Vol.
174,
No.
January
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
15, 1991
197-203
CLATHRIN-COATED VESICLES FROM HUMAN PLACENTA CONTAIN GTP-BINDING PROTEINS James M. Lenhard,
Marilyn
Department Washington
Received
November
20,
Aach
Levy,
and Philip
D. Stahl
of Cell Biology and Physiology University School of Medicine St. Louis, Missouri 63110
1990
Biochemical and morphological techniques were used to investigate the GTP-binding proteins of clathrin-coated vesicles. Binding of [3H]GTP to clathrin coats was demonstrated by electron microscopic autoradiography. Purified coated vesicles bound 5.2 pmol [35S]GTPyS/mg protein. Addition of GTP or GTPyS, but not ATP nor GMP, inhibited binding of [35S]GTP+ to intact coated vesicles and Triton X-loo-extracted coats. A series of 23-24 kDa GTPbinding proteins with isoelectric points between pH 5-8 were detected in coated vesicles. We suggest that the low molecular weight ras-like GTPbinding protein(s) play a role in regulating vesicle-mediated protein transport or signal transduction within intracellular organelles. 0 1991 Academic Press, Inc.
A number eukaryotic yeast,
of steps
cells SARl,
binding
have
YPTl,
secretion
that
membrane vesicles role
in
proteins With
(l-3).
Recent regulate
and the (6). both
inward
this
molecular
weight
low
from
is
our
facilitated have
and outward
membrane
traffic,
GTP-binding
within
the vesicles
proteins. weight
GTP-
stages
indicate
that
endosomes from
In
m-like
different
laboratory
complexes
proteins
Coated
during
of macrophage
GTP-binding with
molecular
a role
fusion
network
of proteins
to GTP-binding
of receptor-ligand
we assayed
proteins.
for
to play
in vitro
be associated
in mind,
code
believed
trans-Golgi Since
transport
to be linked
observations
transport
would
GTP-binding
are
proteins Selective
the vesicular shown
and SEC4 genes
proteins
binding
in been
(4,
of GTP-
5).
the plasma
by clathrin-coated been
identified
it that
purified
clathrin-coated
vesicles
were
found
seemed mediate
as playing likely protein
vesicles to contain
that for
several
a these
transport. associated low
proteins.
METHODS: Coated Vesicle Isolation. All procedures were carried out at 4OC. Human placenta was resuspended in three volumes of 250 mM sucrose, 1 mM MgCl,, 0.5 mM EGTA, 20 mM MES, pH 6.5 (homogenization buffer) and homogenized with a Waring blender. The homogenate was centrifuged at 11,000 x g for 30 minutes and the supernatant was decanted and centrifuged at 29,000 x g for 4.0 hours. The resulting pellet was resuspended in homogenization buffer, layered onto a 30% sucrose cushion, and centrifuged at 80,000 x g for 5 min. The 30% sucrose
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
layer and the pellet were discarded. The supernatant was again layered onto 30% sucrose, centrifuged at 80,000 x g for 5 min, and the sucrose cushion and pellet were discarded. The supernatant was layered onto 10% Ficoll, 45% D,O, 1 mM MgCl,, 20 mM MES, pH 6.5 and centrifuged at 100,000 x g for 120 min. The pellet was resuspended in homogenization buffer and centrifuged at 20,000 x g for 10 min. The resulting supernatant was layered onto 15% Ficoll in 75% D,O, at 140 mM NaCl, 1 mM MgCl,, 0.5 mM EGTA, 20 mM MES, pH 6.5 and centrifuged 80,000 x g for 14 hours. The pellet containing coated vesicles was resuspended in homogenization buffer to 5 mg/ml final protein concentration. Clathrin coats were prepared by diluting the vesicles with 9 volumes of 1% Triton X-100 in homogenization buffer and incubating the sample at 22’C for 30 min. Clathrin coats were collected by centrifugation at 100,000 x g for 60 min. The phospholipid content was measured by following the methods described by Rouser et al., (7). Proteins were measured according to Bradford (8). GTP-binding assavs. Binding of [35S]GTP~S to clathrin-coated vesicles was determined by incubating the vesicles (10 ug total protein/100 ~1) at 22’C for 60 min in 2 uCi [35S]GTPyS (120 Ci/mmol), 250 mM sucrose, 1 mM MgCl,, 0.5 mM EGTA, and 20 mM HEPES, pH 7.0 (binding buffer). The vesicles were collected by centrifugation at 200,000 x g for 30 minutes and washed twice with binding buffer. Competition for [35S]GTP~S binding was determined by adding various concentrations of GTP, GTPrS, ATP, and GMP to the vesicles prior to addition The association of GTP-binding proteins with clathrin coats of [ 35S]GTPyS. was determined by extracting [35S]GTPrS bound to coated vesicles with 1% TX100 for various periods of time. Clathrin coats were collected by centrifugation at 200,000 x g for 30 minutes, washed with binding buffer, and coat-associated radioactivity was determined by liquid scintillation counting. Electron microsconv. Coated vesicles were processed for electron microscopy to monitor purity. The vesicles were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate pH 7.4, post-fixed in 1.5% osmium tetroxide, incubated with 4% uranyl acetate, dehydrated with a graded series of ethanol, infiltrated with propylene oxide , and embedded in Polybed 812 (Polysciences Inc., Washington, Pa.). Tissue was sectioned with a diamond knife, post-stained with uranyl acetate and lead citrate, and examined in a Zeiss 10A electron microscope. Electron microscopic autoradiography was performed by incubating clathrin coats (60 ug total protein) in 240 nl binding buffer containing 10 pCi [3H]GTP (7 Ci/mmol) and 10 uM GTPyS or 10 uCi [3H]GTP and 100 pM ATP. The clathrin coats were washed with binding buffer and processed for electron microscopy as described above. Light gold thin sections were mounted on Formvar carbon-coated nickel grids, coated with Ilford L-4 emulsion using a wire loop, and developed using the gold latensification procedure (9). Developed grids were post-stained in uranyl acetate and lead citrate and viewed in a Zeiss 10A electron microscope. Molecular weight determination. Protein (100 rigs) from purified clathrincoated vesicles and clathrin coats was separated by isoelectric focusing (10) The proteins were transferred to nitrocellulose (12), and SDS-PAGE (11). incubated with 25 uCi [a-32P]GTP, washed, and autoradiographed (13). RESULTS: that
Electron
>99% of
coated
the vesicles
vesicles
a linear
micrographs
centrifuged
for
4 hours
by SDS-PAGE and heavy
the gel.
