3105
Journal of Cell Science 110, 3105-3115 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS9653
Direct measurement of clathrin-coated vesicle formation using a cell-free
assay
Anne Gilbert, Jean-Pierre Paccaud and Jean-Louis Carpentier*
Department of Morphology, CMU, 1, rue Michel Servet, CH-1211 Geneva 4, Switzerland
*Author for correspondence (e-mail: [email protected])
SUMMARY
Factors controlling the last stages of clathrin-coated vesicle
formation were investigated using an assay allowing direct
measurement of the detachment of these vesicles from the
plasma membrane. Plasma membranes from cultured cells
surface-labelled with 125I-α2-macroglobulin (a ligand that
preferentially associates with clathrin-coated pits) were
isolated by sonication of cells attached to a poly-L-lysinecoated substratum and incubated in the presence of
nucleotide(s) ± cytosol. A significant proportion of the
membrane-associated radioactivity was released into the
incubation medium in sedimentable form (14×106 g). The
nucleotide and ligand specificities of this process together
with the results of a series of biochemical, morphological
and gradient analyses, led to the conclusion that measure-
ment of the released sedimentable radioactivity provides a
direct estimate of the formation of clathrin-coated vesicles
from clathrin-coated pits. A morphological analysis of
quick-frozen replicas of these membranes indicated that
only the last stages of clathrin-coated vesicle formation
were studied in the assay. Taking advantage of this cell-free
system, we demonstrate that membrane-associated
cytosolic factors and GTP-binding proteins, noteably
dynamin, play a crucial role. Moreover, although these
events can occur in the absence of ATP and Ca2+, optimal
conditions for the formation of clathrin-coated vesicles
require the presence of ATP, GTP and cytosol.
INTRODUCTION
of the membranes and are therefore not directly accessible to biochemical manipulation. Thus, understanding these mechanisms
requires the reconstitution of individual steps in vitro and the
development of cell-free or broken-cell systems. Two such model
systems have been developed, but their conclusions remain
mostly controversial (Smythe et al., 1989, 1992; Schmid and
Smythe, 1991; Lin et al., 1991, 1992; Podbilewicz and Mellman,
1990; Schmid, 1993). In the present study we report the development of a cell-free assay where the inside of the plasma
membrane is exposed and which allows, for the first time, direct
measurement of the detachment of clathrin-coated vesicles and
also enables the parameters involved in the formation of these
vesicles to be investigated. Taking advantage of this cell-free
system, we followed the uptake of a receptor constitutively
present in clathrin-coated pits (α2-macroglobulin receptor tagged
with 125I-α2-macroglobulin) and showed that clathrin-coated
vesicle formation requires cytosolic factors and is stimulated by
ATP and GTP. GTP-binding proteins (in particular dynamin) participate in the process, which can occur independently of Ca2+.
Surface receptors enable cells to select from the extracellular
medium the exact components they need at each moment. These
ligands, necessary for cell function, bind to high affinity surface
receptors, which are either already present in clathrin-coated pits
in their unbound state, or become localized in these specialized
surface domains as a result of ligand binding (Brown et al., 1983;
Goldstein et al., 1985). This surface interaction between the
ligand and the receptor is followed by the uptake of the ligandreceptor complex into the cell via the formation of clathrincoated vesicles in a process named receptor-mediated endocytosis (Goldstein et al., 1985; Van Deurs et al., 1989; Pearse
and Robinson, 1990; Trowbridge, 1991; Schmid, 1992). In the
case of transport protein receptors, this process allows the
delivery of nutrients or cellular building blocks, such as cholesterol, iron or vitamin B12, inside the cells. In the case of signalling receptors (i.e. polypeptide hormone or growth factor
receptors), receptor-mediated endocytosis does not seem to be
required for the transmission of the biological signal but rather
is involved in the control of surface receptor number (and hence
cell sensitivity to corresponding hormones and growth factors)
and in the removal of ligands from the cell surface to terminate
their action (Brown et al., 1983; Carpentier, 1989, 1993).
Little is known about the mechanisms underlying receptormediated endocytosis, largely because the components of the
machinery are located, for the most part, on the cytoplasmic side
Key words: Clathrin, Endocytosis, G-protein, Dynamin
MATERIALS AND METHODS
Materials
Culture dishes were obtained from Becton Dickinson and Thermanox
coverslips (15 mm diameter) from NUNC (Naperville IL). DMEM
medium, FCS, penicillin/streptomycin, non-essential amino acids and
3106 A. Gilbert, J.-P. Paccaud and J.-L. Carpentier
EDTA were purchased from Gibco laboratories (Grand Island, NY).
Sephadex G-25, Sephacryl S-300 and Sepharose-S-cation exchange
columns were obtained from Pharmacia (Uppsala, Sweden). 125I and
ECL western blotting detection reagents were from Amersham (Buckingham, UK) and IODO-GENTM from Pierce (Rockford, IL); α2
macroglobulin and cholera toxin B-subunit were from CalBiochem (La
Jolla, CA). Nucleotides and analogues, Nycodenz, poly-L-lysine, Hepes,
EDTA, DTT, BSA fraction V, trypsin, soybean trypsin inhibitors, wheat
germ agglutinin (WGA), saturated transferrin, ATP bioluminescent
Assay Kit and ATP/GTP-regenerating system (consisting of creatine
phosphate and creatine phosphokinase) were obtained from Sigma (St
Louis, MO). Triton-X100 was from MERCK (Darmstadt, Germany);
glutaraldehyde from Fluka Chemie (Buchs, Switzerland), agar from
Difco laboratories (Detroit Michigan, USA), nitrocellulose paper from
Bio-Rad laboratories (Richmond, CA) and acrylamide/bisacrylamide
from SERVA (Heidelberg, Germany). Dynamin wild type and K44A
mutant were a generous gift from S. Schmid (Department of Cell
Biology, The Scripps Research Institute, La Jolla, California).
Cell culture
3T3 L1 fibroblasts were grown to confluence in DMEM medium supplemented with 10% FCS, penicillin/streptomycin and non-essential
amino acids. Fibroblasts required for in vitro experiments were
incubated overnight in FCS-depleted medium to remove most of the
cellular bound α2-macroglobulin derived from serum.
Iodination
α2-macroglobulin was radiolabeled with 125I (specific activity 1.7
mCi/mg), using IODO-GEN and according to the manufacturer’s
instructions. The B-subunit of cholera toxin, transferrin and WGA
were iodinated by the same IODO-GEN method. The labeled ligands
were separated from 125I by gel filtration on a PD10 Sephadex G-25
column using 0.1 M sodium phosphate buffer (pH 7.4) as eluent.
