Interactions of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS)-Related Protein with a Novel Solid-Supported Lipid Membrane System (TRANSIL)

Schmitz, Arndt; Schleiff, Enrico; Röhring, Cornelia; Loidl-Stahlhofen, Angelika; Vergères, Guy

doi:10.1006/abio.1998.3080

Cited by 22 publications

(14 citation statements)

References 38 publications

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“…Porous silica beads coated with single phospholipid bilayers were used to further investigate this interaction. These beads (TRANSILs) are excellent tools for analysing protein binding to membranes (Schmitz et al ., 1999). The lipid molecules on these beads are separated from the support by an ultrathin layer of water molecules, rather than being linked by covalent bonds.…”

Section: Resultsmentioning

confidence: 99%

Ca²⁺‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

Racapé

Belbahri

Engelhardt

et al. 2005

Molecular Microbiology

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SummaryPopA is released by type III secretion from the bacterial plant pathogen Ralstonia solanacearum and triggers the hypersensitive response (HR) in tobacco. The function of PopA remains obscure, mainly because mutants lacking this protein are not altered in their ability to interact with plants. In an attempt to identify the site of PopA activity in plant cells, we generated transgenic tobacco plants expressing the popA gene under the control of an inducible promoter. Immunocytologic analysis revealed that the HR phenotype of these plants correlated with the presence of PopA at the plant plasma membrane. Membrane localization was observed irrespective of whether the protein was designed to accumulate in the cytoplasm or to be secreted by the plant cell, suggesting a general lipidbinding ability. We found that the protein had a high affinity for sterols and sphingolipids in vitro and that it required Ca 2+ + + + for both lipid binding and oligomerization. In addition, the protein was integrated into liposomes and membranes from Xenopus laevis oocytes where it formed ion-conducting pores. These characteristics suggest that PopA is part of a system that aims to attach the host cell plasma membrane and to allow molecules cross this barrier.

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Section: Resultsmentioning

confidence: 99%

Ca²⁺‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

Racapé

Belbahri

Engelhardt

et al. 2005

Molecular Microbiology

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“…Control incubations performed with bovine serum albumin showed no binding of this protein to SUV composed of either PC:DOPG or PC:BBPS, indicating the absence of non‐specific protein association with the vesicles. The association of hTH1 with phospholipid bilayers was also studied using TRANSIL, a silica gel coated with a lipid bilayer, with SDS–PAGE‐based protein detection [12,13]. The use of this approach allows the estimation of the partition coefficients between the aqueous phase and the membrane for hTH1, since the separation of the membrane‐bound and free protein required a simple centrifugation step (5 min, 1000× g ) keeping nearly equilibrium binding conditions.…”

Section: Resultsmentioning

confidence: 99%

“…The partition coefficients ( K p ) or effective association constants ( K a ), obtained as described [12], were defined as

and

[P] bound and [P] free being the concentration of membrane‐bound and free protein, respectively, [P] total =[P] bound +[P] free , and [L] is the concentration of lipid. The effective dissociation constant ( K d =1/ K p ) corresponds to the concentration of accessible lipid at which 50% of protein is bound.…”

Section: Methodsmentioning

confidence: 99%

“…In this work we have characterized the interaction of recombinant human TH isoform 1 (hTH1) with phospholipid bilayers of defined composition, as well as the effects of membrane association on the activity and the conformational stability of the enzyme. We have used both liposomes and TRANSIL ® , a solid supported lipid membrane system [12]. TRANSIL beads have characteristics typical of the lipid bilayer of biological membranes and have the advantage of being easily separated from the aqueous phase by sedimentation [12,13].…”

Section: Introductionmentioning

confidence: 99%

“…We have used both liposomes and TRANSIL ® , a solid supported lipid membrane system [12]. TRANSIL beads have characteristics typical of the lipid bilayer of biological membranes and have the advantage of being easily separated from the aqueous phase by sedimentation [12,13]. The use of TRANSIL has allowed us to determine the dissociation constants for the interaction of full‐length and proteolysed forms of hTH1 with membranes of different phospholipid composition.…”

Section: Introductionmentioning

confidence: 99%

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The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N‐terminal region of the enzyme

Thórólfsson¹,

Døskeland²,

Muga³

et al. 2002

FEBS Letters

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Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. We have studied the association of recombinant human TH with model membranes by using either liposomes or silica gel beads coated with single phospholipid bilayers (TRANSIL 0 ). The use of TRANSIL beads has allowed the determination of apparent dissociation constants (K d ) for the binding of the enzyme to negatively charged bilayers (K d = 230^380 W WM, at pH 6.0^7.0). Binding to the bilayers is accompanied by a decrease in enzyme activity. Proteolysed forms of the enzyme show decreased binding affinity and two putative amphipathic N-terminal K K-helices are proposed to be involved in membrane binding. As seen by circular dichroism, binding to the bilayer does not seem to induce significant changes on the secondary structure content of the enzyme, but K K-helical structures appear to be stabilized against thermal denaturation in the membrane-bound state. Thus, amphitropism, a mechanism that regulates the function of peripheral proteins by weak binding to membrane lipids, may add to the factors that regulate both the activity and the stability of TH. ß

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Advances in membrane receptor screening and analysis

Cooper

2004

J of Molecular Recognition

182

143

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During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand-membrane-receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included.

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Interactions of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS)-Related Protein with a Novel Solid-Supported Lipid Membrane System (TRANSIL)

Cited by 22 publications

References 38 publications

Ca²⁺‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

Ca²⁺‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N‐terminal region of the enzyme

Advances in membrane receptor screening and analysis

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Interactions of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS)-Related Protein with a Novel Solid-Supported Lipid Membrane System (TRANSIL)

Cited by 22 publications

References 38 publications

Ca2+‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

Ca2+‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N‐terminal region of the enzyme

Advances in membrane receptor screening and analysis

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Product

Resources

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Ca²⁺‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

Ca²⁺‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco