Cholesterol-dependent localization of NAP-22 on a neuronal membrane microdomain (raft).
A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-beta-cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca(2+)-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.
Department of Biotechnology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, 606-8585, Japan. smaekawa@ipc.kit.ac.jp
S
S
Maekawa
C
C
Sato
K
K
Kitajima
N
N
Funatsu
H
H
Kumanogoh
Y
Y
Sokawa
D000818:Animals; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D008081:Liposomes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D008081:Liposomes; D009419:Nerve Tissue Proteins; C066853:Basp1 protein, rat; D002784:Cholesterol