Structure and assembly of CAV1 8S complexes revealed by single particle electron microscopy

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 of negatively stained recombinant 8S complexes of human caveolin 1. We show that 8S complexes are toroidal structures ~15 nm in diameter that consist of an outer ring, an inner ring, and central protruding stalk. Moreover, we map the position of the N and C termini and determine their role in complex assembly, and visualize the 8S complexes in heterologous caveolae. Our findings provide critical insights into the structural features of 8S complexes and allow us to propose a model for how these highly stable membrane-embedded complexes are generated.

Molecular architecture of the human caveolin-1 complex

“…S4). This agrees with previous reports suggesting 7-14 copies of Cav1 per 8S complex ( 21 , 22 , 26 ). The 11 Cav1 protomers organize into a disc-shaped complex with a diameter of ~140 Å and a height of ~34 Å (Fig.…”

“…Although caveolin proteins are found exclusively in metazoans ( 18 ), when expressed in Escherichia coli , Cav1 forms 8S-like complexes ( 26 ) and induces the formation of heterologous caveolae ( h -caveolae) ( 27 , 28 ). Thus, E. coli -expressed Cav1 forms oligomers and sculpts membranes, two of its essential functions in mammalian cells.…”

“…Thus, E. coli -expressed Cav1 forms oligomers and sculpts membranes, two of its essential functions in mammalian cells. Using single-particle cryo-EM analysis, we determined the structure of E. coli expressed and purified complexes ( 26 ) (Fig. 1, Figs.…”

Reconstitution of Caveolin-1 into Artificial Lipid Membrane: Characterization by Transmission Electron Microscopy and Solid-State Nuclear Magnetic Resonance

“…The CAV1 purity was analyzed by SDS-PAGE and its histidine tag was confirmed by western blot using an anti-penta histidine antibody, as shown in Figure 1B. CAV1 appears to be pure and runs at the correct apparent molecular weight as a monomer (22)(23); however, a significant part of the sample is found as large oligomers-with or without boiling in SDS-in this large-scale preparation. A more systematic investigation of the CAV1 oligomerization was then performed to elucidate this discrepancy.…”

Caveolin-1 Regulates Cellular Metabolism: A Potential Therapeutic Target in Kidney Disease

“…The formation of caveolae mainly involves the following steps: first, caveolins, as integral membrane proteins, are synthesized in the ER, and then, Cav-1 and Cav-2 monomers are cotranslationally inserted into the membrane of the ER and form oligomerized 8S-Cav oligomers, which contain 7-14 caveolins (Han et al, 2020). The complex of oligomerized 8S-Cav oligomers is transported to the Golgi complex in the form of coat protein II (COPII) vesicle-dependent transport machinery, where Cav-1 assembles into a cholesterol-rich complex (a 70s-Cav complex composed of 18-25 8S-Cav subunits), and the 70s-Cav complex is subsequently transported and partially inserted into the plasma membrane and binds to cholesterol on the plasma membrane through its CSD domain, recruiting the cavin-1 complex, EHD2, PACSIN2, and ROR1 to form mature caveolae (Parton and del Pozo, 2013;Busija et al, 2017;Parton et al, 2018;Parton et al, 2020b).…”