125I-α -macroglobulin-trypsin complexes (125I-α -M) were formed
2
2
by incubation for 5 minutes at room temperature of 125I-α2
macroglobulin with a twofold molar excess of trypsin, followed by
addition of soybean trypsin inhibitor to the same final molar concentration as the trypsin. Reaction products were separated from
complexes by filtration on Sephacryl S-300 (Gliemann et al., 1983).
Cytosol preparation
Cytosol was extracted from calf brain and fractionated on a Sepharose
S-cation exchange column as described by Lin et al. (1991) (with
modifications). Briefly, cytosol (30 ml) was applied to a Sepharose-S
cation exchange column (2.5×15 cm) that had been equilibrated with
Hepes buffer (25 mM, pH 6.8) containing 1 mM EDTA. The column
was washed with 30 ml Hepes buffer and eluted with Hepes buffer
containing 0.1 M KCl. 2 ml fractions were collected and proteins
measured by absorbance at 280 nm. Two protein peaks were obtained:
the first one corresponded to the void volume (elution of dextran blue
2000) while the second corresponded to a molecular weight of
670,000 (thyroglobulin) (Keen et al., 1979). Immunoblotting with an
anti-clathrin antibody revealed that peak 2 was enriched in clathrin
(data not shown).
When required, the fractionated and dialysed cytosol was clathrindepleted with antibody CVC7 IgG2a (hybridoma supernatant).
Antibody was concentrated by ammonium sulfate precipitation and
incubated with cytosol (1 mg/ml) for 2 hours at 4°C (1V/10 V
antibody/cytosol). Antibody-antigen complexes were removed from
the cytosol by protein A-Sepharose incubation (2 hours, 4°C) and centrifugation of protein A-antibody-antigen complexes. Control cytosol
was similarly treated with protein A-Sepharose.
ATP present in the perchloric acid-extractable nucleotide pool of
cytosol was assayed using the ATP bioluminescent Assay Kit. Fractionated and dialysed cytosol contained 1.5±0.4 nM ATP, corresponding to 13.6% of the initial ATP concentration in the non-dialysed
cytosolic fraction.
Preparation of 125I-α2-M-labeled membranes
Purified plasma membrane preparations were prepared according to
Lin et al. (1991), with some modifications. Fibroblasts were detached
from culture flasks by incubating cells for 10 minutes at 37°C in
DPBS (2.5 mM KCl, 1.5 mM KH2PO4, 0.15 M NaCl, 10 mM
Na2HPO4, pH 7.4) containing 0.02% EDTA, followed by resuspension in α2-M binding buffer (124 mM NaCl, 4.7 mM KCl, 2.5 mM
CaCl2, 1.25 mM MgCl2, 2.5 mM H2PO4−/HPO42−, 1.25 mM Na2SO4,
25 mM Hepes and 5% albumin, pH 7.6) at 4°C. This buffer, described
by Gliemann and Davidsen (1986), was required for optimal binding
of 125I-α2-M to fibroblasts. When either 125I-transferrin, 125I-WGA or
125I-B subunit of cholera toxin (CT) were used as ligand, cells were
resuspended in 3T3-L1 buffer (100 mM NaCl, 1 mM CaCl2, 10 mM
KCl, 50 mM Hepes, 1% albumin, pH 7.4). Fibroblasts in suspension
were incubated for 2 hours at 4°C in the presence of 125I-α2-M and
unbound 125I-ligand was removed by centrifugation (5 minutes, 1000
rpm). All operations were performed at 4°C. Incubated cells were
sedimented for 1 hour onto coverslips (15 mm diameter) coated with
poly-L-lysine (1 mg/ml) stuck to the bottom of 35 mm Petri dishes.
The medium was aspirated and 1 ml of binding buffer (containing at
least 2% BSA) was added for 30 minutes. Adherent cells were then
washed twice in buffer A (50 mM Hepes, pH 7.4, 100 mM NaCl) and
twice in buffer B (25 mM Hepes-KOH, 25 mM KCl, 2.5 mM
magnesium acetate, 0.2 mM DTT, pH 7.0) before being disrupted in
buffer B by gentle sonication (Branson, sonifier 250). To remove cell
debris, coverslips were immediately rinsed in buffer B.
Plasma membrane budding
Five coverslips with adherent plasma membranes were fixed with
double-sided tape to a plastic support. The support was then inverted
onto an ice-cooled Petri dish containing 3.5 ml of buffer B supplemented with or lacking factors to be tested. Petri dishes containing
the sets of five coverslips were incubated for various periods of time
at 37°C or on ice (which results in a minimal temperature of 10°C,
not far from the membrane level). Two sets of five coverslips were
used for each incubation. At the end of the incubation, the coverslips
were cooled by bringing back the Petri dishes onto ice when needed.
Coverslips were next rinsed in buffer B at 4°C. Incubation mixtures
and rinsing buffer were pooled and first centrifuged at 10×103 g. The
supernatants were collected and centrifugated at 14×106 g in a 70.1Ti
rotor (Beckman ultracentrifuge L8-M, Instrument Co, Fullerton, CA).
Supernatants of this second centrifugation were collected and pellets
of the two centrifugations, as well as adherent membranes, were solubilized in 0.5 M NaOH. The radioactive content of each fraction was
measured in a Beckman Gamma 5500 counter. The radioactivity
present in each fraction (collected and pooled from the two sets of
five coverslips) was expressed as a percentage of the total radioactivity associated with these ten coverslips. When required, alkaline phosphodiesterase enzymatic activity was measured as described by
Beaufay et al. (1974).
Electron microscopy
Thin section electron microscopy of intact cells
Fibroblasts incubated in the presence of α2-M coupled to colloidal
gold (15 nm) (α2-M-gold) and prepared as previously described (Tran
et al., 1987), were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2, for 30 minutes at room temperature. They were next
washed several times in the cacodylate buffer and further processed
for electron microsocopy as previously described (Tran et al., 1987).
Quick frozen rotatory replicas of membrane
3T3-L1 fibroblasts surface-labeled at 4°C with α2-M-gold were sedimented on poly-L-lysine (1 mg/ml)-coated nickel grids. Purified
plasma membranes were prepared as described before (Moore et al.,
1987; Lin et al., 1991; Carpentier et al., 1993; Krischer et al., 1993).
Clathrin-coated vesicle formation in vitro 3107
Replicas were performed by the quick-freeze rotary shadowed
technique of Heuser (1980).
Vesicles
To visualize the content of the pellet obtained through plasma
membrane budding, coverslips with adherent membranes (12×5 coverslips) were incubated with 1 mg/ml cytosol and 1 mM ATP for 10
minutes at 37°C and the incubation medium was subjected to high
speed centrifugation as described above. The pellet was then fixed
with 0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.0,
embedded in agar, stained by tannic acid Kellenberger and embedded
in Epon. Thin sections of pellet obtained from the cytosol fraction
were used as a control.