probing exposing
chain
the gel
in each
Low molecular
Western-blots the
GTP-binding
blots
of proteins to X-ray
film.
proteins, x g.
the
Frctaion
was stained
fraction
weight
(fig
vesicles 1).
To test
vesicles
9% D,O to 20% Ficoll
at 80,000
clathrin
clathrin-coated
clathrin-coated
made of 2% Ficoll
analyzed of
were
contained
gradient
of purified
from
for
GTP-binding separated The amount 198
proteins
whether
were
90% D,O
layered were
The amount
by densitometer were
by SDS-PAGE with of GTP-binding
onto
and
the gradient
protein.
was determined
illustrate
detected [a-32P]GTP protein
of tracings by and in each
Vol.
174,
No.
Figure clathrin purity
1.
Electron microscopy Clathrin-coated coats. by electron microscopy
was quantitated
fraction
that
clathrin
(Fig
Receptors 100-extracted 1% Triton vesicles
for
X-100,
purified vesicles
weight
RESEARCH
clathrin-coated were isolated
as described
molecular
IgG,
smooth with
transferrin, coats
removed
extracted
of
BIOPHYSICAL
and
in the text
(bar
COMMUNICATIONS
vesicles analyzed
= 0.1 micron).
The densitometric GTP-binding
and for
tracings
proteins
revealed
co-fractionate
in
2).
clathrin were
AND
by densitometry.
and low
the gradient
coats
BIOCHEMICAL
1, 1991
(14).
vesicle (compare
Triton
and ferritin When coated
contamination fig
X-100
1 with
are vesicles
insoluble were
and membranes fig
3; 14).
revealed
no qualitative
4
n6umber
in Triton extracted
within
Xwith
coated
SDS-PAGE of clathrinnor
quantitative
40-
2
0
Bottom
Fraction
8
10 TOP
Co-fractionation of GTP-binding proteins and clathrin in Ficoll Figure 2. density gradients. Purified clathrin-coated vesicles (ZOO ug protein) were The distribution of clathrin centrifuged through 10 ml Ficoll gradients. heavy chain and total low molecular weight GTP-binding proteins in the gradient was determined by densitometer tracings of protein stained gels and autoradiographs of Western blots probed with [u-~*P]GTP, respectively. 199
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 3. Electron microscopic autoradiography of [3H]GTP-binding proteins Purified clathrin coats were incubated in the presence of clathrin coats. GTPyS (panel a) or ATP (panel b) and [‘HJGTP, washed, and processed for electron microscopic autoradiography (bar = 0.1 micron). difference (data
in
not
shown;
of coated test
protein
content This
15).
vesicles
whether
bound
the
with
1% Triton
100,
60% and 30% of
After the
The specificity by measuring
and GMP.
Both
[35S]GTP~S; also
the
specific
activity
The coated
vesicles
lipid-associated
GTPyS/mg In order
bound protein
5.2
to confirm
binding
proteins,
electron
coats
was performed.
detected
by the
formation
of GTPyS (fig generated
transferring “P]GTP like
the
not
using
in
vesicles
grains.
This
process
ATP (fig
3b).
Less
lacking
coated
clathrin
vesicles
two dimensional
The blot Partial
under
these 200
were gel
to nitrocellulose. occurs
to clathrin
bound
8.1 phosphate).
[3H]GTP
GTPbound
10% of
to
was
was inhibited
by the
silver
coats. further
characterized
electrophoresis
and
was incubated
renaturation
conditions
placenta.
coats
than
We
with
contained
using
[3H]GTP
40%.
picomol
lipid-associated
autoradiography
by autoradiography.
proteins
(13.0 membranes
of
in areas
proteins proteins
and analyzed
but
from
total
clathrin-coated
of silver
prepared
ATP,
uM
than
associated
protein
GTPyWumol
was
of 0.1
by less
proteins
membranes
X-
GTP, GTPyS,
binding
binding
and the
Binding
present
the proteins
GTP-binding
of competing
GTPyWmg
microscopic
3a),
were
The GTP-binding by separating
respectively.
to total
the
1% Triton
clathrin-coats
inhibited
To was
and extracted
to purified
picomol
that
clathrin addition
with
presence
picomol
protein.
