Dataset Information

Transmembrane protein (perfringolysin o) association with ordered membrane domains (rafts) depends upon the raft-associating properties of protein-bound sterol.

ABSTRACT: Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3?-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts.

SUBMITTER: Lin Q

PROVIDER: S-EPMC3882507 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Transmembrane protein (perfringolysin o) association with ordered membrane domains (rafts) depends upon the raft-associating properties of protein-bound sterol.

Lin Qingqing Q London Erwin E

Biophysical journal 20131201 12

Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds bo ...[more]

PMID: 24359745

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Project description:BACKGROUND: The importance of membrane compartmentalization into specific membrane microdomains has been shown in many biological processes such as immunoreceptor signaling, membrane trafficking, pathogen infection, and tumor progression. Microdomains like lipid rafts, caveolae and tetraspanin enriched microdomains are relatively resistant to solubilization by some detergents. Large detergent-resistant membrane fragments (DRMs) resulting from such membrane solubilization can be conveniently isolated by density gradient ultracentrifugation or gel filtration. Recently, we described a novel type of raft-like membrane microdomains producing, upon detergent Brij98 solubilization, "heavy DRMs" and containing a number of functionally relevant proteins. Transmembrane adaptor protein LAX is a typical "heavy raft" protein. The present study was designed to identify the molecular determinants targeting LAX-derived constructs to heavy rafts. METHODOLOGY/PRINCIPAL FINDINGS: We prepared several constructs encoding chimeric proteins containing various informative segments of the LAX sequence and evaluated their effects on targeting to heavy rafts. Replacement of the polybasic membrane-proximal part of LAX by CD3?-derived membrane-proximal part had no effect on LAX solubilization. Similarly, the membrane-proximal part of LAX, when introduced into non-raft protein CD25 did not change CD25 detergent solubility. These results indicated that membrane-proximal part of LAX is not important for LAX targeting to heavy rafts. On the other hand, the replacement of transmembrane part of CD25 by the transmembrane part of LAX resulted in targeting into heavy rafts. We also show that LAX is not S-acylated, thus palmitoylation is not involved in LAX targeting to heavy rafts. Also, covalent dimerization was excluded as a cause of targeting into heavy rafts. CONCLUSIONS/SIGNIFICANCE: We identified the transmembrane domain of LAX as a first motif targeting transmembrane protein constructs to detergent-resistant heavy rafts, a novel type of membrane microdomains containing a number of physiologically important proteins.

Project description:How lipid asymmetry impacts ordered lipid domain (raft) formation may yield important clues to how ordered domain formation is regulated in vivo. Under some conditions, a sphingomyelin (SM) and cholesterol-rich ordered domain in one leaflet induces ordered domain formation in the corresponding region of an opposite leaflet composed of unsaturated phosphatidylcholine (PC) and cholesterol. In other conditions, the formation of ordered domains in a SM and cholesterol-rich leaflet can be suppressed by an opposite leaflet containing unsaturated PC and cholesterol. To explore how PC unsaturation influences the balance between these behaviors, domain formation was studied in asymmetric and symmetric lipid vesicles composed of egg SM, cholesterol, and either unsaturated dioleoyl PC (DOPC) or 1-palmitoyl 2-oleoyl PC (POPC). The temperature dependence of ordered domain formation was measured using Förster resonance energy transfer, which detects nanodomains as well as large domains. In cholesterol-containing asymmetric SM+PC outside/PC inside vesicles, the PC-containing inner leaflet tended to destabilize ordered domain formation in the SM+PC-containing outer leaflet relative to ordered domain stability in cholesterol-containing symmetric SM/PC vesicles. Residual ordered domain formation was detected in cholesterol-containing asymmetric SM+DOPC outside/DOPC inside vesicles, but ordered domain formation was completely or almost completely suppressed by asymmetry in cholesterol-containing SM+POPC outside/POPC inside vesicles over the entire temperature range measured. Suppression of ordered domain formation in the latter vesicles was confirmed by fluorescence anisotropy measurements. Because mixtures of SM, POPC, and cholesterol form domains in symmetric vesicles, and this lipid composition mimics plasma membranes to a significant degree, it is possible that under some conditions in vivo the loss of lipid asymmetry could trigger ordered domain formation.

Dataset Information

Transmembrane protein (perfringolysin o) association with ordered membrane domains (rafts) depends upon the raft-associating properties of protein-bound sterol.

Publications

Transmembrane protein (perfringolysin o) association with ordered membrane domains (rafts) depends upon the raft-associating properties of protein-bound sterol.

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