Single Particle Analysis Reveals the Organization of the Membrane Remodeling Protein Caveolin-1 within Disc-Shaped Complexes

Abstract

Highly stable oligomeric complexes of the monotopic membrane protein caveolin serve as fundamental building blocks of caveolae. Current evidence suggests these complexes are disc shaped, but the details of their structural organization and how they assemble are poorly understood. Here, we address these questions using single particle electron microscopy (EM) of negatively-stained recombinant 8S complexes of human caveolin-1 (CAV1). We show that 8S complexes are toroidal structures ∼15 nm in diameter that consist of an outer ring composed of distinct globular domains, an inner ring, and a central protruding stalk. Based on the stain distribution along the outer ring, each 8S complex appears to contain approximately 10 CAV1 monomers. To determine the orientation of monomers within the complex, we labeled the N- and C-terminus with EM-recognizable tags. We found that the C-terminus of CAV1 monomers are positioned in the center of the complex whereas the N-terminus is orientated toward the edge of the toroidal structure. Next, we performed sequence truncation analysis to investigate the role of the N-and C-terminus in disc formation. These studies revealed both the N- and C-termini are required to maintain the size and disc shape of 8S complexes. Finally, we used negative staining to visualize 8S complexes in heterologous caveolae (h-caveolae) induced by the expression of CAV1 in E. coli. We found that h-caveolae do in fact appear to be composed of multiple copies of 8S complexes and the complexes can partially retain an arc-like organization when h-caveolae were disassembled by detergent. Taken together, these findings provide critical insights into the structural features of 8S complexes and allow us to propose a new model for how these highly stable membrane-embedded complexes are generated.