[35S]GTPyS
of extraction
phosphate) (10.4
proteins
total washed
of GTP-binding
vesicles
GTPyWumol
grains
the
vesicles
was preserved,
binding
in
the
were
100 uM ATP or GMP inhibited
clathrin-coated
picomol
of
proteins,
the vesicles
1 uM GTP or GTPyS completely
however,
compared
purified
percentage
activity
[35S]GTPyS
coated
the detergent-extractable
4 and 16 hours
binding
to normal
GTP-binding and
binding
of
that
retained
vesicles
X-100.
compared
a small
coats
to clathrin-coated
determined
indicates
constitutes
clathrin
when
in
(13).
with
of some rasFigure
4
[u-
Vol.
174,
No.
BIOCHEMKAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
36.029.0
-
24.0-
20.1-
14.2-+
I
I
I
I
I
I
8.0 7.0 5.0 10.0 9.0 6.0 Figure 4. Two dimensional gels blotted for [a-32P]GTP-binding proteins. Proteins of purified clathrin-coated vesicles (100 pg) were separated by isoelectric nitrocellulose. X-ray film. markers and
illustrates
form
focusing
followed by SDS-PAGE and subsequently transferred to The blot was incubated in [a-32P]GTP, washed, and exposed to The numbers on the left side of the gel represent molecular mass those below the gel represent the p1 of the proteins.
a spot
pH 5-8.
at
proteins
the origin
of the
The same GTP-binding
extracted
clathrin
It proteins SDS-PAGE trimers
coats
G, or G,)
(16).
Cholate coated placental
commonly
used
proteins
eluted
with from
of 50 kDa (data molecular
not
weights
GTP-binding
the
by gel
association
DISCUSSION:
(data
The results
coated
vesicles
Since
preparations
contain
high
molecular
detergent
(18).
for
high
This
proteins
lower
23-24
kDa
of spots
between
in TX-lOO-
mobility
(50 kDa)
The
[35S]GTP~S
column
exhibited
a major
peak of We also
antibodies
failed
kDa)
molecular to SDS-PAGE due of
a molecular
activity
mass
at higher
to detect
code A-569,
trimeric
GTP-binding
activity
peak near
to
permeation
a higher
binding
used
This
(115
compared
binding
(serum
by gel
ras-like
have
cholate.
GTP-binding
was therefore
mobility
apparently
after
clathrin
weight
(Pharmacia).
chromatography
A second
extracting
separated
column
GTP-binding
denaturation
detergent
the higher
observed.
weight
molecular
were
proteins
by using not
identified
to irreversible
filtration
shown).
was not
proteins
0: subunit
the
also
12 HR lo/30
The GTP-binding
(19).
were that
to separate
from
from
and a series
and active
and the
a Superose
when analyzed
fractions
(17)
vesicles
weights
dimension
an effective
membranes
using
is
GTP-binding
is
molecular
shown).
suggested
vesicles
coated
to their
not
may be susceptible
solubilize
weight
proteins
(data
(eg.
chromatography
proteins
first
been
from
technique
with
has previously
from
proteins
the
GTP-binding
that
the 20)
trimeric
that
recognize
shown). presented multiple
of clathrin-coated
in
this
paper
low molecular vesicles 201
demonstrate weight are
that
GTP-binding commonly
clathrinproteins.
contaminated
with
Vol.
174,
No.
smooth
1, 1991
vesicles,
these
nucleotide
clathrin
coats.
coated rather
than
with
with
contain evidence
that
low
vesicles
and
initially
Detergent less
than
5% of
that
the
with
proteins of
proteins
were
the
the
It
anchored protein
is
that
(eg.,
or plasma
determined. may reflect The low w-like
their
intracellular
implicated
weight
proteins
whose
&
secretory
vitro consist
assay
GTP-binding
a role
gene
to show
that
of clathrin-coated
the
size multiple
that
to the COOH-
the GTP-binding
also
possible
that
proteins.
protein
gene
for
pool.
products.
by the
same gene
and
or
proteins
are
vesicles
also
proteins
identified
of coated proteins that
they
play These
study
have
granules transport
vesicles,
however
organellar
Recently,
in vesicular
early
in
(25).
