Structure and Function of Transmembrane Segment XII in Osmosensor and Osmoprotectant Transporter ProP of <i>Escherichia coli</i>

Liu, Feng; Culham, Doreen E.; Vernikovska, Yaroslava I.; Keates, Robert A. B.; Boggs, Joan M.; Wood, Janet M.

doi:10.1021/bi062198r

Cited by 19 publications

(78 citation statements)

References 53 publications

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“…Replacement of these residues with Cys had little effect on ProP activity (28,48). DTME cross-linking of these Cys (but not of Cys at other positions) was previously shown to occur when ProP was expressed in E. coli cells cultivated to exponential phase at low osmolality (28,48). The degree of cross-linking remained essentially constant when each variant was expressed in cls ϩ or cls Ϫ bacteria that were grown in low or high osmolality medium to exponential or stationary phase (Fig.…”

Section: Subcellular Locations Of CL Lacymentioning

confidence: 82%

“…A, the schematic illustrates dimerization and C-terminal coiled-coil formation by ProP. Extracts of bacteria expressing ProP*-P420C were analyzed by SDS-PAGE and Western blotting as described previously (28,48). Monomeric and dimeric ProP migrate with apparent molecular masses of ϳ40 and 90 kDa in this system (arrows).…”

Section: Discussionmentioning

confidence: 99%

“…To determine whether membrane composition and subcellular localization influence ProP dimerization or coiled-coil formation, we used DTME to cross-link ProP variants with cysteine (Cys) only at positions 420 (at the periplasmic end of TM XII), 439 (in the cytoplasm, just beyond TM XII), or 480 (in the coiled-coil). Replacement of these residues with Cys had little effect on ProP activity (28,48). DTME cross-linking of these Cys (but not of Cys at other positions) was previously shown to occur when ProP was expressed in E. coli cells cultivated to exponential phase at low osmolality (28,48).…”

Section: Subcellular Locations Of CL Lacymentioning

confidence: 96%

“…The osmolality at which ProP activity is half-maximal (⌸ 1/2 /RT) depends on the structure of the transporter (21,27,28,34,48) and the solvent to which it is exposed (42). The positive correlations among growth medium osmolality, the CL content of E. coli, and ⌸ 1/2 /RT for ProP suggested that ⌸ 1/2 /RT is also elevated by a CL-rich membrane environment (7).…”

Section: Subcellular Locations Of CL Lacymentioning

confidence: 99%

“…The dimer interface includes transmembrane (TM) helix XII and the C-terminal coiled-coil (28,48). Residues 419 -437 of ProP constitute the membrane-embedded portion of TM XII (48) and residues 468 -497 form antiparallel ␣-helical coiledcoils (25,26,28) (Figs. 1 and 6A).…”

Section: Subcellular Locations Of CL Lacymentioning

confidence: 99%

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Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

Romantsov

Stalker

Culham

et al. 2008

Journal of Biological Chemistry

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125

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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 coiledcoil 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.Osmotic pressure changes elicit transmembrane water fluxes that concentrate or dilute the cytoplasm of living cells, disrupting their structure and function. Cells respond by actively adjusting the distributions of selected solutes across the cytoplasmic membrane and water follows, restoring cellular hydration and volume (1). Bacteria can use K ϩ as an osmoregulatory solute but they prefer protein-stabilizing organic osmolytes like proline, glycine, glycine betaine, and ectoine (1, 2). These compounds are also called osmoprotectants because, when provided exogenously, they stimulate bacterial growth in high (but not low) osmotic pressure media. Well characterized, functionally redundant transporters, enzymes, and channels modulate the osmolyte composition of Gram-negative bacterium Escherichia coli (3-5). We are exploiting that system to learn how osmotic pressure is sensed, how resulting signals are transduced, and how cells respond by modulating their own structure, growth, and division.The mole fractions of the major phospholipids in the cytoplasmic membrane of E. coli are usually cited as 0.75 for phosphatidylethanolamine (PE), 3 0.20 for phosphatidylglycerol (PG), and 0.05 for ...

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Section: Subcellular Locations Of CL Lacymentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Subcellular Locations Of CL Lacymentioning

confidence: 96%

Section: Subcellular Locations Of CL Lacymentioning

confidence: 99%

Section: Subcellular Locations Of CL Lacymentioning

confidence: 99%

See 3 more Smart Citations

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

Romantsov

Stalker

Culham

et al. 2008

Journal of Biological Chemistry

Self Cite

125

View full text Add to dashboard Cite

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Structural characterization of the osmosensor ProP

Sayeed

Baenziger

2009

Biochimica et Biophysica Acta (BBA) - Biomembranes

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ProP, an osmoprotectant symporter from the major facilitator superfamily was expressed, purified and reconstituted into proteoliposomes that are amenable to structural characterization using infrared spectroscopy. Infrared spectra recorded in both (1)H(2)O and (2)H(2)O buffers reveal amide I band shapes that are characteristic of a predominantly alpha-helical protein, and that are similar to those recorded from the well-characterized homolog, lactose permease (LacY). Curve-fit analysis shows that ProP and LacY both exhibit a high alpha-helical content. Both proteins undergo extensive peptide hydrogen-deuterium exchange after exposure to (2)H(2)O, but are surprisingly thermally stable with denaturation temperatures greater than 60 degrees C. 25-30% of the peptide hydrogens in both ProP and LacY are resistant to exchange after 72 h in (2)H(2)O at 4 degrees C. Surprisingly, these exchange resistant peptide hydrogens exchange completely for deuterium at temperatures below those that lead to denaturation. Our results show that ProP adopts a highly alpha-helical fold similar to that of LacY, and that both transmembrane folds exhibit unusually high temperature-sensitive solvent accessibility. The results provide direct evidence that ProP adopts a structure consistent with other major facilitator superfamily members.

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Cardiolipin and the osmotic stress responses of bacteria

Romantsov

Guan

Wood

2009

Biochimica et Biophysica Acta (BBA) - Biomembranes

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207

166

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Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls– bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H+-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.

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Structure and Function of Transmembrane Segment XII in Osmosensor and Osmoprotectant Transporter ProP of Escherichia coli

Cited by 19 publications

References 53 publications

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

Structural characterization of the osmosensor ProP

Cardiolipin and the osmotic stress responses of bacteria

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