Negative staining
3T3-L1 fibroblasts were sedimented on poly-L-lysine (1 mg/ml)coated nickel grids. Purified plasma membranes were prepared as
described before (Moore et al., 1987; Lin et al., 1991; Carpentier et
al., 1993; Krischer et al., 1993) and the membranes were incubated
under various conditions. At the end of the 10 minute incubation
period, membranes were washed in buffer B at 4°C and fixed for 15
minutes at 4°C followed by 15 minutes at room temperature in 4%
glutaraldehyde in buffer B. Tannic acid and uranyl acetate staining
were performed according to Sanan and Anderson (1991).
Nycodenz density gradient centrifugation
Pellets resulting from high-speed centrifugation of the incubation
medium (see above) or microsomal fractions prepared according to
de Duve et al. (1955) were resuspended by Dounce homogenization
in 1 ml buffer B. The suspension was then overlaid (in a tube for the
SW55 rotor) on a discontinous gradient of Nycodenz consisting of 0.9
ml Nycodenz (1.13, 1.17, 1.25, 1.30 g/ml) or on a continuous
Nycodenz gradient (1.07-1.30 g/ml). The suspension from 125I-CT
budding reactions was deposited in the bottom of the tube and overlaid
with a discontinuous gradient consisting of 0.9 ml of Nycodenz (1.25,
1.17, 1.13, 1.02 g/ml). After centrifugation at 39,000 rpm for 16 hours,
the gradient was cut and the fractions were collected. The radioactivity present in each fraction was measured.
EDTA washes
125I-α
2-M internalization in 3T3L1 fibroblasts
Fibroblasts were detached from culture flasks and resuspended in
binding buffer as described above. Duplicate samples of 1.5×106
cells/ml were incubated in the presence of 125I-α2-M for 2 hours at
4°C. The medium was removed by centrifugation (5 minutes, 1000
rpm) and replaced by fresh incubation medium and a second incubation was carried out for various periods of time (3, 5, 10, 20 minutes)
at 37°C in the absence of 125I-α2-M. Incubation was terminated by
diluting samples in ice-cold PBS and centrifugation at 4°C. Pellets
were resuspended three times in cold 50 mM EDTA (in PBS solution)
for 20 minutes to remove cell-surface-bound ligand. The pellet (internalized 125I-α2-M) and the three supernatants were counted for
radioactivity. Internalization was expressed as a percentage of the
radioactivity in cell-bound plus EDTA washes.
125I-α -M latency in vesicles
2
Pellets resulting from high speed centrifugation were resuspended by
Dounce homogenization in PBS supplemented with EDTA (50 mM)
at 4°C for 30 minutes in order to remove accessible 125I-α2-M
(Gliemann and Davidsen, 1986). The vesicle suspension was then
loaded on a discontinuous Nycodenz gradient as described above and
the radioactivity present in each fraction determined.
Depletion of cellular ATP
Fibroblasts were detached from culture flasks in DPBS containing
0.02% EDTA as described previously, washed and resuspended at 4°C
in DPBS containing 10 mM NaN3 and 2 mM NaF (Schmid and Carter,
1990). Cells were then incubated at 37°C for 30 minutes to deplete
cellular ATP. Ligand incubation was performed as described previously,
except that binding buffer was supplemented with NaN3 and NaF.
RESULTS
125I-α -M
2
is segregated in clathrin-coated structures
on isolated plasma membranes
Previous studies have shown that following a 2 hour incubation
at 4°C, α2-M either coupled to colloidal gold or radiolabeled
with 125I preferentially associated with clathrin-coated pits
(>80%), and when the cells were warmed at 37°C, the ligand
was rapidly internalized (Dickson et al., 1981; Tran et al., 1987;
Krischer et al., 1993) (Fig. 1A,B). The goal of the present study
was to develop a cell-free system allowing the formation of
clathrin-coated vesicles from clathrin-coated pits to be tracked,
so we made use of 3T3L1 fibroblast surface-labelled with this
ligand and isolated plasma membranes according to the methodology of Lin et al. (1991). 3T3-L1 fibroblasts were surfacelabelled in suspension with gold-α2-M or 125I-α2-M at 4°C,
sedimented on poly-L-lysine-coated grids or coverslips,
sonicated and extensively washed so that only plasma
membranes remained adhered to the coverslips or the grids. As
shown on rapid-freeze deep-etched replicas, the inner surfaces
of plasma membranes were cleaned of most neighboring cytoplasmic organelles except some cytoskeleton elements; by
contrast, clathrin-coated pits and flat clathrin lattices remained
intact (Fig. 1C,D) and the organization of the clathrin coat was
preserved (Heuser, 1980). Following a 2 hour incubation at 4°C
in the presence of α2-M coupled to colloidal gold (15 nm),
labelling remained low, but >80% of the tagged ligands were
found in clathrin-coated pits or associated with flat clathrin
lattices when observed by transparency on quick-frozen rotatory
replicas (Fig. 1C,D). Thus, under the conditions studied, only
stages of clathrin-coated vesicle formation subsequent to the
organization of the clathrin lattices were investigated.
125I-α -M
2
is released into the incubation medium in a
sedimentable form
When coverslips with associated 125I-α2-M-labelled
membranes were incubated for various periods of time at 37°C
in the presence of fractionated cytosol (1 mg/ml of protein)
and/or nucleotides, radioactivity appeared in the incubation
medium in a time- and nucleotide-dependent fashion (Fig. 2).
Control conditions, including low temperature incubations at
approx. 10°C in the presence of cytosol, or at 37°C in buffer
alone, showed a drastic reduction in radioactivity release (Fig.
2). The radioactivity released into the medium in the presence
of nucleotides and/or cytosol was not associated with cellular
debris and fragments since a low speed centrifugation (10×103
g) sedimented only a small percentage (<3%) of the initially
bound radioactivity in all conditions tested (Fig. 2).
To distinguish the proportion of the released radioactivity
that corresponded to 125I-α2-M dissociation and the proportion
that could potentially be associated with released organelles,
the supernatant of the above-mentioned low speed centrifugation was submitted to high speed centrifugation (14×106 g).
Under these conditions, approx. 10-20% of the radioactivity
initially associated with the membranes remained soluble independently of time, temperature and exposure to nucleotides
3108 A. Gilbert, J.-P. Paccaud and J.-L. Carpentier
and/or cytosol (Fig. 2). Note, when free 125I-α2-M was
submitted to the same centrifugation 95% of the material
remained soluble (data not shown). By contrast, the proportion
of radioactivity that sedimented at high speed ranged from
approx. 3% to 60%, depending on the experimental conditions
(Fig. 2). On the basis of these observations, we considered that
high speed sedimentable radioactivity was potentially representative of the formation of clathrin-coated vesicles, and in all
subsequent experiments this value was recorded as a measure
of the specific release of radioactivity.