Our lab
30
unknown, proteins
(26,
with
this
approximately roles
implicated
can fuse
to be in
largely
by chromaffin
membrane-derived
in Golgi-
vesicles.
vesicles 27).
present
remains
include are
(23).
secretion
202
finding
and charge
pathways vesicles,
the
coat
has been
plasma
was
likely
by their
polyisoprenylation,
reticulum-derived
family
and endocytic
is
in post-translational
interactions during
is
that
bilayer it
GTP-binding
functions
suggests
we found
of
it
weight
the origin
cellular
intracellular
the
represent
coated
distribution
endoplasmic
of
same GTP-binding
in
these
in a 70%
attached
amount
may be coded
in GTP-binding
resulted
with
with
differences
membrane-derived
as playing between
m-related the
the
in
the lipid
moieties
associated
processing,
molecular
and other
fusion
from
present
vesicles
coats,
molecular
arise
may
definitive
intact,
consistent
by lipid
proteins
the heterogeneity
known traffic
low
proteins
Heterogeneity
is
extracted
these
Whether
Since remained
in coated
the source
proteolytic
phosphorylation). derived
the
are
16 hours
a significant
are
these
could
modifications
Since
detergent
in
some of
variations
This
to access
observed
Alternatively,
bilayer.
proteins
difficult
One explanation
retained
are vesicles
same conditions
proteins
to membranes in
for
was extracted. coat
results
we provide
proteins
these
are
(22).
retained
heterogeneity
these
lipid
GTP-binding
is
protein and the
the
proteins.
vesicles Under
proteins the
are
terminus some of
total
detergent
GTP-binding
association ras
of coated
to
pure
in
clathrin-coated
importantly,
these
was used
coated-vesicles
These
that
GTP with
with
present
with
with
bind
proteins
proteins part
GTP-binding
activity.
the
with
More
weight
that
autoradiography
vesicles.
suggesting
characterize
in GTP-binding
extracted
smooth
(21).
extraction
in
COMMUNICATIONS
by extraction
proteins
of GTP-binding
associated
report
molecular
removed
microscopy
contaminating proteins
were
the GTP-binding
are
a previous
GTP-binding
decrease
that
RESEARCH
retained
association
preparations
only
consistent
the
We conclude
vesicles
coats
Electron
demonstrate
BIOPHYSICAL
vesicles
clathrin
specificity.
conclusively
AND
contaminating
The remaining
detergent. high
BIOCHEMICAL
(24)
endosomes
and
the through
has developed which
been
an h
largely (28).
This
Vol.
174,
fusion
No.
event
possibility vesicles
1, 1991
is inhibited that
binding
these
transduction. proteins
disseminated
during proteins Thus,
AND
by GTPyS (4).
the GTP-binding
may function
Alternatively, signal
BIOCHEMICAL
that
COMMUNICATIONS
support the clathrin-coated
by mediating
in other
generated
may not be confined
by the vesicles
transport
may function signals
RESEARCH
These observations associated with
proteins
vesicular
BIOPHYSICAL
cellular
by low molecular
vesicle roles
fusion. such as
weight
GTP-
to the plasma membrane and may be
regulate
receptor-mediated
endocytosis.
Acknowledgments. This work was supported by the National Institute of Health (AI20015 and GM42259-19). Dr. Lenhard is a recipient of a research fellowship from the Cancer Research Institute. We would like to thank Dr. Janice Blum for helpful discussion during the course of this work. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Nakano, A., and Muramatsu, M. (1989) J. Cell Biol. 109, 2677-2691. Segev, N., Mulholland, J. and Botstein, D. (1988) Cell, 52, 915-924. Salminen, A., and Novick, P. J. (1987) Cell 49, 527-538. Mayorga, L. S., Diaz, R., and Stahl, P. D. (1989a) Science 244, 1474-1477. Mayorga, L. S., Diaz, R., Colombo, M. I., and Stahl, P. D., (1989b) Cell Regulation 1, 113-124. Brodsky, F. M. (1988) Science 242, 1396-1402. Rouser, B., Siakstos, A. N., Fleischer, S. (1981) Lipids 1: 85-86. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Wisse, E., and Tates, A. D. (1968) Proc. 4th Europ. Reg. Conf. Electron Micros., Rome. OrFarrell, P. H. (1975) J. Biol. Chem. 250, 4007-4111. Laemmli, U. K. (1970) Nature (Lond). 227, 680-685. Towbin, H., Staebelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4355. Schmitt, H. D., Wagner, P., Pfaff, E., and Gallwitz, D. (1986) Cell 47, 401-412. Pearse, B. M. F. (1982) Proc Nat1 Acad Sci USA 79, 451-455. Pearse, B. M. F. (1983) Methods in Enzymology 98, 320-326. Bhullar, R. P., and Haslam, R. J. (1987) Biochem. J. 245, 617-620. Pearse, B. M. F. (1978) J. Mol. Biol. 126, 803-812. Evans, T., Brown, M. L., Fraser, E. D., Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059 Wolfman, A., Moscucci, A., and Macara, I. A. (1989) J. Biol. Chem. 264, 10820-10827. Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. 83, 265-269. Bielinski, D. F., Morin, P. J., Dickey, B. F., and Fine, R. E. (1989) J. Biol. Chem. 264, 18363-18367. Hancock, J. F., Magee, A. I., Childs, J. E., and Marshall, C. J. (1989) Cell 57, 1167-1177. Hall, A. (1990) Science 249, 635-640. Burgoyne, R. D., and Morgan, A. (1989) FEBS. Lett. 245, 122-126. Lanoix, J., Roy, L., and Paiement, J. (1989) Biochem. J. 262, 497-503. Plutner, H., Schwaninger, R., Pind, S., and Balch, W. E. (1990) EMBO J. 9, 2375-2383. Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., Zerial, M. (1990) Cell 62, 317-329. Mayorga, L., S., Diaz, R., and Stahl, P. D. (1988) J. Biol. Chem. 263, 17213-17216.
