Mechanism of caveolin filament assembly
Mechanism of caveolin filament assembly
Published online before print August 7, 2002
Imma Fernandez , Yunshu Ying , Joseph Albanesi , and Richard G. W. Anderson
PNAS | August 20, 2002 | vol. 99 | no. 17
Departments of Cell Biology and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9039
Edited by David D. Sabatini, New York University School of Medicine, New York, NY, and approved June 27, 2002 (received for review April 3, 2002)
Caveolin-1 was the first protein identified that colocalizes with the 10-nm filaments found on the inside surface of caveolae membranes. We have used a combination of electron microscopy (EM), circular dichroism, and analytical ultracentrifugation to determine the structure of the oligomers that form when the first 101 aa of caveolin-1 (Cav1?101) are allowed to associate. We determined that amino acids 79?96 in this caveolin-1 fragment are arranged in an -helix. Cav1?101 oligomers are 11 nm in diameter and contain seven molecules of Cav1?101. These subunits, in turn, are able to assemble into 50 nm long x 11 nm diameter filaments that closely match the morphology of the filaments in the caveolae filamentous coat. We propose that the heptameric subunit forms in part through lateral interactions between the -helices of the seven Cav1?101 units. Caveolin-1, therefore, appears to be the structural molecule of the caveolae filamentous coat.
Abbreviations: EM, electron microscopy
A unique membrane coat is found on the inside surface of fibroblast (1) and endothelial cell (2) caveolae. Each coat consists of 4?6, concentrically arranged, 10-nm-wide filaments that are embedded in the membrane. Sometimes the filaments are curved or spiral, whereas other times they run parallel to each other. This filamentous coat decorates both planar and deeply invaginated regions of membrane. The full extent of the coat is best appreciated, however, in the planar coats, where they can measure up to 125 nm in diameter. This coat is not removed by washing the membrane with high-salt solutions, suggesting it is not constructed from peripheral membrane proteins. By contrast, treatment of the membrane with cholesterol-binding drugs like nystatin or filipin cause the coat to disassemble. During disassembly, the filaments disintegrate into particles that measure 10 nm in diameter (1).
The only known protein component of the caveolae coat is caveolin-1, which has been localized by immunogold to individual filaments in the coat (1). There are three caveolin genes (designated 1, 2, and 3), coding for five distinct proteins (3). Each share in common a long hydrophobic region, which is probably inserted in the membrane, and N-terminal and C-terminal portions that most likely are in the cytoplasm. Whether caveolin-1 is the structural subunit of the filament has not been determined.
The apparent molecular mass of caveolin-1 is 22?24 kDa. Therefore, a 10-nm-wide filament made from pure caveolin-1 would most likely be constructed from caveolin-1 oligomers. The ability of caveolin-1 to oligomerize is well documented. There appear to be two aspects to the oligomerization process. A glutathione S-transferase (GST)-fusion peptide of the N-terminal cytoplasmic portion of caveolin-1 (amino acids 1?101) spontaneously forms oligomers in vitro (4). Removing amino acids 81?101 from the peptide abolishes oligomerization. Oligomerization of the full-length protein, on the other hand, appears to be stabilized by the palmitoylation of cysteine residues located at positions 133, 143, and 156 (5). Indeed, deletion of amino acids 134?154 impairs oligomerization as well as transport of caveolin-1 from the Golgi apparatus to the cell surface (6). Exactly how oligomeric caveolin-1 forms the filamentous caveolae coat, however, cannot be determined without high-resolution structural information.
Materials and Methods
Construction of Caveolin Bacterial Expression Systems. All caveolin constructs were generated by PCR from cDNAs encoding the indicated caveolin isoform by using the Expand High Fidelity PCR system (Roche Applied Science, Indianapolis). The PCR products were subcloned into the multicloning site (BamHI?EcoRI) of the vector pGEX-KT. Plasmids were constructed to encode different caveolin-1, caveolin-2, and caveolin-3 N-terminal cytoplasmic fragments. To prepare a caveolin-1 mutant corresponding to the caveolin-3 mutation found in patients with limb-girdle muscular dystrophy, 1C, the plasmid encoding caveolin-1 wild-type 1?101 was mutagenized by using the QuickChange site-directed mutagenesis protocol (Stratagene) to delete amino acids 91?93 (TFT). The 66A70 mutant Cav1?101 was described (6). EcoRI and BamHI restriction sites were added to the PCR products of all constructs and subcloned into pGEX-KT vectors. Caveolin-1 fragment 80?101 was synthesized by solid-phase methods, purified by HPLC, and characterized by mass spectrometry (MS). Escherichia coli (BL21 strain, Novagen) were transfected with pGEX-KT plasmids, and expression was induced by the addition of 0.1 mM isopropyl -D-thiogalactopyranoside (IPTG; Sigma) at 25?C overnight. Cells were harvested, resuspended in PBS, and lysed by passing three times through an EmulsiFlex-C5 cell disrupter (Avestin, Ottawa) at 14,000 psi (1 psi = 6.9 kPa). Supernatants were separated from insoluble pellet by spinning the lysate at 28,000 x g for 30 min. The supernatants were then incubated with a slurry of glutathione agarose beads (Sigma). The beads were extensively washed with PBS until there was no detectable UV absorption in the eluate. The sample was equilibrated with buffer A (50 mM Tris, pH 8.0/0.2 M NaCl/2.5 mM CaCl2) before thrombin (12 units/liter; Sigma) was added and the sample incubated for 1.5 h at 25?C. Cleaved products were eluted and further purified by anion-exchange chromatography on MonoQ (Amersham Biosystems, Piscataway, NJ).