Sedimentable 125I-α2-M is released via the formation
of clathrin-coated vesicles
Information about the nature of the structures with which sedimentable 125I-α2-M was associated was obtained by gradient
centrifugation and electron microscopic analysis of the pellet.
Fig. 1. Electron microscopic localization of α2-M receptors in clathrin-coated pits. Cultured 3T3 L1 fibroblasts were incubated for 2 hours at
4°C in the presence of α2-M-gold (15 nm) and prepared for either thin-section electron microscopy (A,B) or quick-freeze rotary shadowing
(C,D), as described in Materials and methods. (A,B) Gold particles (black) are associated with clathrin-coated pits (cp) on the cell surface.
(C,D) View of the inner aspect of the plasma membrane showing the typical honeycomb structure of the clathrin coat, which either decorates
the cytoplasmic surface of an invagination (C) or appears as a planar lattice (D). Colloidal gold particles (seen by transparency and appearing
white because the negative was reversed) are associated with membrane segments decorated with clathrin. Bars, 0.2 µm (A,B,D); 0.1 µm (C).
A
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AAAA
AA
AA
AA
AA
AA
AA AA
AA
AA
AA
AA
AA
AA
AA
AA
AAAA
AA
AA
AA
AA
AA AA
sed (14×106 g)
80
A Incubation medium pelleted and resuspended
sed (10×103 g)
100
soluble
100
α2M
(cytosol + ATP)
80
80
α2M
(GTP)
Fig. 2. Release of 125I-α2-M from adhered plasma membranes.
Coverslips coated with 125I-α2-M membranes were prepared as
described in Materials and methods and incubated in buffer B
containing CaCl2 (1 mM) and supplemented with or lacking
fractionated cytosol (1 mg/ml), ATP (1 mM) or GTP (1 mM) for the
indicated periods of time, at 8-10°C or 37°C. The incubation medium
was centrifuged at low speed (10×103 g) and the radioactive content of
the pellet was measured. The supernatant resulting from the first
centrifugation was then centrifuged at high speed (14×106 g) and
radioactivity present in the resulting supernatant and pellet was
measured. Sedimentable (low and high speed) and soluble radioactivity
are expressed as a percentage of the total radioactivity initially
associated with plasma membranes (= total sedimentable radioactivity
+ soluble radioactivity + radioactivity remaining associated with the
membranes at the end of the incubation period). Values are expressed as
mean ± s.e.m. (applicable to values obtained at the end of the high
speed centrifugation); n=3, 3, 3, 3, 6, 5, 4, 14, 6, 3 (from left to right).
Following centrifugation in a discontinous Nycodenz gradient,
sedimentable 125I-α2-M equilibrated at a density of 1.17/1.25
g/ml (Fig. 3). When a continuous Nycodenz gradient was used,
125I-α2-M-containing structures were recovered at a median
density of 1.196 g/ml, corresponding to the density of the
clathrin-coated vesicles (data not shown) (Pearse, 1983; Simion
et al., 1983; Daiss and Roth, 1983). Moreover when transferrin,
a ligand classically internalized via clathrin-coated pits and
currently used in broken cell systems as a marker of clathrincoated pits (Schmid and Smythe, 1991), was applied in its
iodinated form, 125I-transferrin-containing vesicles equilibrated
at the same density as those containing 125I-α2-M (Fig. 3). In
both cases, more than 80% of the sedimentable radioactivity was
recovered in the 1.17/1.25 g/ml fraction. Such a distribution was
clearly distinct from that of plasma membrane markers (alkaline
phosphodiesterase and 125I-WGA) (Fig. 3). Similar conclusions
were reached following exposure to either GTP, cytosol+ATP or
GTPγS (Fig. 3 and data not shown). Use of 125I-B-subunit of
cholera toxin, which does not segregate in clathrin-coated pits
but associates with and is internalized through caveolae (Tran et
al. 1987), resulted in the equilibration of 75% of the sedimentable (at 14×106 g) radioactive material in the lower density
fraction of the gradient (<1.13 g/ml), and this radioactive
material remained sedimentable at 14×106 g (Fig. 3).
40
40
1.25/1.30
1.13/1.17
load/1.13
1.25/1.30
1.17/1.25
1.13/1.17
load/1.13
60
20
20
0
0
density (g/ml)
1.25/1.30
15
60
Transferrin
(GTP)
density (g/ml)
B Microsomal fractions
100
100
Phosphodiesterase
80
60
40
20
0
density (g/ml)
WGA-4°C.
80
60
40
20
0
1.25/1.30
15
10
80
1.17/1.25
-
10
80
1.17/1.25
-
10
100
CT
(cytosol + ATP)
1.13/1.17
-
10
100
1.13/1.17
-
+
+
+
load/1.13
min at 10°C
15
+
+
load/1.13
10
+
Percent recovered
radioactivity
5
-
1.25/load
2
+
-
1.25/1.30
min at 37°C
+
+
-
0
1.17/1.25
+
+
-
20
0
1.17/1.25
+
+
-
20
1.13/1.17
+
+
-
40
1.13/1.17
+
+
-
40
1.02/1.13
0
cytosol
ATP
GTP
60
load/1.13
20
Percent recovered radioactivity
40
60
1.17/1.25
60
Percent recovered
phosphodiesterase
Released radioactivity (% of total)
Clathrin-coated vesicle formation in vitro 3109
density (g/ml)
Fig. 3. Density distribution in Nycodenz gradient of the sedimentable
radioactivity resulting from the membrane budding reaction.
(A) Fibroblasts were incubated with radiolabelled α2-M, transferrin or
the B-subunit of cholera toxin (CT) at 4°C. Plasma membrane-coated
coverslips were prepared and incubated for 10 minutes at 37°C in buffer
B containing cytosol+ATP (1 mM) or GTP (1 mM). Pellets resulting
from high speed centrifugation of incubation medium were resuspended
by Dounce homogenization in 1 ml of buffer B and loaded onto
Nycodenz discontinous gradients, as described in Materials and
methods. For the CT gradient, incubation medium was deposited in the
bottom of the tube and overlaid with the gradient. Similar results were
obtained when the incubation medium was deposited on the top of the
gradient. Data show the percentage of radiolabelled ligand recovered in
each fraction of the gradient at the end of the centrifugation.