203
174,
No.
January
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
15, 1991
197-203
CLATHRIN-COATED VESICLES FROM HUMAN PLACENTA CONTAIN GTP-BINDING PROTEINS James M. Lenhard,
Marilyn
Department Washington
Received
November
20,
Aach
Levy,
and Philip
D. Stahl
of Cell Biology and Physiology University School of Medicine St. Louis, Missouri 63110
1990
Biochemical and morphological techniques were used to investigate the GTP-binding proteins of clathrin-coated vesicles. Binding of [3H]GTP to clathrin coats was demonstrated by electron microscopic autoradiography. Purified coated vesicles bound 5.2 pmol [35S]GTPyS/mg protein. Addition of GTP or GTPyS, but not ATP nor GMP, inhibited binding of [35S]GTP+ to intact coated vesicles and Triton X-loo-extracted coats. A series of 23-24 kDa GTPbinding proteins with isoelectric points between pH 5-8 were detected in coated vesicles. We suggest that the low molecular weight ras-like GTPbinding protein(s) play a role in regulating vesicle-mediated protein transport or signal transduction within intracellular organelles. 0 1991 Academic Press, Inc.
A number eukaryotic yeast,
of steps
cells SARl,
binding
have
YPTl,
secretion
that
membrane vesicles role
in
proteins With
(l-3).
Recent regulate
and the (6). both
inward
this
molecular
weight
low
from
is
our
facilitated have
and outward
membrane
traffic,
GTP-binding
within
the vesicles
proteins. weight
GTP-
stages
indicate
that
endosomes from
In
m-like
different
laboratory
complexes
proteins
Coated
during
of macrophage
GTP-binding with
molecular
a role
fusion
network
of proteins
to GTP-binding
of receptor-ligand
we assayed
proteins.
for
to play
in vitro
be associated
in mind,
code
believed
trans-Golgi Since
transport
to be linked
observations
transport
would
GTP-binding
are
proteins Selective
the vesicular shown
and SEC4 genes
proteins
binding
in been
(4,
of GTP-
5).
the plasma
by clathrin-coated been
identified
it that
purified
clathrin-coated
vesicles
were
found
seemed mediate
as playing likely protein
vesicles to contain
that for
several
a these
transport. associated low
proteins.
METHODS: Coated Vesicle Isolation. All procedures were carried out at 4OC. Human placenta was resuspended in three volumes of 250 mM sucrose, 1 mM MgCl,, 0.5 mM EGTA, 20 mM MES, pH 6.5 (homogenization buffer) and homogenized with a Waring blender. The homogenate was centrifuged at 11,000 x g for 30 minutes and the supernatant was decanted and centrifuged at 29,000 x g for 4.0 hours. The resulting pellet was resuspended in homogenization buffer, layered onto a 30% sucrose cushion, and centrifuged at 80,000 x g for 5 min. The 30% sucrose
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
layer and the pellet were discarded. The supernatant was again layered onto 30% sucrose, centrifuged at 80,000 x g for 5 min, and the sucrose cushion and pellet were discarded. The supernatant was layered onto 10% Ficoll, 45% D,O, 1 mM MgCl,, 20 mM MES, pH 6.5 and centrifuged at 100,000 x g for 120 min. The pellet was resuspended in homogenization buffer and centrifuged at 20,000 x g for 10 min. The resulting supernatant was layered onto 15% Ficoll in 75% D,O, at 140 mM NaCl, 1 mM MgCl,, 0.5 mM EGTA, 20 mM MES, pH 6.5 and centrifuged 80,000 x g for 14 hours. The pellet containing coated vesicles was resuspended in homogenization buffer to 5 mg/ml final protein concentration. Clathrin coats were prepared by diluting the vesicles with 9 volumes of 1% Triton X-100 in homogenization buffer and incubating the sample at 22’C for 30 min. Clathrin coats were collected by centrifugation at 100,000 x g for 60 min. The phospholipid content was measured by following the methods described by Rouser et al., (7). Proteins were measured according to Bradford (8). GTP-binding assavs. Binding of [35S]GTP~S to clathrin-coated vesicles was determined by incubating the vesicles (10 ug total protein/100 ~1) at 22’C for 60 min in 2 uCi [35S]GTPyS (120 Ci/mmol), 250 mM sucrose, 1 mM MgCl,, 0.5 mM EGTA, and 20 mM HEPES, pH 7.0 (binding buffer). The vesicles were collected by centrifugation at 200,000 x g for 30 minutes and washed twice with binding buffer. Competition for [35S]GTP~S binding was determined by adding various concentrations of GTP, GTPrS, ATP, and GMP to the vesicles prior to addition The association of GTP-binding proteins with clathrin coats of [ 35S]GTPyS. was determined by extracting [35S]GTPrS bound to coated vesicles with 1% TX100 for various periods of time. Clathrin coats were collected by centrifugation at 200,000 x g for 30 minutes, washed with binding buffer, and coat-associated radioactivity was determined by liquid scintillation counting. Electron microsconv. Coated vesicles were processed for electron microscopy to monitor purity. The vesicles were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate pH 7.4, post-fixed in 1.5% osmium tetroxide, incubated with 4% uranyl acetate, dehydrated with a graded series of ethanol, infiltrated with propylene oxide , and embedded in Polybed 812 (Polysciences Inc., Washington, Pa.). Tissue was sectioned with a diamond knife, post-stained with uranyl acetate and lead citrate, and examined in a Zeiss 10A electron microscope. Electron microscopic autoradiography was performed by incubating clathrin coats (60 ug total protein) in 240 nl binding buffer containing 10 pCi [3H]GTP (7 Ci/mmol) and 10 uM GTPyS or 10 uCi [3H]GTP and 100 pM ATP. The clathrin coats were washed with binding buffer and processed for electron microscopy as described above. Light gold thin sections were mounted on Formvar carbon-coated nickel grids, coated with Ilford L-4 emulsion using a wire loop, and developed using the gold latensification procedure (9). Developed grids were post-stained in uranyl acetate and lead citrate and viewed in a Zeiss 10A electron microscope. Molecular weight determination. Protein (100 rigs) from purified clathrincoated vesicles and clathrin coats was separated by isoelectric focusing (10) The proteins were transferred to nitrocellulose (12), and SDS-PAGE (11). incubated with 25 uCi [a-32P]GTP, washed, and autoradiographed (13). RESULTS: that
Electron
>99% of
coated
the vesicles
vesicles
a linear
micrographs
centrifuged
for
4 hours
by SDS-PAGE and heavy
the gel.
