Biochemical characterization of detergent-resistant membranes: a systematic approach.
Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.
Babiychuk
Eduard B
EB
Department of Cell Biology, Institute of Anatomy, University of Bern, Bern 9, CH 3000, Switzerland. edik@ana.unibe.ch
Draeger
Annette
A
D000199:Actins / Q000737:chemistry; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008861:Microsomes / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry; D012995:Solubility; D013270:Stomach / Q000648:ultrastructure; D013395:Sucrose / Q000737:chemistry; D013552:Swine
D000199:Actins; D003902:Detergents; D008563:Membrane Lipids; D008565:Membrane Proteins; D013395:Sucrose; D002784:Cholesterol