(B) Microsomal fractions were obtained from cells incubated with 125IWGA (wheat germ agglutinin) for 2 hours at 4°C and loaded onto the
same Nycodenz discontinous gradient as above and subjected or not to
alkaline phosphodiesterase detection (Beaufay et al., 1974). Values are
the mean ± range of two experiments.
Evidence for the presence of 125I-α2-M within sealed
vesicles was obtained by washing the vesicles in the presence
of EDTA (Glieman and Davidsen, 1986). Following such
washes, more than 80% of the sedimentable 125I-α2-M was
3110 A. Gilbert, J.-P. Paccaud and J.-L. Carpentier
recovered in the 1.17/1.25 g/ml fraction of the Nycodenz
gradient, similar to what was observed in control conditions
(data not shown). These results indicated that the radioactive
material was not accessible to EDTA treatment and was thus
located inside the vesicles. Similar EDTA treatment of intact
cells surface-labelled with 125I-α2-M removed >90% of the
radioactivity from the cell surface.
Direct observation, at the EM level, of the pellet collected at
the end of an incubation in the presence of nucleotides+cytosol
or in the presence of GTP alone indicated that it contained
vesicles, some being decorated with clathrin (Fig. 4). (Note that
the low binding of α2-M coupled to colloidal gold and the relatively low amount of clathrin-coated vesicles observed (in particular due to the very small size of the pellet) hampered visualization of gold particles within the released clathrin-coated
vesicles.) The fuzzy material present between vesicles corresponded to the cytosol added to the incubation medium and
required to obtain a visible pellet at the end of the ultracentrifugation. Indeed ultracentrifugation of cytosol revealed the
uniform presence of fuzzy material and the absence of vesicular
structures (data not shown). Clathrin-coated vesicles present in
the pellet showed a mean diameter of 88.7 nm, whereas nonclathrin-coated vesicles had a mean diameter of 65.1 nm, consistent with the diameters of vesicles derived from clathrincoated pits and caveolae respectively (van Deurs et al., 1989).
Biochemical attempts to reveal the presence of clathrin in
the vesicular pellet by immunoblotting were unsucessful
because the amount of membrane available was too low,
hampering any protein detection and hence leading to an
inability to detect clathrin in the original membrane prepara-
Fig. 4. EM views of the pellet obtained by high speed sedimentation
of the incubation medium. Plasma membrane-coated coverslips were
incubated for 10 minutes at 37°C in buffer B containing cytosol (1
mg/ml), CaCl2 (1 mM) and ATP (1 mM). Incubation medium was
then subjected to high speed centrifugation (14×106 g). The pellet
was fixed with 2.5% glutaraldehyde in cacodylate buffer (pH 7.0)
and processed for thin section electron microscopy as described in
Materials and methods. The pellet contains vesicles, among which
some are delineated by a clathrin coat. Βar, 0.1 µm.
tion and a fortiori in the pellet. Such failure cannot however be
attributed to the absence of clathrin coats since: (1) the incubation medium contained clathrin-coated vesicles (see above);
and (2) clathrin-coated structures were present on the cytoplasmic side on isolated membrane preparations, as directly
shown morphologically (Figs 1, 5A). Moreover, a detailed
morphological quantification of negatively stained isolated
plasma membranes exposed to various experimental conditions
revealed a good parallel between the release of 125I-α2-M into
the incubation medium and the loss of clathrin-coated structures from the isolated plasma membranes. The percentage of
plasma membrane inner surface decorated with clathrin coats
indeed dropped by 57% and 90% following exposure to GTPγS
or ATP+GTP+cytosol, respectively (Fig. 5B). Eventually,
following pretreatment of isolated membrane with 0.5 M Tris
to remove clathrin (see below) (Keen et al., 1979), incubation
of these membranes in the presence of cytosol restored the
release of sedimentable radioactivity (Fig. 6). Importantly, this
recovery of function was reduced by 70% if the incubation was
carried out in the presence of clathrin-depleted cytosol (Fig. 6).
Taken together, these data demonstrate that sedimentable
125I-α -M recovered in the incubation medium is released via
2
the formation of clathrin-coated vesicles.
Cytosol and nucleotide requirements for the
formation of 125I-α2-M-containing vesicles
The presence of cytosol in the incubation medium stimulated the
formation of 125I-α2-M-containing vesicles (Fig. 7A). This effect
was significantly potentiated by ATP or GTP and the maximal
stimulation was reached when both nucleotides were added
together (Fig. 7A). Stimulation was also observed in the presence
of either ATP or GTP even in the absence of added cytosol (Fig.
7A). At all concentrations tested, GTP was found to be more
efficient than ATP (Fig. 7B). In the absence of added cytosol,
however, ATP and GTP effects could have been mediated by
cytosolic factors adhered to the membranes. To investigate this
possibility, experiments were repeated after treatment of the
membranes with 0.5 M Tris or 0.25 M NaCl, pH 7.0, two conditions known to remove membrane-associated proteins
including or not clathrin, respectively (Keen et al., 1979; Peeler
et al., 1993), and the effects of ATP and GTP were abolished (Fig.
6). By contrast, the effects of the two nucleotides were restored
by supplementing the incubation medium with fractionated
cytosol (which contained clathrin, as verified by immunoblotting
with anti-clathrin antibody) (Fig. 6 and data not shown). Thus,
cytosolic factors associated with membranes are implicated in
clathrin-coated vesicle formation.
Given that GTP stimulated vesicle formation (Fig. 7A,B),
membrane-associated GTP-binding proteins could be involved
in the process. This seemed to be the case since, in the absence
of cytosol, two non-hydrolyzable analogues of GTP (GTPγS
and GMP-PNP) stimulated vesicle formation to the same
extent as GTP (Fig. 8). This was even more obvious at lower
nucleotide concentration, where both analogues were similarly
more efficient than GTP in inducing vesicle formation (Fig. 8).
Under these conditions, GTP degradation by membrane-associated GTPases could, at least in part, be responsible for the
weak effect of GTP, since a GTP-regeneration system
enhanced this effect (Fig. 8) and extensive washing of the
membranes (which cleaned the membrane of associated
GTPases) improved GTP stimulatory effects (Fig. 6). These
Clathrin-coated vesicle formation in vitro 3111
observations provided evidence for the ‘locking’ of a regulatory GTP-binding protein(s) in an active form in the presence
of non-hydrolysable GTP analogues. Further evidence for the
involvement of GTP-binding protein(s) in the process was the
weak stimulation of the formation of 125I-α2-M-containing
vesicles in the presence of GDP (Fig. 8).