probing exposing
chain
the gel
in each
Low molecular
Western-blots the
GTP-binding
blots
of proteins to X-ray
film.
proteins, x g.
the
Frctaion
was stained
fraction
weight
(fig
vesicles 1).
To test
vesicles
9% D,O to 20% Ficoll
at 80,000
clathrin
clathrin-coated
clathrin-coated
made of 2% Ficoll
analyzed of
were
contained
gradient
of purified
from
for
GTP-binding separated The amount 198
proteins
whether
were
90% D,O
layered were
The amount
by densitometer were
by SDS-PAGE with of GTP-binding
onto
and
the gradient
protein.
was determined
illustrate
detected [a-32P]GTP protein
of tracings by and in each
Vol.
174,
No.
Figure clathrin purity
1.
Electron microscopy Clathrin-coated coats. by electron microscopy
was quantitated
fraction
that
clathrin
(Fig
Receptors 100-extracted 1% Triton vesicles
for
X-100,
purified vesicles
weight
RESEARCH
clathrin-coated were isolated
as described
molecular
IgG,
smooth with
transferrin, coats
removed
extracted
of
BIOPHYSICAL
and
in the text
(bar
COMMUNICATIONS
vesicles analyzed
= 0.1 micron).
The densitometric GTP-binding
and for
tracings
proteins
revealed
co-fractionate
in
2).
clathrin were
AND
by densitometry.
and low
the gradient
coats
BIOCHEMICAL
1, 1991
(14).
vesicle (compare
Triton
and ferritin When coated
contamination fig
X-100
1 with
are vesicles
insoluble were
and membranes fig
3; 14).
revealed
no qualitative
4
n6umber
in Triton extracted
within
Xwith
coated
SDS-PAGE of clathrinnor
quantitative
40-
2
0
Bottom
Fraction
8
10 TOP
Co-fractionation of GTP-binding proteins and clathrin in Ficoll Figure 2. density gradients. Purified clathrin-coated vesicles (ZOO ug protein) were The distribution of clathrin centrifuged through 10 ml Ficoll gradients. heavy chain and total low molecular weight GTP-binding proteins in the gradient was determined by densitometer tracings of protein stained gels and autoradiographs of Western blots probed with [u-~*P]GTP, respectively. 199
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 3. Electron microscopic autoradiography of [3H]GTP-binding proteins Purified clathrin coats were incubated in the presence of clathrin coats. GTPyS (panel a) or ATP (panel b) and [‘HJGTP, washed, and processed for electron microscopic autoradiography (bar = 0.1 micron). difference (data
in
not
shown;
of coated test
protein
content This
15).
vesicles
whether
bound
the
with
1% Triton
100,
60% and 30% of
After the
The specificity by measuring
and GMP.
Both
[35S]GTP~S; also
the
specific
activity
The coated
vesicles
lipid-associated
GTPyS/mg In order
bound protein
5.2
to confirm
binding
proteins,
electron
coats
was performed.
detected
by the
formation
of GTPyS (fig generated
transferring “P]GTP like
the
not
using
in
vesicles
grains.
This
process
ATP (fig
3b).
Less
lacking
coated
clathrin
vesicles
two dimensional
The blot Partial
under
these 200
were gel
to nitrocellulose. occurs
to clathrin
bound
8.1 phosphate).
[3H]GTP
GTPbound
10% of
to
was
was inhibited
by the
silver
coats. further
characterized
electrophoresis
and
was incubated
renaturation
conditions
placenta.
coats
than
We
with
contained
using
[3H]GTP
40%.
picomol
lipid-associated
autoradiography
by autoradiography.
proteins
(13.0 membranes
of
in areas
proteins proteins
and analyzed
but
from
total
clathrin-coated
of silver
prepared
ATP,
uM
than
associated
protein
GTPyWumol
was
of 0.1
by less
proteins
membranes
X-
GTP, GTPyS,
binding
binding
and the
Binding
present
the proteins
GTP-binding
of competing
GTPyWmg
microscopic
3a),
were
The GTP-binding by separating
respectively.
to total
the
1% Triton
clathrin-coats
inhibited
To was
and extracted
to purified
picomol
that
clathrin addition
with
presence
picomol
protein.
