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/D2
O 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.
This article describes a purification protocol for
bovine brain-coated vesicles that includes a sucrose/
O 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
Ficoll 400 (Cat. No. 17-0400-02) is from Pharmacia;
sucrose (Cat. No. BP 220-1) is from Fisher Scientific;
O (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
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)
, 1.9 mM
, pH 7.0. To make 1 liter of 10× PBS (stock
solution), dissolve 80g NaCl, 2 g KCl, 2.58g KH2
, and 0.2g NaN3
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
- Separate the cerebellum and the lower part of the
brain from the cortex.
- Take a hemisphere of the cortex, place it in an ice
bucket covered with plastic wrap, and remove the
meninges along with blood vessels contained therein
- Collect the cleaned cortex hemispheres in a
preweighed beaker on ice. Determine the weight of the
tissue and estimate the volume of homogenization
buffer needed in Section III,B (approximately 1 liter
per kg of tissue).
- Wash the cortex hemispheres with cold PBS
several times to remove the remaining blood. To do
this, fill the beaker containing the hemispheres with
PBS, mix gently, and pour the liquid off. It is helpful
to use a household sieve at this step. Repeat the washes
until the blood is removed.
- If you want to store the brain tissue for later processing,
cut it into small pieces after the washing step
and freeze it in liquid nitrogen. Store frozen material
at -80°C. Otherwise continue with Section III,B.
- Buffer A: 0.1M MES, 1.0mM EGTA, 0.5mM MgCl2, 0.02% NaN3, pH 6.5. To make 1 liter of 10x
buffer A (stock solution), dissolve 195.2g MES, 3.8g
EGTA, and 1.0g MgCl2 in double distilled water.
Adjust the pH with 10N NaOH to 6.5, add 2 g NaN3,
and bring the volume to 1 liter. Do not add NaN3 prior
to the adjustment of the pH because MES is acidic and
may release HN3. To prepare 1× buffer A for homogenization,
dilute the stock solution 1:10 with doubledistilled
water, chill to 4°C, and supplement with
0.1 mM PMSF prior to use. Prepare about 3 liters buffer
A per kilogram tissue.
- Protease inhibitor (PMSF stock solution, 1000× =
0.1 M). To make 10 ml, dissolve 174 mg PMSF in 10 ml
pure methanol, aliquot, and store at -20°C. Dilute 1:
1000 to get a working concentration of 0.1 mM. Note
that PMSF will hydrolyze in water and that it is not
soluble in this solvent at high concentrations.
C. Differential Centrifugations
1. Preparation of Postmitochondrial Supernatants
- Fill the cup of a Waring commercial blender with
300-400 g of washed tissue and an equivalent amount
of cold buffer A containing 0.1mM freshly added PMSE Do not fill the cup up to the top, but leave space
below the rim.
- Homogenize the tissue by three to six bursts of
10-15s duration with the setting on maximum speed.
The number of bursts required to give a good homogenization
varies: for brain, usually three bursts are
sufficient, whereas other organs, especially adrenal
glands, require more (see Section IV). Do not increase
the length of the bursts but their number to prevent
heating of the homogenate.
2. Preparation of Microsomal Pellets
- Pour the homogenate into buckets of a Sorvall
GS-3 or GSA rotor, balance the buckets, and centrifuge
them in the precooled rotor at 7000rpm (about 8000g in both types of rotors) for 50min at 4°C.
- Pour the turbid supernatants through a funnel
with several layers of gauze to separate floating lipids
from the supernatant.
- The bulky pellets (up to a third of the total
volume) should be resuspended with an at least equivalent
volume of cold homogenization buffer and recentrifuged
under the aforementioned conditions.
- Discard the pellets after the second centrifugation
and keep the combined supernatants on ice.
3. Preparation of Crude Clathrim-Coated Vesicles
from Microsomal Pellets
- Fill the postmitochondrial supernatant into the
tubes for a Beckman Ti 35 or Ti 45 rotor, balance the
tubes, and ultracentrifuge them at 32,000rpm at 4°C. for 1.6 h (Ti 35) or at 40,000rpm for 1 h (Ti 45) in precooled
- After centrifugation, discard the clear supernatant
and either refill the tubes with postmitochondrial
supernatant for the next spin (leave the first pellet
in the tube without resuspending it to save time) or
remove the microsomal pellets when all the postmitochondrial
supernatant has been centrifuged or pellets
from three to four ultracentrifugations have been collected
in one tube.