A role for dynamin in clathrin-coated vesicle
formation
Dynamin has recently been demonstrated to be involved in the
last stages of the pinching off of clathrin-coated pits (Herskovits
et al., 1993; van der Bliek et al., 1993; Damke et al., 1994,
1995). To determine whether this GTP-binding protein could
participate in the process under study, isolated membranes
were exposed to 0.25 M NaCl, a condition that is known to
clean the membranes of associated proteins but which does not
affect clathrin-coated pit organization (see above). Following
such treatment, GTPγS, similar to GTP (see above and Fig. 9),
was without effect on clathrin-coated vesicle formation (Fig.
9). By contrast, the addition of dynamin to the incubation
medium restored this effect, whereas a mutated form of
dynamin, previously described to be deficient in GTP binding
(van der Bliek et al., 1993; Damke et al., 1994, 1995), was
without significant effect (Fig. 9). These observations demon50
Sedimentable radioactivity (%)
40
A
AA
A
AA
AA
AA
AA
AA
30
20
10
0
control
Fig. 5. Negative staining of isolated plasma membrane and
quantification of the surface occupied by clathrin-coated structures.
(A) Plasma membranes incubated for 10 minutes at 37°C in buffer
B, were fixed, stained with uranyl acetate and processed as
described in Materials and methods. This preparation allows one to
distinguish between flat clathrin lattices (cl) or coated pits (cp), and
invaginated clathrin-coated domains of the plasma membrane or
coated buds (cb). (B) Plasma membranes which had been incubated
for 10 minutes at 37°C in buffer B supplemented with or lacking
GTPγS (0.2 mM) or a mixture of cytosol, ATP (1 mM) and GTP
(1 mM), and prepared as described in (A), were used to estimate the
relative proportion of flat clathrin lattices and clathrin-coated pits,
as well as the loss of clathrin-coated structures from the plasma
membrane (production of clathrin-coated vesicles). The total
clathrin-coated surfaces in control conditions were normalized to
100% and were used as reference for values obtained in the two
other experimental conditions. Under these conditions, the decrease
in the percentages of clathrin-coated pits and lattices from the 100%
in control conditions represented the loss of clathrin-coated
structures and, hence, the formation of clathrin-coated vesicles.
Quantifications were carried out on a total of 50 plasma membranes
collected in the course of two different experiments. Values are
expressed as mean ± range.
AA
AA
AAA
AA
A AA
AAA
AA
A AA
AAA
AA
A
A AAA
extensive
washings
ATP
cytosol + ATP
AA
A
AAAA
AAAA
AA
TRIS
0.5M, pH7
NaCl
0.25M
AA
GTP
cytosol + GTP
clathrin-depleted cytosol
+ GTP
Fig. 6. Effect of Tris and NaCl on clathrin-coated vesicle formation.
125I-α -M-labelled plasma membranes, prepared as described in
2
Materials and methods, were washed at 4°C with: buffer B for 2
minutes (control); buffer B for 15 minutes followed by buffer B (2×2
minutes) (extensive washings); 0.25 M NaCl for 15 minutes followed
by buffer B (2×2 minutes) (NaCl); 0.5 M Tris, pH 7.0, for 15 minutes
followed by buffer B (2×2 minutes) (TRIS). At the end of each pretreatment, coverslips with adherent membranes were incubated in
buffer B for 10 minutes at 37°C supplemented with or lacking
fractionated cytosol, ATP (1 mM) or GTP (1 mM). At the end of the
incubation period, medium was centrifuged (14×106 g) and the
radioactivity present in the pellet was measured and expressed as a
percentage of 125I-α2-M initially associated with plasma membranes.
When indicated, the cytosol was depleted of clathrin by
preincubation with anti-clathrin antibodies. Values are expressed as
mean ± s.e.m. (n=4, except in the case of clathrin-depleted cytosol
where n=3).
3112 A. Gilbert, J.-P. Paccaud and J.-L. Carpentier
strate that, as previously proposed on the basis of experiments
carried out in other assays, dynamin is a crucial protein
involved in clathrin-coated vesicle formation.
Clathrin-coated vesicle formation can occur in the
absence of ATP and Ca2+
At all concentrations studied, GTP was more rapid and more
potent than ATP in promoting cytosol-independent clathrincoated vesicle formation and acted through a GTP-binding
protein (Fig. 7A,B). Experiments carried out with membranes
isolated from ATP-depleted cells (incubations with NaF and
NaN3, depleting >98% of cellular ATP) revealed that the
number of 125I-α2-M-containing vesicles formed in response
Sedimentable radioactivity (%)
A
60
50
ATP + GTP + cytosol
to GTP (in the presence or absence of fractionated cytosol),
was not affected by ATP depletion (data not shown). Thus,
clathrin-coated vesicles can form in the absence of ATP and,
in the absence of cytosol, GTP is the most efficient nucleotide.
Nevertheless, even in the absence of added cytosol, ATP
alone induced clathrin-coated vesicle formation (Fig. 7). Based
on the observation that the addition of ATP+GTP+cytosol to
the incubation medium was the most effective mixture
promoting clathrin-coated vesicle formation, it is clear,
however, that both ATP and GTP play complementary roles in
the formation of clathrin-coated vesicles, but their respective
implication remains to be elucidated.
Addition of 1 mM EGTA to the incubation medium did not
affect the release of sedimentable radioactivity (Fig. 10).
Moreover, clathrin-coated vesicle formation remained constant
at the various Ca2+ concentrations tested (up to 1 mM) (Fig.
10). Thus, Ca2+ is not required at the stages of clathrin-coated
vesicle formation studied by our assay.
GTP + cytosol
40
A
A
A
30
20
10
A
A
A
0
0
5
10
Time (min at 37°C)
ATP + cytosol
DISCUSSION
GTP
Elucidating the molecular mechanisms governing receptormediated endocytosis forms an important cornerstone upon
which to unravel how cell growth and maintenance is controlled. Addressing such questions requires cell-free systems
such as those which allowed a breakthrough in our understanding of how transport vesicles bud and fuse along the
biosynthetic pathway (Malhotra et al., 1989; Rothman and
Orci, 1992; Barlowe et al., 1994).
Two model systems have been developed to reconstitute
clathrin-coated vesicle formation in vitro (Lin et al., 1991;
ATP + GTP
ATP
cytosol
buffer
15
B
1.00
Nucleotide concentration (mM)
Fig. 7. Effect of cytosol, ATP and GTP on clathrin-coated vesicle
formation. (A) 125I-α2-M-labelled plasma membranes were
incubated for various periods of time at 37°C in buffer B containing
CaCl2 (1 mM) and supplemented with or lacking fractionated
cytosol, ATP (1 mM) or GTP ( 1mM), as indicated. At the end of the
incubation period, the medium was centrifuged (14×106 g) and the
radioactivity present in the pellet was measured and expressed in
terms of percent of 125I-α2-M initially associated with plasma
membranes. (B) The same incubation condition were used as in A
(without cytosol) except that the incubation time was constant (10
minutes at 37°C) and that the concentration of the two nucleotides
tested and of the GTP analogue (GTPγS) ranged between 0 mM and
1.0 mM. Values are expressed as mean ± s.e.m. (n=4).
