Expression and characterization of recombinant caveolin. Purification by polyhistidine tagging and cholesterol-dependent incorporation into defined lipid membranes.
Caveolin, a 22-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin has been proposed to function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Because of its unusual membrane topology, both the N- and C-terminal domains of caveolin remain entirely cytoplasmic and are not subject to luminal modifications that are accessible to other integral membrane proteins. Under certain conditions, caveolin also exists in a soluble form as a cytosolic protein in vivo. These properties make caveolin an attractive candidate for recombinant expression in Escherichia coli. Here, we successfully expressed recombinant full-length caveolin in E.coli. A polyhistidine tag was placed at its extreme C terminus for purification by Ni(2+)-nitrilotriacetic acid affinity chromatography. Specific antibody probes demonstrated that recombinant caveolin contained a complete N and C terminus. Recombinant caveolin remained soluble in solutions containing the detergent octyl glucoside and formed high molecular mass oligomers like endogenous caveolin. By electron microscopy, recombinant caveolin homo-oligomers appeared as individual spherical particles that were indistinguishable from endogenous caveolin homo-oligomers visualized by the same technique. As recombinant caveolin behaved as expected for endogenous caveolin, this provides an indication that recombinant caveolin can be used to dissect the structural and functional interaction of caveolin with other protein and lipid molecules in vitro. Recombinant caveolin was efficiently incorporated into lipid membranes as assessed by floatation in sucrose density gradients. This allowed us to use defined lipid components to assess the possible requirements for insertion of caveolin into membranes. Using a purified synthetic form of phosphatidylcholine (1,2-dioleoylphosphorylcholine), we observed that incorporation of caveolin into membranes was cholesterol-dependent; the addition of cholesterol dramatically increased the incorporation of caveolin into these phosphatidylcholine-based membranes by approximately 25-30-fold. This fits well with in vivo studies demonstrating that cholesterol plays an essential role in maintaining the structure and function of caveolae. Further functional analysis of these reconstituted caveolin-containing membranes showed that they were capable of recruiting a soluble recombinant form of G(i)2 alpha. This is in accordance with previous studies demonstrating that caveolin specifically interacts directly with multiple G protein alpha-subunits. Thus, recombinant caveolin incorporated into defined lipid membranes provides an experimental system in which the structure, function, and biogenesis of caveolin-rich membrane domains can be dissected in vitro.
Li
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S
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, USA.
Song
K S
KS
Lisanti
M P
MP
D051242:Caveolin 1; D022461:Caveolins; D002784:Cholesterol / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D051242:Caveolin 1; D022461:Caveolins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D002784:Cholesterol; D005982:Glutathione Transferase