- Resuspend the pellets with 1-2 volumes of buffer
A supplemented with 0.1 mM PMSF using a 10-ml
glass pipette in the reverse orientation (the wide top
end down) and then homogenize thoroughly by 10-15
strokes in a Potter-Elvehjem device (overhead drive at
: 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
- Mix the well-homogenized microsomes with the
same volume of Ficoll-sucrose solution and fill into
open tubes suitable for an SS-34 Sorvall rotor. Centrifuge
the tubes at 43,000g (19,000rpm) and 4°C for
40 min in a precooled rotor.
- After centrifugation, pour the supernatants containing
the clathrin-coated vesicles through a funnel
with gauze to remove floating lipids. The compact sediment
(about a fifth of the total volume) will stay in the
tube and can be discarded.
- Dilute the combined supernatants with 3-4
volumes of cold buffer A containing 0.1 mM PMSE
5. Removal of Aggregated Material
- Ultracentrifuge the coated vesicles in the diluted
Ficoll-sucrose solution as described in Section III,C,2.
The pellets obtained in this step are considerably
smaller than the microsomal pellets. The color usually
varies from yellowish-white to brown and is most
likely due to contaminating ferritin (Kedersha and
- Pour off the supernatants and carefully remove
the pellets from the tubes with a spatula. Use a small
volume of buffer A + PMSF to wash off material still
attached to the walls of the tube.
- Homogenize the combined pellets in about 20
volumes of buffer A + PMSF per bovine brain using
first a 10-ml glass pipette in the reverse orientation (the
wide top end down) and then homogenize very thoroughly
by 10-15 strokes in a Potter-Elvehjem device
(overhead drive at slow rotation).
6. Removal of Smooth Membrane Contaminants
- Fill the crude clathrin-coated vesicles into open
tubes for an SS-34 Sorvall rotor. Centrifuge the tubes
at 20,000g (13,000rpm) at 4°C for 10min in a precooled
- After centrifugation, retrieve, pool and place the
supernatants on ice. Discard the pellets.
- Buffer A/D2O: 0.1M MES, 1.0mM EGTA, 0.5mM MgCl2, 0.02% NAN3, pH 6.5. To make 1 liter of 1x buffer
A in D2O, dissolve 19.5 g MES, 0.38 g EGTA, and 0.10 g
MgCl2 in D2O. Adjust the pH with 5-10 N NaOH solution
(prepared in D2O as well) to 6.5, add 2 g NAN3, and
bring the volume to 1 liter using D2O. Do not add
NAN3 prior to the adjustment of the pH because MES
is acidic and may release HN3. Chill to 4°C and supplement
with 0.1 mM PMSF prior to use.
- Sucrose/D2O solution: 8% (w/v) sucrose in buffer
A/D2O. To make 200 ml, dissolve 16g sucrose in buffer
A/D2O and adjust the volume with the same buffer.
Keep on ice.
- Precool an SW 28 rotor and the buckets.
- Pour 30ml of the sucrose/D2O solution per SW 28
centrifugation tube (open top, ultraclear, Beckman
#344058). Place the tubes on ice. Carefully overlay
5 ml of the coated vesicles from Section III,C,5 per
- Balance the tubes and assemble the buckets and
the rotor. Spin the rotor in an ultracentrifuge at
110,000g (25,000rpm) at 4°C for 2h.
- Remove the supernatants and resuspend the pellets
in 5-10 volumes of buffer A plus PMSF using a
pipette and a suitable homogenizer.
- Material obtained after this step can either be used
directly or be frozen in liquid nitrogen and stored
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.
|FIGURE 1 Purification scheme for clathrin-coated vesicles.
|FIGURE 2 Coat proteins obtained by Tris extraction of a
clathrin-coated vesicle preparation from bovine
brain as described
in the text. Coated vesicles were
incubated in 0.5M Tris, pH 7.0,
to release the peripheral
membrane proteins and were then
remove the membranes. Approximately
12µg Tris extract
was electrophoresed in a low-Bis SDS-polyacrylamide
gel (for details, see Lindner and Ungewickell, 1992).
that in this gel system the brain-specific, clathrin-
AP180 is well resolved from the
clathrin heavy chain. Both AP180
and auxilin are only
detectable in brain-derived, clathrin-coated
nonneuronal tissues, homologues are expressed (CALM
for AP180 and GAK/auxilin2 for auxilin).
For the preparation of various coat proteins, such
as clathrin or the adaptor complexes, crude clathrincoated
vesicles obtained in Section III,C,4 are a good
starting material of sufficient purity (80-90% pure,
100-120mg/kg bovine brain). For a typical electrophoresis
pattern of a Tris extract of crude bovine
brain clathrin-coated vesicles as described in Section
III,C,4, see Fig. 2.
