Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets (original) (raw)
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The Journal of Cell Biology, 2001
Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-⌬ N2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed cave-olin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.
Caveolin-2 Is Targeted to Lipid Droplets, a New "Membrane Domain" in the Cell
The Journal of cell …, 2001
Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its  isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH 2 -terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH 2 -and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.
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
Journal of Biological Chemistry, 2000
Caveolin-1 serves as the main coat protein of caveolae membranes, as an intracellular cholesterol shuttle, and as a regulator of diverse signaling molecules. Of the 12 residues conserved across all caveolin isoforms from all species examined to date, only Ser 80 and Ser 168 could serve as phosphorylation sites. We show here that mimicking chronic phosphorylation of Ser 80 by mutation to Glu (i.e. Cav-1(S80E)), blocks phosphate incorporation. However, Cav-1(S168E) is phosphorylated to the same extent as wild-type caveolin-1. Cav-1(S80E) targets to the endoplasmic reticulum membrane, remains oligomeric, and maintains normal membrane topology. In contrast, Cav-1(S80A), which cannot be phosphorylated, targets to caveolae membranes. Some exocrine cells secrete caveolin-1 in a regulated manner. Cav-1(S80A) is not secreted by AR42J pancreatic adenocarcinoma cells even in the presence of dexamethasone, an agent that induces the secretory phenotype. Conversely, Cav-1(S80E) is secreted to a greater extent than wildtype caveolin-1 following dexamethasone treatment. We conclude that caveolin-1 phosphorylation on invariant serine residue 80 is required for endoplasmic reticulum retention and entry into the regulated secretory pathway.
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
Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum
The Journal of biological chemistry, 2002
Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expressio...
Journal of Biological Chemistry, 1998
Caveolae are vesicular invaginations of the plasma membrane. The chief structural proteins of caveolae are the caveolins. Caveolins form a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules. Because the responsibilities assigned to caveolae continue to increase, this review will focus on: (i) caveolin structure/function and (ii) caveolae-associated signal transduction. Studies that link caveolae to human diseases will also be considered. The Caveolin Gene Family: Caveolin-1,-2, and-3 Molecular cloning has identified three distinct caveolin genes (1-6), caveolin-1, caveolin-2, and caveolin-3. Two isoforms of caveolin-1 (Cav-1␣ and Cav-1) are derived from alternate initiation during translation. Caveolin-1 and-2 are most abundantly expressed in adipocytes, endothelial cells, and fibroblastic cell types, whereas the expression of caveolin-3 is muscle-specific. Caveolin proteins interact with themselves to form homo-and hetero-oligomers (7-9), which directly bind cholesterol (10) and require cholesterol for insertion into model lipid membranes (10, 11). Caveolin oligomers may also interact with glycosphingolipids (12). These protein-protein and protein-lipid interactions are thought to be the driving force for caveolae formation (7). In addition, the caveolin gene family is structurally and functionally conserved from worms (Caenorhabditis elegans) to man (13), supporting the idea that caveolins play an essential role. Caveolin-1 assumes an unusual topology. A central hydrophobic domain (residues 102-134) is thought to form a hairpin-like structure within the membrane. As a consequence, both the N-terminal domain (residues 1-101) and the C-terminal domain (residues 135-178) face the cytoplasm. A 41-amino acid region of the N-terminal domain (residues 61-101) directs the formation of caveolin homooligomers (7), whereas the 44-amino acid C-terminal domain acts as a bridge to allow these homo-oligomers to interact with each other, thereby forming a caveolin-rich scaffold (14). Recent co-immunoprecipitation and dual labeling experiments directly show that caveolin-1 and-2 form a stable hetero-oligomeric complex and are strictly co-localized (9). Caveolin-2 localization corresponds to caveolae membranes as visualized by immunoelectron microscopy (9). Thus, caveolin-2 may function as an "accessory protein" in conjunction with caveolin-1. Caveolin-interacting Proteins A number of studies support the hypothesis that caveolin proteins provide a direct means for resident caveolae proteins to be sequestered within caveolae microdomains. These caveolin-interacting proteins include G-protein ␣ subunits, Ha-Ras, Src family tyrosine kinases, endothelial NOS, 1 EGF-R and related receptor tyrosine kinases, and protein kinase C isoforms (11, 15-18, 20-32). Heterotrimeric G-proteins-G-proteins are dramatically enriched within caveolae membranes, where caveolin-1 directly interacts with the ␣ subunits of G-proteins (18). Mutational or pharmacological activation of G s ␣ prevents its co-fractionation with caveolin-1 and blocks its direct interaction with caveolin-1 in vitro, indicating that the inactive GDP-bound form of G s ␣ preferentially interacts with caveolin-1. G-protein binding activity is located within a 41-amino acid region of the cytoplasmic N-terminal domain of caveolin-1 (residues 61-101). A polypeptide derived from this region of caveolin-1 (residues 82-101) effectively suppresses the basal GTPase activity of purified G-proteins by inhibiting GDP/ GTP exchange. In contrast, the analogous region of caveolin-2 possesses GTPase-activating protein activity with regard to heterotrimeric G-proteins (3). However, both of these activities (GDI and GAP) actively hold or place G-proteins in the inactive GDP-liganded conformation (3). Ha-Ras-Ha-Ras and Src family tyrosine kinases also directly interact with caveolin-1 (20, 23). Using a detergent-free procedure and a polyhistidine-tagged form of caveolin-1 for affinity purification of caveolin-rich membranes, G-proteins, Src family kinases, and Ha-Ras were all found to co-fractionate and co-elute with caveolin-1. Wild-type Ha-Ras also interacted with recombinant caveolin-1 in vitro. Ras binding activity was localized to a 41-amino acid membrane-proximal region (61-101) of the cytosolic N-terminal domain of caveolin-1, i.e. the same caveolin-1 region responsible for interacting with G-protein ␣ subunits. Reconstituted caveolinrich membranes interacted with a soluble recombinant form of wild-type Ha-Ras but failed to interact with mutationally activated soluble Ha-Ras (G12V) (23). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents this interaction. Recombinant overexpression of caveolin in intact cells was sufficient to functionally recruit a non-farnesylated mutant of Ras (C186S) onto membranes (23). This is consistent with the hypothesis that direct interaction with caveolin-1 promotes the sequestration of inactive Ha-Ras within caveolae microdomains. Src Family Tyrosine Kinases-Caveolin-1 interacts with wildtype Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src) (20). Thus, caveolin prefers the inactive conformation of G␣ subunits, Ha-Ras and c-Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 61-101. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the autoactivation of purified recombinant c-Src tyrosine kinase and a related Src family kinase, Fyn. Co-expression of caveolin-1 with c-Src shows that caveolin-1 dramatically suppresses the tyrosine kinase activity of c-Src. Thus, it appears that caveolin-1 functionally interacts with wild-type c-Src via caveolin residues 82-101. Endothelial Nitric Oxide Synthase (eNOS)-Several independent co-immunoprecipitation and domain-mapping studies demonstrate that eNOS interacts directly with caveolin-1 residues 82-101 (30, 34, 36-38). In support of these data, recombinant co-expres
Journal of Biological Chemistry
Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein α subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein α subunits, including G, G, and G. Mutational or pharmacologic activation of G subunits prevents the interaction of caveolin with G proteins, indicating that inactive G subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro b...
Journal of Biological Chemistry, 1997
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein ␣ subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolinscaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules.
The Role of Proline in the Membrane Re-entrant Helix of Caveolin-1
Journal of Biological Chemistry, 2010
Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro 110 residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.