Purification of Clathrin-Coated Vesicles from Bovine Brain, Liver, and Adrenal Gland
Clathrin-coated vesicles are intermediates in several selective membrane transport processes in eukaryotic cells (for review, see Bonifacino and Lippincott- Schwartz, 2003; Brodsky et al., 2001; Schmid, 1997). They are derived from clathrin-coated membrane regions on the plasma membrane, the TGN, endosomes (Stoorvogel et al., 1996), and possibly also lysosomes (Traub et al., 1996) by a process of invagination and fission. In addition to the main structural protein clathrin, a three-legged molecule composed of one heavy and one light chain per leg, a number of other proteins have been isolated from clathrin-coated vesicles: most prominent are the adaptor protein (AP) complexes, heterotetramers consisting of two heavy chains, an intermediate chain, and a light chain (for review, see Kirchhausen, 1999). AP-1 and AP-2 complexes link clathrin to specific membrane areas by binding to clathrin N-terminal domains and to cytosolic tails of transmembrane receptors. The recently discovered AP-3 and AP-4 complexes appear to function independently of clathrin and are not enriched in clathrin-coated vesicles. Furthermore, clathrin-coated vesicles contain monomeric adaptor proteins, such as AP 180 and members of the epsin family. These interact with coat components via their poorly structured C-terminal regions (Kalthoff et al., 2002) and with phosphoinositides of the membrane via their N-terminal ENTH domain (for review, see De Camilli et al., 2002). Also present are accessory proteins such as auxilins that provide a J-like domain for the recruitment of Hsc70 to clathrin, thereby serving to initiate the removal of the clathrin coat (Ungewickell et al., 1995). A novel bilayered clathrin coat without curvature has been described on multivesicular endosomes (Raiborg et al., 2002; Sachse et al., 2002). It contains the monomeric clathrin adaptor protein HRS and appears to be involved in the sorting of ubiquitinated membrane proteins to internal vesicles. This flat structure probably does not to give rise to coated vesicles.
Since the first report on the purification of clathrincoated vesicles (Kanaseki and Kadota, 1969), a variety of protocols have been published. Most recent protocols are based on a purification procedure introduced by Campbell et al. (1984). This procedure rapidly provides crude clathrin-coated vesicles that are well suited for the preparation of various coat proteins (Ahle et al., 1988; Ahle and Ungewickell, 1990; Lindner and Ungewickell, 1991). This basic protocol has been further improved by the introduction of a centrifugation step involving a sucrose/D2O cushion (Maycox et al., 1992). By this step, contaminations of smooth membrane vesicles are reduced further. A similar procedure has been used to purify rat brain clathrincoated vesicles for proteomic characterization (Wasiak et al., 2002).
This article describes a purification protocol for bovine brain-coated vesicles that includes a sucrose/ D2O step gradient at the end. With appropriate volume adjustments, the protocol can also be used for the purification of coated vesicles from other species such as pigs or rats. For the preparation of clathrin-coated vesicles from other organs (adrenal gland, liver), some modifications in the protocol are suggested.
II. MATERIALS AND INSTRUMENTATION
Ficoll 400 (Cat. No. 17-0400-02) is from Pharmacia; sucrose (Cat. No. BP 220-1) is from Fisher Scientific; D2O (Cat. No. 15,188-2) is from Aldrich; EGTA (Cat. No. E-3889), MES (Cat. No. M-3023), and phenylmethylsulfonyl fluoride (PMSF, Cat. No. P-7626) are from Sigma. All other reagents are from Sigma or Fisher in analytical grade.
Biological material is obtained from an local abattoir within 1h of slaughter and is kept on ice until further processing (1-2h). Fresh and cleaned material can be frozen in liquid nitrogen and stored at -80°C for several months. It is helpful to cut the tissue into small pieces before freezing. This supports a rapid drop of temperature in the tissue and minimizes ice crystal formation. The latter process reduces yield and purity of the following preparation, probably due to the destruction of membrane vesicles and liberation of proteolytic activities.
For homogenization of the tissue, a Waring commercial blender (VWR International, Cat. No. 58977- 169) is used. Membrane pellets are resuspended with Potter-Elvehjem homogenizers of various sizes (10- 55ml) obtained from Fisher Scientific (Cat. No. 08414- 14 A to D). The metal shaft of the larger homogenizers is attached to a variable-speed overhead drive so that the pestle can be rotated.
Low-speed centrifugations are done in a Sorvall RC- 5B centrifuge using GS-3, GSA, or SS-34 heads. Highspeed centrifugations are performed with Beckman Ti 45, Ti 35, and SW 28 rotors in conventional ultracentrifuges. For fixed angle ultracentrifugation rotors, Beckman polycarbonate bottles with screw caps (Cat. No. 355622) are used and for the SW 28 rotor open-top ultraclear tubes (Cat. No. 344058) are used.
A. Cleaning of Bovine Brain Cortices
Phosphate-buffered saline (PBS): 137mM NaCl, 2.7 mM KCl, 8.2mM Na2HPO4, 1.9 mM KH2PO4, 0.02% NaN3, pH 7.0. To make 1 liter of 10× PBS (stock solution), dissolve 80g NaCl, 2 g KCl, 2.58g KH2PO4, 11.64g Na2HPO4, and 0.2g NaN3 in double-distilled water. Adjust the pH to 7.0 (if necessary) and bring the volume to 1 liter. Dilute this stock solution 1:10 with double-distilled water and chill to 4°C prior to use. Approximately 2-3 liters of 1× PBS are needed per kilogram tissue.
C. Differential Centrifugations
1. Preparation of Postmitochondrial Supernatants
2. Preparation of Microsomal Pellets
3. Preparation of Crude Clathrim-Coated Vesicles from Microsomal Pellets
12.5% Ficoll-sucrose: To make 1 liter, dissolve 125g sucrose and 125g Ficoll 400 in buffer A, pH 6.5, and stir overnight in the cold room. Ficoll 400 dissolves only slowly! Keep at 4°C.
4. Concentration of Crude Clathrim-Coated Vesicles by Pelleting
5. Removal of Aggregated Material
6. Removal of Smooth Membrane Contaminants
Following this protocol (for summary, see Fig. 1), near homogeneous preparations of clathrin-coated vesicles are obtained from bovine brain. The yields usually are about 50-100mg clathrin-coated vesicles/ kg brain cortex.
This basic protocol can also be used with other organs rich in clathrin-coated membranes, such as adrenal glands, liver, or placenta. For adrenal glands, a more thorough homogenization (usually 6 or more 10-s bursts in a Waring commercial blender) is required to break up the very resistant capsule material.
Clathrin-coated vesicle preparations from liver usually contain a considerable amount of ribonucleoprotein complexes termed vaults. It is advisable to remove these structures by velocity centrifugation on 5-40% sucrose gradients as an additional step after the preparation described earlier (for details, see Kedersha and Rome, 1986). Vaults are at least partially dissociated by the conventional extraction methods for clathrin coat structures and thus contaminate coat protein extracts.
For the preparation of fusion-competent, clathrincoated vesicle from cell culture cells, a more elaborate protocol has been described (Woodman and Warren, 1991)
Do not mix the Ficoll-sucrose solution to microsomal pellets obtained in Section III,C,3 before thorough homogenization of the pellets. The high viscosity of the Ficoll-sucrose solution prevents proper homogenization.
In order to quantitatively pellet the clathrin-coated vesicles from the supernatant after centrifugation in Ficoll-sucrose, dilute with a minimum of 3 volumes of buffer A + PMSF to decrease both the density and the viscosity of the supernatant.
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