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
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
For the preparation of fusion-competent, clathrincoated
vesicle from cell culture cells, a more elaborate
protocol has been described (Woodman and Warren,
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
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.
Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988).
Structural relationships between clathrin assembly proteins from
the Golgi and the plasma membrane. EMBO J
Ahle, S., and Ungewickell, E. (1990). Auxilin, a newly identified
clathrin-associated protein in coated vesicles from bovine brain. J. Cell Biol
Bonifacino, J. S., and Lippincott-Schwartz, J. (2003). Coat proteins:
Shaping membrane transport. Nature Rev. Mol. Cell Biol
Brodsky, E M., Chen, C. Y., Knuehl, C., Towler, M. C., and Wakeham,
D. E. (2001). Biological basket weaving: Formation and function
of clathrin-coated vesicles. Annu. Rev. Cell Dev. Biol
Campbell, C., Squicciarini, J., Shia, M., Pilch, R E, and Fine, R. E.
(1984). Identification of a protein kinase as an intrinsic component
of rat liver coated vesicles. Biochemistry 23
De Camilli, P., Chen, H., Hyman, J., Panepucci, E., Bateman, A.,
and Brunger, A. T. (2002). The ENTH domain. FEBS Lett
Kanaseki, T., and Kadota, K. (1969). The "vesicle in a basket". A
morphological study of the coated vesicle isolated from the nerve
endings of the guinea pig brain, with special reference to the
mechanism of membrane movements. J. Cell Biol
Kalthoff, C., Alves, J., Urbanke, C., Knorr, R., and Ungewickell, E. J.
(2002). Unusual structural organization of the endocytic proteins
AP180 and epsin 1. J. Biol. Chem
Kedersha, N. L., and Rome, L. H. (1986). Isolation and characterization
of a novel ribonucleoprotein particle: Large structures
contain a single species of small RNA. J. Cell Biol
Kirchhausen, T. (1999). Adaptors for clathrin-mediated traffic. Annu.
Rev. Cell Dev. Biol
Lindner, R., and Ungewickell, E. (1991). Light-chain-independent
binding of adaptors, AP180 and auxilin to clathrin. Biochemistry 30
Lindner, R., and Ungewickell, E. (1992). Clathrin-associated proteins
from bovine brain coated vesicles: An analysis of their number
and assembly-promoting activity. J. Biol. Chem
Maycox, P. R., Link, E., Reetz, A., Morris, S. A., and Jahn, R. (1992).
Clathrin-coated vesicles in nervous tissue are involved primarily
in synaptic vesicle recycling. J. Cell Biol
Raiborg, C., Bache, K. G., Gillooly, D. J., Madshus, I. H., Stang, E.,
and Stenmark, H. (2002). Hrs sorts ubiquitinated proteins into
clathrin-coated microdomains of early endosomes. Nature Cell
Sachse, M., Urbe, S., Oorschot, V., Strous, G. J., and Klumperman, J.
(2002). Bilayered clathrin coats on endosomal vacuoles are
involved in protein sorting toward lysosomes. Mol. Biol. Cell 13
Schmid, S. L. (1997). Clathrin-coated vesicle formation and protein
sorting: An integrated process. Annu. Rev. Biochem
Stoorvogel, W., Oorschot, V., and Geuze, H. J. (1996). A novel class
of clathrin-coated vesicles budding from endosomes. J. Cell Biol
Traub, L. M., Bannykh, S. I., Rodel, J. E., Aridor, M., Balch, W. E., and
Kornfeld, S. (1996). AP-2-containing clathrin coats assemble on
mature lysosomes. J. Cell Biol
Ungewickell, E., Ungewickell, H., Holstein, E. H., Lindner, R.,
Prasad, K., Barouch, W., Martin, B., Greene, L. E., and Eisenberg,
E. (1995). Role of auxilin in uncoating clathrin-coated vesicles. Nature 378
Wasiak, S., Legendre-Guillemin, V., Puertollano, R., Blondeau, E,
Girard, M., de Heuvel, E., Boismenu, D., Bell, A. W., Bonifacino,
J. S., and McPherson, P. S. (2002). Enthoprotin: A novel clathrinassociated
protein identified through subcellular proteomics. J. Cell Biol
Woodman, P. G., and Warren, G. (1991). Isolation and characterization
of functional clathrin-coated vesicles. J. Cell Biol