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

Abstract

Defining the molecular composition of caveolae is essential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-1alpha and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homogenates without using the silica-coating technology and (ii) 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, therefore, requires immunoisolations to purify caveolae. Shearing of silica-coated plasma membranes provides a homogeneous population of caveolae whose constituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphosphatidylinositol-anchored proteins, beta-actin, and angiotensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-1alpha. Caveolin-1beta 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 contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent preparation for use in the molecular analysis of caveolae.