI:http://dx.doi.org/10.7554/eLife.19042.017
Mentions: We next examined whether MlaB plays additional roles in the activity of the ABC transporter. To test this, we constructed a variant with a mutation in the STAS domain of MlaB at position 52, which in two other STAS domain proteins have been shown to be important for function (Aravind and Koonin, 2000; Diederich et al., 1994; Rouached et al., 2005). This mutation does not affect the assembly of the MlaFEB complex (Figure 5A), allowing us to compare the steady-state ATP hydrolysis rates of purified MlaFEBWT and MlaFEBT52A complexes. We demonstrate that MlaFEBWT exhibits high intrinsic ATPase activity in detergent micelles (kcat = 1.8 ± 0.5 μmol ATP s-1/μmol complex) (Figure 5A), comparable to known ABC importers (Reich-Slotky et al., 2000; Tal et al., 2013). In addition, ATP binding is cooperative (Hill coefficient = 1.5 ± 0.5). Remarkably, no activity is detected with the MlaFEBT52A complex, similar to a variant of the complex containing a predicted non-functional mutation in the MlaF Walker A motif (MlaFK47REB) (Walker et al., 1982). Accordingly, the mlaBT52A or mlaFK47R alleles do not complement SDS-EDTA sensitivity observed in ΔmlaB and ΔmlaF strains, respectively (Figure 5—figure supplement 1). These results imply that MlaB assumes critical role(s) during catalysis, and validate this STAS domain protein as an essential and functional component of the IM ABC transporter.10.7554/eLife.19042.015Figure 5.MlaD and MlaB modulate ATP hydrolytic activity of the IM 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.