0
Sedimentable radioactivity (%)
10
0
GDP
0.75
20
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AA
AAAA
AAAA
AAAA
AA
GMP-PNP
0.50
30
GTP γ s
0.25
GDP
0
0.00
AA
AAAA
AAA
A AAAA
AA
10
GMP-PNP
ATP
10
20
GTP γ s
20
30
GTP + RS
GTP
[Nucleotide]=1mM
40
GTP
30
B
[Nucleotide]=0.1mM
40
GTP
A
GTP γ S
Sedimentable radioactivity (%)
Sedimentable radioactivity (%)
40
Fig. 8. Effect of GTP analogues on clathrin-coated vesicle formation.
125I-α -M-labelled plasma membranes adhered to coverslips were
2
incubated for 10 minutes at 37°C in buffer B supplemented with
GTP ± regenerating system (5 mM creatine phosphate + 10 U/ml
creatine phosphokinase), GTPγS, GMP-PNP or GDP, as indicated. In
(A) the nucleotide concentration was 0.1 mM whereas in (B) it was 1
mM. At the end of the incubation period, the medium was
centrifuged (14×106 g) and the radioactivity present in the pellet was
measured and expressed as a percentage of 125I-α2-M initially
associated with plasma membranes. Values are expressed as mean ±
s.e.m. (n=4).
Clathrin-coated vesicle formation in vitro 3113
25
Sedimentable radioactivity (%)
Sedimentable radioactivity (%)
20
15
10
5
20
15
ATP (1mM) + cytosol
GTP (1mM)
10
5
0
0.00
0
Dynamin
GTPγ S 0.25mM
0.25
0.50
0.75
1.00
CaCl2 (mM)
+
K44A
WT
+
+
0.25M NaCl
+
Buffer
Fig. 9. Effect of wild type dynamin (WT) and of the dynamin mutant
(K44A) on clathrin-coated vesicle formation. 125I-α2-M-labeled
plasma membranes were washed at 4°C with 0.25M NaCl as
described in Fig. 6. At the end of this pre-treatment, membranes
were incubated for 10 minutes at 37°C in buffer B containing 0.25
mM GTPγS supplemented with or lacking dynamin (30 µg/ml) or
K44A (30 µg/ml). The control condition corresponds to plasma
membranes washed at 4°C with buffer B instead of NaCl. Values are
expressed as mean ± s.e.m. (n=3).
Schmid and Smythe, 1991; Schmid, 1993). The first system
involves the use of perforated cells washed of their cytosol and
whose cytoplasmic domain is freely accessible to reagents
(Smythe et al., 1989, 1992). The coupling of this assay to the
use of probes with different degrees of accessibility from outside
the cell allowed different stages of clathrin-coated vesicle
formation to be distinguished and specifically analysed (Schmid
and Smythe, 1991; Carter et al., 1993). This broken-cell system
has the major advantage of reflecting very closely the in vivo
situation but, on the other hand, the results generated could raise
interpretation problems because the system remains highly
complex, addresses a broad spectrum of events, and cannot be
cleaned of all residual cytosol. The second assay takes advantage
of a virtually pure plasma membrane preparation obtained by
sonication of cells attached to a poly-L-lysine-coated substratum
(Lin et al., 1991, 1992). The adherent membranes are then
exposed to a medium supplemented with various reagents and
the status of clathrin-coated pits on the membrane is assayed
using either morphological or biochemical methods (Lin et al.,
1991). This cell-free assay has allowed a convincing reconstitution of most features of clathrin-coated pit assembly (Moore et
al., 1987; Mahaffey et al., 1989; Peeler et al., 1993). However,
the results generated in terms of clathrin-coated vesicle
formation are controversial, mainly because they give an indirect
estimate of this event through a measure of the disappearance of
endogenous clathrin from plasma membranes (Lin et al., 1991).
Since the results obtained by these two model systems are mostly
conflicting (Schmid, 1993), we have developed in the present
study an in vitro assay which preserves the advantages of Lin’s
cell-free assay but abrogates its major disadvantage of measuring
the loss of an object. Our assay directly estimates clathrin-coated
EGTA 1mM
Fig. 10. Role of Ca2+ in clathrin-coated vesicle formation. 125I-α2M-labelled plasma membranes were incubated for 10 minutes at
37°C in buffer B, supplemented with either GTP (1 mM) or cytosol
and ATP (1 mM) in the presence of EGTA (1 mM) or various
concentrations of CaCl2 as indicated. At the end of the incubation
period, the medium was centrifuged (14×106 g) and the radioactivity
present in the pellet was measured and expressed as a percentage of
125I-α -M initially associated with plasma membranes. Values are
2
expressed as mean ± range of 2 experiments.
vesicle formation through a measure of the release of 125I-α2-M
initially present in clathrin-coated pits.
Radioactivity released into the incubation medium is shown
to be intact 125I-α2-M contained in sealed clathrin-coated
vesicles for the following reasons: (1) it is sedimentable at
14×106 g, demonstrating that it is not soluble but associated
with membranous material; (2) it is not sedimentable at 10×103
g, indicating that it is not associated with cell fragments or large
membranous segments; (3) morphological analysis of the pellet
reveals that it contains clathrin-coated vesicles of the appropriate size; (4) following treatment with 0.5 M Tris (which
removes clathrin), radioactivity release is restored by cytosol
but not by clathrin-depleted cytosol addition; (5) the distribution profile of 125I-α2-M-containing structures in a continuous
Nycodenz gradient indicates a median density (1.196 g/ml),
which agrees with that of clathrin-coated vesicles; (6) more than
85% of the radioactivity present in the pellet is recovered in a
1.17/1.25 g/ml fraction following centrifugation in a Nycodenz
gradient; (7) 80% of the sedimentable radioactivity is inaccessible to EDTA treatment, indicating that it is within sealed
vesicles; (8) the release of the radioactivity is nucleotide specific
and requires cytosolic factors associated with the membranes;
(9) EM analysis of the membrane preparation reveals the
presence of various forms of clathrin-coated structures, and in
experimental conditions optimal for the release of 125I-α2-M
into the incubation medium, they can be seen to disappear; and
(10) dynamin, which specifically induces clathrin-coated
vesicle formation, promotes the release of 125I-α2-M, while an
inactive mutant of this GTP-binding protein has no effect.