Column Chromatography. Purified samples of the indicated recombinant protein (0.2?5.0 mg/ml) were analyzed by using a HiLoad 16/60 Superdex 75 or on a Superdex 200 gel filtration column (Amersham Pharmacia Biotech). The column was preequilibrated with buffer B (200 mM sodium phosphate/300 mM NaCl, pH 6.3) and calibrated with standard proteins of known molecular size (Amersham Pharmacia Biotech). Samples were eluted at a flow rate of 1 ml/min and the fractions were monitored by absorbance at 280 nm.
Analytical Ultracentrifugation. Analytical ultracentrifugation experiments were performed by using a Beckman XL-I analytical ultracentrifuge. For sedimentation equilibrium experiments, samples were loaded in an An60Ti rotor with an equilibrium six-channel centerpiece (path length 1.2 cm) and run at 9,000, 12,000, and 15,000 rpm, at 4?C. Data were collected at a wavelength of either 280 or 305 nm. Background absorbance was estimated by overspeeding at 42,000 rpm until a flat baseline was obtained. Analysis of the data, including estimation of average molecular weight as a function of protein concentration (Fig. 4A), was carried out by using Beckman software. The calculated molecular weight of Cav11?101 monomers was 11,725. Partial specific volumes, estimated by using the SEDIMENTATION INTERPRETATION PROGRAM V.1.03 (Biomolecular Interaction Technologies Center, Durham, NH) based on the method of Cohn and Edsall (7), were 0.7209 at 4?C and 0.7277 at 20?C. Extinction coefficients were calculated as 19,060 M?1? cm?1 at 280 nm by the method of Gill and von Hippel (8). Sedimentation velocity data were collected at 280 nm, 20?C, and 40,000 rpm by using the 12-mm charcoal-filled double-sector centerpiece. Data were analyzed by using the g(s*) method in the Beckman software. In this method, an apparent sedimentation coefficient distribution function [g(s*)] is calculated from the absorbance curves collected at successive intervals (9, 10). Molecular weights (M) and diffusion coefficients (D) were calculated by using the relations s = M(1?)/Naf and D = kT/f, where s is the sedimentation coefficient, f is the frictional coefficient, T is the absolute temperature, k is Boltzmann's constant, is the partial specific volume, is the solvent density, and NA is Avogadro's number. Hydrodynamic radii (R) were calculated by using the Stokes equation: f = 6R.
Fig 4. Analytical ultracentrifugation suggests that Cav11?101 self-assembles into at least two oligomeric states. (A) Sedimentation equilibrium analysis. Cav11?101 peptides were purified as described. Various concentrations of the peptide were subjected to equilibrium centrifugation at various speeds in an An60Ti rotor. The plot shows the distribution of average molecular weights as a function of peptide concentration calculated from multiple runs at 9,000 rpm. At this speed, molecular weights of 400,000?450,000 were consistently observed at the highest concentrations, near the bottom of the centrifuged cell. However, the average molecular weight of Cav11?101 oligomers at each concentration decreased progressively with increasing rotor speed (not shown), suggesting pressure-dependent dissociation. (B) Sedimentation velocity analysis. The symmetric nature of the sedimentation coefficient distribution function is indicative of a relatively homogenous population of oligomers. Thus, at the high speed used (55,000 rpm in the experiment shown), Cav11?101 has presumably undergone pressure-induced dissociation to a stable oligomeric state. Calculations based on sedimentation and diffusion constants obtained here suggest that this stable oligomer has a molecular weight of approximately 83,000.
Negative Staining. Samples of the indicated caveolin peptide (5 ?l at 0.5 mg/ml) were processed for negative staining by using glow-discharged, Formvar-coated, 400-mesh nickel grids. We used 1% aqueous uranyl acetate to stain all samples before viewing with the JEOL 1200 CX electron microscope at
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