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Abstract
The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.
Highlights
Plasmic membrane and water follows, restoring cellular hydration and volume [1]
The CL synthase encoded by the osmotically inducible cls gene is responsible for most CL synthesis but an alternate pathway for CL synthesis is evident in CL synthase-deficient bacteria [6, 8]
We showed that the osmosensory transporter protein included the N-terminal FlAsH tag MVCCPGCC (ProP) co-localizes with CL at E. coli cell poles and that this polar localization is less pronounced in clsϪ than in clsϩ bacteria [8]
Summary
Culture Media and Growth Conditions—E. coli strains were cultivated at 37 °C in LB medium [30] or in NaCl-free MOPS medium, a variant of the MOPS medium described by Neidhardt et al [31] from which all NaCl was omitted. The compositions of the PRLs were further adjusted by mixing them with an appropriate quantity of CL or PG liposomes and extruding 21 times through polycarbonate filters with 0.4-m pores (Avanti Polar Lipids, Inc.) as described by van der Heide et al [41] To determine their size distributions, extruded (proteo)liposomes were diluted to achieve a concentration of ϳ0.1 mg lipid/ml buffer, transferred to square cuvettes, and analyzed with a Zetasizer Nano Series Dynamic Light Scattering Spectrometer (Malvern Instruments, Inc., Westborough, MA). Where ⌸ is the osmotic pressure of the transport assay medium, a0 is the initial rate of proline uptake measured with medium osmolality ⌸/RT, Amax is the uptake rate that would be observed at infinite medium osmolality, R is the gas constant, T is the temperature, ⌸1/2/RT is the medium osmolality yielding half-maximal activity, and B is a constant inversely proportional to the slope of the response curve. Protein Assays—The protein concentrations of cell suspensions and lysates were determined by the bicinchoninic acid assay [44] using the BCA Kit from Pierce (Rockford, IL) with bovine serum albumin as standard
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