The Membrane-spanning Domains of Caveolins-1 and -2 Mediate the Formation of Caveolin Hetero-oligomers. IMPLICATIONS FOR THE ASSEMBLY OF CAVEOLAE MEMBRANES IN VIVO (original) (raw)
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Oligomeric structure of caveolin: implications for caveolae membrane organization
Proceedings of the National Academy of Sciences of the United States of America, 1995
A 22-kDa protein, caveolin, is localized to the cytoplasmic surface of plasma membrane specializations called caveolae. We have proposed that caveolin may function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Here, we show that caveolin interacts with itself to form homooligomers. Electron microscopic visualization of these purified caveolin homooligomers demonstrates that they appear as individual spherical particles. By using recombinant expression of caveolin as a glutathione S-transferase fusion protein, we have defined a region of caveolin's cytoplasmic N-terminal domain that mediates these caveolin-caveolin interactions. We suggest that caveolin homooligomers may function to concentrate caveolin-interacting molecules within caveolae. In this regard, it may be useful to think of caveolin homooligomers as "fishing lures" with multiple "hooks" or attachment sites for caveolin-interacting molecules.
Journal of Biological Chemistry, 2000
Caveolins form interlocking networks on the cytoplasmic face of caveolae. The cytoplasmically directed N and C termini of caveolins are separated by a central hydrophobic segment, which is believed to form a hairpin within the membrane. Here, we report that the caveolin scaffolding domain (CSD, residues 82-101), and the C terminus (residues 135-178) of caveolin-1 are each sufficient to anchor green fluorescent protein (GFP) to membranes in vivo. We also show that the first 16 residues of the C terminus (i.e. residues 135-150) are necessary and sufficient to attach GFP to membranes. When fused to the caveolin-1 C terminus, GFP co-localizes with two trans-Golgi markers and is excluded from caveolae. In contrast, the CSD targets GFP to caveolae, albeit less efficiently than full-length caveolin-1. Thus, caveolin-1 contains at least two membrane attachment signals: the CSD, dictating caveolar localization, and the C terminus, driving trans-Golgi localization. Additionally, we find that caveolin-1 oligomer/oligomer interactions require the distal third of the caveolin-1 C terminus. Thus, the caveolin-1 C-terminal domain has two separate functions: (i) membrane attachment (proximal third) and (ii) protein/protein interactions (distal third). Caveolae are flask-shaped invaginations of the plasma membrane that are found in most cell types. However, caveolae are most abundant in endothelial cells, adipocytes, epithelial cells, fibroblasts, and myocytes (1). These structures participate in three main areas of cell physiology: endocytosis (2), cholesterol traffic (3), and signal transduction (4). They are coated on their cytoplasmic face by a family of proteins, the caveolins. Three mammalian caveolin genes (caveolin-1,-2, and-3) have been identified and characterized (5). Whereas caveolin-1 and-2 have overlapping tissue distributions (6), caveolin-3 is limited to muscle and neuroglial cells (7-10). Although expression of caveolin-1 or-3 is sufficient to form caveolae in cells lacking these structures (11-14), caveolins are
Molecular Characterization of Caveolin-Induced Membrane Curvature
The Journal of biological chemistry, 2015
The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain (OD) and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryo-electron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane-association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the fun...
Journal of Biological Chemistry, 1997
Caveolin is a principal structural component of caveolae membranes in vivo. Recently, a family of caveolinrelated proteins has been identified; caveolin has been retermed caveolin-1. Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes. Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties. Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1⌬C) or the N-terminal domain (Cav-1⌬N); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel. Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1. However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes. These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process. To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants. These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer. This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3. Homo-typic interaction of the Cterminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homooligomers while excluding other caveolin family members. This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1,-2, and-3.
Biogenesis of Caveolae: Stepwise Assembly of Large Caveolin and Cavin Complexes
Traffic, 2010
We analyzed the assembly of caveolae in CV1 cells by following the fate of newly synthesized caveolin-1 (CAV1), caveolin-2 and polymerase I and transcript release factor (PTRF)/cavin-1 biochemically and using live-cell imaging. Immediately after synthesis in the endoplasmic reticulum (ER), CAV1 assembled into 8S complexes that concentrated in ER exit sites, due to a DXE sequence in the N-terminal domain. The coat protein II (COPII) machinery allowed rapid transport to the Golgi complex. Accumulating in the medial Golgi, the caveolins lost their diffusional mobility, underwent conformational changes, associated with cholesterol, and eventually assembled into 70S complexes. Together with green fluorescent protein-glycosyl-phosphatidylinositol (GFP-GPI), the newly assembled caveolin scaffolds underwent transport to the plasma membrane in vesicular carriers distinct from those containing vesicular stomatitis virus (VSV) G-protein. After arrival, PTRF/cavin-1 was recruited to the caveolar domains over a period of 25 min or longer. PTRF/cavin-1 itself was present in 60S complexes that also formed in the absence of CAV1. Our study showed the existence of two novel large complexes containing caveolar coat components, and identified a hierarchy of events required for caveolae assembly occurring stepwise in three distinct locations-the ER, the Golgi complex and the plasma membrane.
American journal of physiology. Cell physiology, 2017
Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae ("little caves") require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring...
Journal of Biological Chemistry, 1999
Here, we have created a series of caveolin-1 (Cav-1) deletion mutants to examine whether the membrane spanning segment is required for membrane attachment of caveolin-1 in vivo. One mutant, Cav-1-(1-101), contains only the cytoplasmic N-terminal domain and lacks the membrane spanning domain and the C-terminal domain. Interestingly, Cav-1-(1-101) still behaves as an integral membrane protein but lacks any known signals for lipid modification. In striking contrast, another deletion mutant, Cav-1-(1-81), behaved as a soluble protein. These results implicate caveolin-1 residues 82-101 (also known as the caveolin scaffolding domain) in membrane attachment. In accordance with the postulated role of the caveolin-1 scaffolding domain as an inhibitor of signal transduction, Cav-1-(1-101) retained the ability to functionally inhibit signaling along the p42/44 mitogen-activated protein kinase cascade, whereas Cav-1-(1-81) was completely ineffective. To rule out the possibility that membrane attachment mediated by the caveolin scaffolding domain was indirect, we reconstituted the membrane binding of caveolin-1 in vitro. By using purified glutathione S-transferase-caveolin-1 fusion proteins and reconstituted lipid vesicles, we show that the caveolin-1 scaffolding domain and the C-terminal domain (residues 135-178) are both sufficient for membrane attachment in vitro. However, the putative membrane spanning domain (residues 102-134) did not show any physical association with membranes in this in vitro system. Taken together, our results provide strong evidence that the caveolin scaffolding domain contributes to the membrane attachment of caveolin-1. The plasma membrane of many cell types is stippled with
Cell-free formation and interactome analysis of caveolae
The Journal of cell biology, 2018
Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on () extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1)-stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the earl...