The cell-free system that we have developed has three major
advantages. (1) It has a high yield, since the percentage of
radioactivity initially associated with plasma membranes and
which is recovered in the pellet at the end of the incubation ranges
from less 2% (in the absence of added nucleotide and cytosol) to
more than 50% (in the presence of ATP+GTP+cytosol). (2) It
3114 A. Gilbert, J.-P. Paccaud and J.-L. Carpentier
addresses limited stages of clathrin-coated vesicle formation
since, as shown morphologically, following a 2 hour incubation
at 4°C, α2-M is essentially associated with flat clathrin lattices
and clathrin-coated invaginations on the cell surface. Thus, in the
absence of cytosol, the assay addresses neither the question of
clathrin coat assembly nor the process of receptor sequestration
in clathrin-coated pits. By contrast, invagination of the lattice,
pinching off of pits to form buds and detachment of clathrincoated vesicles are events which can be studied. (3) In addition
to a dissection of the last stages of clathrin-coated vesicle
formation, this assay tracks ligand-receptor complexes, allowing
a direct comparison of the mechanisms governing the uptake of
different ligands, such as those which bind to class I and class II
receptors internalized through clathrin-coated pits (Carpentier et
al., 1982), or those which internalize via non-clathrin-coated pits
(Tran et al., 1987; Sandvig and Van Deurs, 1991).
This novel assay demonstrates that cytosolic elements associated with the plasma membrane are involved in the process under
study. Among these, GTP-binding proteins are good candidates
and, more specifically, dynamin may play a key role. Indeed, the
formation of clathrin-coated vesicles was stimulated not only by
GTP but also by two non-hydrolyzable analogues of GTP
(GTPγS and GMP-PNP), while GDP was inactive. GTP was
close to fully effective even in the absence of added cytosol indicating that the GTP-binding protein(s) concerned were associated
with the plasma membrane. These GTP-binding proteins are also
likely to be cytosolic since, following membrane pretreatment
with 0.25 M NaCl, which removed membrane-associated
proteins without affecting clathrin (Keen et al., 1979), the effect
of GTP was abrogated. This inhibition could than be reversed by
the addition of cytosol. The participation of GTP-binding proteins
in clathrin-coated vesicle-mediated endocytosis has been
suggested previously (Carter et al., 1993, Damke et al., 1994),
but in these experiments, while GTP was stimulatory, GTPγS was
inhibitory, suggesting that GTP hydrolysis was required for the
process to be completed. The difference in the conception of the
two assays could explain these apparent discrepancies. Indeed,
the perforated-cell assay may preserve regulatory events which
were not taken into account in the present assay due to the
extensive cleaning of the membrane preparation used. Nevertheless, it must be stressed that our observations agree with those
obtained in studies of COP-coated vesicle formation. Here,
addition of GTPγS to in vitro intra-Golgi transport assays caused
accumulation of COPI-coated vesicles (Malhotra et al., 1989) and
addition of the non-hydrolyzable GTP analogue GMP-PNP to in
vitro ER budding assay resulted in the accumulation of COPIIcoated vesicles (Barlowe et al., 1994).
Previous data concerning the ATP-dependence of clathrincoated vesicle formation in vivo are controversial: while several
groups have reported that receptor-mediated endocytosis was
blocked in ATP-depleted cells (Ciechanover et al., 1983; Hertel
et al., 1986; Schmid and Carter, 1990), others have suggested
that at least a single round of endocytosis can occur in ATPdepleted cells (Clarke and Weigel, 1985; Larkin et al., 1985).
Results from in vitro experiments were similarly inconsistent: on
the one hand, ATP hydrolysis appeared to be necessary for the
scission of deeply invaginated clathrin-coated pits in perforated
A431 cells (Schmid and Smythe, 1991), while on the other hand,
internalization of 125I-transferrin occurred efficiently in broken
MDCK cells in the absence of added ATP or cytosol (Podbilewicz and Mellman, 1990) and ATP hydrolysis was not
required in Lin’s assay (Lin et al., 1991). Our data indicate that
clathrin-coated vesicle formation can occur independently of
ATP. These results are again analogous to those described during
COPI- and COPII-coated vesicle formation, where budding
occurs without an absolute requirement for ATP and where the
only nucleotide required is GTP (Orci et al., 1993; Ostermann
et al., 1993; Barlowe et al., 1994). Nevertheless, ATP together
with cytosol are required, in addition to GTP, for the optimal
release of clathrin-coated vesicles in the experimental conditions
tested. Indeed, as shown both biochemically and morphologically, the most rapid and efficient release of clathrin-coated
vesicles is obtained in the presence of these three components.
The ATP-and GTP-dependences observed in the presence of
cytosol for optimal clathrin-coated vesicle formation are in
agreement with results obtained in perforated cells.
In previous studies where intracellular [Ca2+]i was modulated
by quin2 in the presence or absence of extracellular calcium,
we showed that the rates of internalization of 125I-insulin and
125I-transferrin were independent of [Ca2+] (Iacopetta et al.,
i
1986; Carpentier et al., 1992). Results obtained with the present
cell-free system support these observations, since removal of
calcium from the incubation medium did not affect clathrincoated vesicle formation. These conclusions are similar to those
of Schmid (1993), but differ from those of Lin et al. (1991),
who not only found that 150 µM Ca2+ was required for the
fusion of the adjoining membrane segments at the neck of the
invaginated pit but, in addition, suggested a role for the Ca2+dependent, phospholipid-binding protein annexin VI in
clathrin-coated vesicle formation (Lin et al., 1991, 1992).
Recent observations, however, have questioned the role of
annexin VI in endocytosis (Smythe et al., 1994).
In conclusion, we have developed a cell-free system that
directly measures clathrin-coated vesicle formation and allows
the mechanisms governing the last stages of this process to be
dissected. We demonstrate the involvement of GTP-binding
proteins in this process, and the requirement of ATP and
cytosol for maximal and rapid release. By contrast, Ca2+ does
not appear to be required for clathrin-coated vesicles to form.
Moreover, our results suggest that analogies exists between
COP-coated vesicle and clathrin-coated vesicle formation,
while it is clear that each system also has its own specificities.
Given the potential of this assay, it will prove highly useful in
the identification of cytosolic factors, as well as in the dissection of the mechanisms involved in the formation of clathrincoated vesicles, and in the comparison of the requirements for
the internalization of different ligand-receptor complexes.
We wish to thank Ms G. Porcheron-Berthet for skilled technical
assistance and J. Gruenberg for critical reading of the manuscript and
helpful discussions. This work has been supported by grant
31.43409.95 from the Swiss National Science Foundation.
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(Accepted 13 October 1997)