Immunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin cage - Toward understanding the basis of purification

Defining the molecular composition of caveolae is essential in establishing their molecular architecture and functions. Here, we identify a high affin ity monoclonal antibody that is specific for caveolin-1 alpha and rapidly b inds caveolin oligomerized around intact caveolae, We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cel l and tissue homogenates without using the silica-coating technology and (i i) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechanical disruption . Sonication of plasma membranes, isolated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, ther efore, requires immunoisolations 60 purify caveolae, Shearing of silica-coa ted plasma membranes provides a homogeneous population of caveolae whose co nstituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunois olated after sonication. The caveolae immunoisolated from different low den sity fractions are quite similar in molecular composition. They contain a s ubset of key signaling molecules (i.e. G protein and endothelial nitric oxi de synthase) and are markedly depleted in glycosylphosphatidylinositol-anch ored proteins, P-actin, and angiotensin-converting enzyme. All caveolae iso lated from the cell surface of lung microvascular endothelium in vivo appea r to be coated with caveolin-1 alpha, Caveolin-1 beta and -2 can also exist in these same caveolae, The isolation and analytical procedures as well as the time-dependent dissociation of signaling molecules from caveolae contr ibute to key compositional differences reported in the literature for caveo lae. This new, rapid, magnetic immunoisolation procedure provides a consist ent preparation for use in the molecular analysis of caveolae.