[35S]GTPyS
of extraction
phosphate) (10.4
proteins
total washed
of GTP-binding
vesicles
GTPyWumol
grains
the
vesicles
was preserved,
binding
in
the
were
100 uM ATP or GMP inhibited
clathrin-coated
picomol
of
proteins,
the vesicles
1 uM GTP or GTPyS completely
however,
compared
purified
percentage
activity
[35S]GTPyS
coated
the detergent-extractable
4 and 16 hours
binding
to normal
GTP-binding and
binding
of
that
retained
vesicles
X-100.
compared
a small
coats
to clathrin-coated
determined
indicates
constitutes
clathrin
when
in
(13).
with
of some rasFigure
4
[u-
Vol.
174,
No.
BIOCHEMKAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
36.029.0
-
24.0-
20.1-
14.2-+
I
I
I
I
I
I
8.0 7.0 5.0 10.0 9.0 6.0 Figure 4. Two dimensional gels blotted for [a-32P]GTP-binding proteins. Proteins of purified clathrin-coated vesicles (100 pg) were separated by isoelectric nitrocellulose. X-ray film. markers and
illustrates
form
focusing
followed by SDS-PAGE and subsequently transferred to The blot was incubated in [a-32P]GTP, washed, and exposed to The numbers on the left side of the gel represent molecular mass those below the gel represent the p1 of the proteins.
a spot
pH 5-8.
at
proteins
the origin
of the
The same GTP-binding
extracted
clathrin
It proteins SDS-PAGE trimers
coats
G, or G,)
(16).
Cholate coated placental
commonly
used
proteins
eluted
with from
of 50 kDa (data molecular
not
weights
GTP-binding
the
by gel
association
DISCUSSION:
(data
The results
coated
vesicles
Since
preparations
contain
high
molecular
detergent
(18).
for
high
This
proteins
lower
23-24
kDa
of spots
between
in TX-lOO-
mobility
(50 kDa)
The
[35S]GTP~S
column
exhibited
a major
peak of We also
antibodies
failed
kDa)
molecular to SDS-PAGE due of
a molecular
activity
mass
at higher
to detect
code A-569,
trimeric
GTP-binding
activity
peak near
to
permeation
a higher
binding
used
This
(115
compared
binding
(serum
by gel
ras-like
have
cholate.
GTP-binding
was therefore
mobility
apparently
after
clathrin
weight
(Pharmacia).
chromatography
A second
extracting
separated
column
GTP-binding
denaturation
detergent
the higher
observed.
weight
molecular
were
proteins
by using not
identified
to irreversible
filtration
shown).
was not
proteins
0: subunit
the
also
12 HR lo/30
The GTP-binding
(19).
were that
to separate
from
from
and a series
and active
and the
a Superose
when analyzed
fractions
(17)
vesicles
weights
dimension
an effective
membranes
using
is
GTP-binding
is
molecular
shown).
suggested
vesicles
coated
to their
not
may be susceptible
solubilize
weight
proteins
(data
(eg.
chromatography
proteins
first
been
from
technique
with
has previously
from
proteins
the
GTP-binding
that
the 20)
trimeric
that
recognize
shown). presented multiple
of clathrin-coated
in
this
paper
low molecular vesicles 201
demonstrate weight are
that
GTP-binding commonly
clathrinproteins.
contaminated
with
Vol.
174,
No.
smooth
1, 1991
vesicles,
these
nucleotide
clathrin
coats.
coated rather
than
with
with
contain evidence
that
low
vesicles
and
initially
Detergent less
than
5% of
that
the
with
proteins of
proteins
were
the
the
It
anchored protein
is
that
(eg.,
or plasma
determined. may reflect The low w-like
their
intracellular
implicated
weight
proteins
whose
&
secretory
vitro consist
assay
GTP-binding
a role
gene
to show
that
of clathrin-coated
the
size multiple
that
to the COOH-
the GTP-binding
also
possible
that
proteins.
protein
gene
for
pool.
products.
by the
same gene
and
or
proteins
are
vesicles
also
proteins
identified
of coated proteins that
they
play These
study
have
granules transport
vesicles,
however
organellar
Recently,
in vesicular
early
in
(25).
