Abstract
BackgroundThe 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 FindingsWe 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 CD3ε-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/SignificanceWe 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.
Highlights
According to a presently widely accepted model, the plasma membrane is naturally organized into several types of microdomains differing in their lipid and protein composition and resistance to solubilization by detergents (e.g. Triton X-100, CHAPS, Brij series, dodecylmaltoside)
This finding casted doubts on the real physiological role of rafts in T cell receptor (TCR) signaling. We demonstrated that these results could be explained by the existence of a novel type of membrane raft-like microdomains, producing upon detergent solubilization ‘‘heavy detergent-resistant membrane fragments (DRMs)’’ [12]. We showed that these microdomains share some properties with classical membrane rafts but in contrast to them, upon solubilization by Brij98 they produce DRMs of higher density not floating up during density gradient ultracentrifugation
The size of the heavy DRMs, as determined by gel filtration on Sepharose 4B, is apparently similar to that of the DRMs derived from classical lipid rafts or tetraspanin enriched microdomains (TEMs)
Summary
According to a presently widely accepted model, the plasma membrane is naturally organized into several types of microdomains differing in their lipid and protein composition and resistance to solubilization by detergents (e.g. Triton X-100, CHAPS, Brij series, dodecylmaltoside). We demonstrated that these results could be explained by the existence of a novel type of membrane raft-like microdomains, producing upon detergent solubilization ‘‘heavy DRMs’’ [12] We showed that these microdomains (provisionally called ‘‘heavy rafts’’) share some properties with classical membrane rafts (resistance vs sensitivity to particular detergents, cholesterol dependence) but in contrast to them, upon solubilization by Brij they produce DRMs of higher density not floating up during density gradient ultracentrifugation. They contain a different set of proteins, and are more dependent on protein-protein interactions. The present study was designed to identify the molecular determinants targeting LAX-derived constructs to heavy rafts
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