Requirements for Osmosensing and Osmotic Activation of Transporter ProP from<i>Escherichia coli</i>

Racher, Kathleen I.; Culham, Doreen E.; Wood, Janet M.

doi:10.1021/bi002331u

Cited by 58 publications

(118 citation statements)

References 41 publications

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“…Isolation of Genes proPCg and proPAt-A BglII site overlapping the proPEc stop codon in pDC79 was introduced by site-directed mutagenesis, the resulting plasmid (pYT6) was cleaved with EcoRI and BglII to excise proPEc, and the vector fragment was purified. The gene encoding ProPCg was PCR-amplified using plasmid pHP5 (22) (33). Osmolalities of culture media and buffers were adjusted with NaCl or sucrose, as specified, and measured with a Wescor vapor pressure osmometer (Wescor, Logan, UT).…”

Section: Methodsmentioning

confidence: 99%

The Osmotic Activation of Transporter ProP Is Tuned by Both Its C-terminal Coiled-coil and Osmotically Induced Changes in Phospholipid Composition

Tsatskis

Khambati

Dobson

et al. 2005

Journal of Biological Chemistry

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119

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Transporter ProP of Escherichia coli (ProPEc) senses extracellular osmolality and mediates osmoprotectant uptake when it is rising or high. A replica of the ProPEc C terminus (Asp 468 -Arg 497 ) forms an intermolecular ␣-helical coiled-coil. This structure is implicated in the osmoregulation of intact ProPEc, in vivo. Like that from Corynebacterium glutamicum (ProPCg), the ProP orthologue from Agrobacterium tumefaciens (ProPAt) sensed and responded to extracellular osmolality after expression in E. coli. The osmotic activation profiles of all three orthologues depended on the osmolality of the bacterial growth medium, the osmolality required for activation rising as the growth osmolality approached 0.7 mol/kg. Thus, each could undergo osmotic adaptation. The proportion of cardiolipin in a polar lipid extract from E. coli increased with extracellular osmolality so that the osmolality activating ProPEc was a direct function of membrane cardiolipin content. Group A ProP orthologues (ProPEc, ProPAt) share the C-terminal coiled-coil domain and were activated at low osmolalities. Like variant ProPEc-R488I, in which the C-terminal coiled-coil is disrupted, ProPEc derivatives that lack the coiled-coil and Group B orthologue ProPCg required a higher osmolality to activate. The amplitude of ProPEc activation was reduced 10-fold in its deletion derivatives. The coiled-coil structure is not essential for osmotic activation of ProP per se. However, it tunes Group A orthologues to osmoregulate over a low osmolality range. Coiled-coil lesions may impair both coiled-coil formation and interaction of ProPEc with amplifier protein ProQ. Cardiolipin may contribute to ProP adaptation by altering bulk membrane properties or by acting as a ProP ligand.

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Section: Methodsmentioning

confidence: 99%

The Osmotic Activation of Transporter ProP Is Tuned by Both Its C-terminal Coiled-coil and Osmotically Induced Changes in Phospholipid Composition

Tsatskis

Khambati

Dobson

et al. 2005

Journal of Biological Chemistry

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119

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“…Transport Assays-Bacteria were cultivated and transport assays performed on them as described by Culham et al (42,43). Transport assays were performed with PRLs as described by Racher et al (22).…”

Section: Brown (Mcmaster University)mentioning

confidence: 99%

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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“…Recently, there has been significant progress in our understanding of how osmoregulatory transporters become activated in bacterial membranes [24,25,[33][34][35][36][37]. Osmoregulatory transporters import osmoprotectants into the cell under hyperosmotic stress and thereby assist in the survival of bacteria.…”

Section: Clues From Bacterial Osmosensorsmentioning

confidence: 99%

Electrochemical structure of the crowded cytoplasm

Spitzer¹,

Poolman

2005

Trends in Biochemical Sciences

110

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Requirements for Osmosensing and Osmotic Activation of Transporter ProP fromEscherichia coli

Cited by 58 publications

References 41 publications

The Osmotic Activation of Transporter ProP Is Tuned by Both Its C-terminal Coiled-coil and Osmotically Induced Changes in Phospholipid Composition

The Osmotic Activation of Transporter ProP Is Tuned by Both Its C-terminal Coiled-coil and Osmotically Induced Changes in Phospholipid Composition

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

Electrochemical structure of the crowded cytoplasm

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