Our lab
30
unknown, proteins
(26,
with
this
approximately roles
implicated
can fuse
to be in
largely
by chromaffin
membrane-derived
in Golgi-
vesicles.
vesicles 27).
present
remains
include are
(23).
secretion
202
finding
and charge
pathways vesicles,
the
coat
has been
plasma
was
likely
by their
polyisoprenylation,
reticulum-derived
family
and endocytic
is
in post-translational
interactions during
is
that
bilayer it
GTP-binding
functions
suggests
we found
of
it
weight
the origin
cellular
intracellular
the
represent
coated
distribution
endoplasmic
of
same GTP-binding
in
these
in a 70%
attached
amount
may be coded
in GTP-binding
resulted
with
with
differences
membrane-derived
as playing between
m-related the
the
in
the lipid
moieties
associated
processing,
molecular
and other
fusion
from
present
vesicles
coats,
molecular
arise
may
definitive
intact,
consistent
by lipid
proteins
the heterogeneity
known traffic
low
proteins
Heterogeneity
is
extracted
these
Whether
Since remained
in coated
the source
proteolytic
phosphorylation). derived
the
are
16 hours
a significant
are
these
could
modifications
Since
detergent
in
some of
variations
This
to access
observed
Alternatively,
bilayer.
proteins
difficult
One explanation
retained
are vesicles
same conditions
proteins
to membranes in
for
was extracted. coat
results
we provide
proteins
these
are
(22).
retained
heterogeneity
these
lipid
GTP-binding
is
protein and the
the
proteins.
vesicles Under
proteins the
are
terminus some of
total
detergent
GTP-binding
association ras
of coated
to
pure
in
clathrin-coated
importantly,
these
was used
coated-vesicles
These
that
GTP with
with
present
with
with
bind
proteins
proteins part
GTP-binding
activity.
the
with
More
weight
that
autoradiography
vesicles.
suggesting
characterize
in GTP-binding
extracted
smooth
(21).
extraction
in
COMMUNICATIONS
by extraction
proteins
of GTP-binding
associated
report
molecular
removed
microscopy
contaminating proteins
were
the GTP-binding
are
a previous
GTP-binding
decrease
that
RESEARCH
retained
association
preparations
only
consistent
the
We conclude
vesicles
coats
Electron
demonstrate
BIOPHYSICAL
vesicles
clathrin
specificity.
conclusively
AND
contaminating
The remaining
detergent. high
BIOCHEMICAL
(24)
endosomes
and
the through
has developed which
been
an h
largely (28).
This
Vol.
174,
fusion
No.
event
possibility vesicles
1, 1991
is inhibited that
binding
these
transduction. proteins
disseminated
during proteins Thus,
AND
by GTPyS (4).
the GTP-binding
may function
Alternatively, signal
BIOCHEMICAL
that
COMMUNICATIONS
support the clathrin-coated
by mediating
in other
generated
may not be confined
by the vesicles
transport
may function signals
RESEARCH
These observations associated with
proteins
vesicular
BIOPHYSICAL
cellular
by low molecular
vesicle roles
fusion. such as
weight
GTP-
to the plasma membrane and may be
regulate
receptor-mediated
endocytosis.
Acknowledgments. This work was supported by the National Institute of Health (AI20015 and GM42259-19). Dr. Lenhard is a recipient of a research fellowship from the Cancer Research Institute. We would like to thank Dr. Janice Blum for helpful discussion during the course of this work. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Nakano, A., and Muramatsu, M. (1989) J. Cell Biol. 109, 2677-2691. Segev, N., Mulholland, J. and Botstein, D. (1988) Cell, 52, 915-924. Salminen, A., and Novick, P. J. (1987) Cell 49, 527-538. Mayorga, L. S., Diaz, R., and Stahl, P. D. (1989a) Science 244, 1474-1477. Mayorga, L. S., Diaz, R., Colombo, M. I., and Stahl, P. D., (1989b) Cell Regulation 1, 113-124. Brodsky, F. M. (1988) Science 242, 1396-1402. Rouser, B., Siakstos, A. N., Fleischer, S. (1981) Lipids 1: 85-86. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Wisse, E., and Tates, A. D. (1968) Proc. 4th Europ. Reg. Conf. Electron Micros., Rome. OrFarrell, P. H. (1975) J. Biol. Chem. 250, 4007-4111. Laemmli, U. K. (1970) Nature (Lond). 227, 680-685. Towbin, H., Staebelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4355. Schmitt, H. D., Wagner, P., Pfaff, E., and Gallwitz, D. (1986) Cell 47, 401-412. Pearse, B. M. F. (1982) Proc Nat1 Acad Sci USA 79, 451-455. Pearse, B. M. F. (1983) Methods in Enzymology 98, 320-326. Bhullar, R. P., and Haslam, R. J. (1987) Biochem. J. 245, 617-620. Pearse, B. M. F. (1978) J. Mol. Biol. 126, 803-812. Evans, T., Brown, M. L., Fraser, E. D., Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059 Wolfman, A., Moscucci, A., and Macara, I. A. (1989) J. Biol. Chem. 264, 10820-10827. Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. 83, 265-269. Bielinski, D. F., Morin, P. J., Dickey, B. F., and Fine, R. E. (1989) J. Biol. Chem. 264, 18363-18367. Hancock, J. F., Magee, A. I., Childs, J. E., and Marshall, C. J. (1989) Cell 57, 1167-1177. Hall, A. (1990) Science 249, 635-640. Burgoyne, R. D., and Morgan, A. (1989) FEBS. Lett. 245, 122-126. Lanoix, J., Roy, L., and Paiement, J. (1989) Biochem. J. 262, 497-503. Plutner, H., Schwaninger, R., Pind, S., and Balch, W. E. (1990) EMBO J. 9, 2375-2383. Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., Zerial, M. (1990) Cell 62, 317-329. Mayorga, L., S., Diaz, R., and Stahl, P. D. (1988) J. Biol. Chem. 263, 17213-17216.
203
