(Received for publication, April 28, 1995; and in revised form, July 13, 1995)
From the
We have utilized a rabbit reticulocyte lysate coupled
transcription-translation system to express the large subunits of the
clathrin associated protein-2 (AP-2) complex so that their individual
functions may be studied separately. Appropriate folding of each
subunit into N-terminal core and C-terminal appendage domains was
confirmed by limited proteolysis. Translated 2 subunit bound to
both assembled clathrin cages and immobilized clathrin trimers,
confirming and extending earlier studies with preparations obtained by
chemical denaturation-renaturation. Translated
exhibited rapid, reversible and specific binding to clathrin
cages. As with native AP-2, proteolysis of
bound to
clathrin cages released the appendages, while cores were retained.
Further digestion revealed a
29-kDa
clathrin-binding fragment that remained tightly cage-associated.
Translated
also bound to immobilized clathrin
trimers, although with greater sensitivity to increasing pH than the
translated
2 subunit. Clathrin binding by both the
and
subunits is consistent with a bivalent cross-linking model for lattice
assembly (Keen, J. H.(1987) Cell Biol. 105, 1989). It also
[Abstract]
raises the possibility that the
-clathrin interaction may have
other consequences, such as modulation of lattice stability or shape,
or other
functions.
Receptor-mediated endocytosis is a multi-step process involving
membrane invagination, coated pit formation, and budding of these pits
to form coated vesicles(2) . A major protein implicated in
endocytosis is clathrin, a triskelion-shaped protein that forms the
structural basis for the regular polygonal lattice of coated pits and
vesicles(1, 3) . These coated membranes also contain
additional protein components that have been referred to as assembly,
adaptor, or associated proteins (APs). ()One probable
function of APs is to promote polymerization of the clathrin lattice at
defined sites and times. APs are also likely to interact with receptor
cytoplasmic tails resulting in the selective inclusion of various
receptors into coated pits (reviewed in Refs. 2, 4, 5).
APs vary in
structure and intracellular localization. The best characterized
examples include AP-1, a Golgi-associated heterotetramer consisting of
,
1, AP47, and AP19 polypeptides; AP-2, a plasma
membrane-associated heterotetramer of
,
2, AP50, and AP17
polypeptides; and AP-3/AP180, a neuronspecific
monomer(2, 4) . This study concerns the AP-2 complex
and focuses on the interactions of its
subunit with clathrin. Two
genetically distinct isoforms of
subunit exist:
, an isoform which is expressed ubiquitously, and
, an isoform believed to be expressed primarily in
neurons. The isoforms are 84% identical and differ predominantly in
their C-terminal portions. The
isoform contains a
unique 42 amino acid insert beginning at position 704(6) .
Although AP-2-clathrin interactions have been studied in
detail(1, 7, 8, 9, 10, 11, 12, 13) ,
it has been difficult to ascertain the contributions of individual AP-2
subunits. Fractionation of AP-2 polypeptides with urea and guanidinium
chloride was used to study these interactions, indicating that the
and
2 subunits alone were necessary and sufficient for coat
assembly activity(13) . Ahle and Ungewickell(7) , using
mild denaturation to purify
2 subunit from AP-2, demonstrated that
the former was capable of competitively inhibiting AP-2 binding to
preassembled clathrin cages. This work was extended by Gallusser and
Kirchhausen (14) who demonstrated that recombinant
2
subunit purified by denaturation-renaturation from Escherichia coli inclusion bodies was capable of promoting clathrin assembly.
Collectively, these results support the hypothesis that the
2
subunit plays an important role in AP-2-driven clathrin assembly in
vivo, but the role of the
subunit remains undefined.
We
have previously reported that an /AP50/AP17 complex prepared by
mild denaturation-renaturation was capable of binding to preformed
clathrin cages, suggesting that one or more of the other subunits, most
likely the
subunit, also recognizes and binds
clathrin(13) . We adopt the approach of in vitro translation of the individual large AP-2 subunits to further
explore this issue. The findings reported here indeed demonstrate that
the
subunit can bind tightly to clathrin, consistent with a role
in coat assembly or other coat-associated functions.
The TT rabbit reticulocyte lysate
transcription-translation kit and pSP65 cloning vector were purchased
from Promega. Translabel was obtained from ICN Biomedicals, Inc.
Sepharose CL-4B and Superose 6B resins were from Sigma, and CN-Br
activated Sepharose CL-4B was purchased from Pharmacia Biotech Inc.
Clathrin and assembly proteins were prepared from calf brains as
described previously(1, 15) . L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-trypsin was
from Worthington Biochemical, Inc. HEPES was purchased from Boehringer
Mannheim. All other chemicals were reagent grade or better.
Buffers
used are as follows: Buffer A: 0.1 M sodium MES, 1.0 mM EGTA, 0.5 mM MgCl, 0.02% NaN
, pH
6.50; Buffer B, Buffer A: 1.0 M Tris-HCl, pH 7.0, glycerol
(4.5:4.5:1 (v/v/v)); Buffer C, 5 mM sodium MES, 2 mM CaCl
, pH 6.15; Buffer T, 100 mM dipotassium
tartrate, 10 mM HEPES, 1 mM EGTA, 0.5 mM MgCl
, pH 7.0
The cDNA
template for the C-terminal deletion mutant was produced by restricting the pSP65
sense
construct with AvaI (
nucleotide 2021). The
mutant was then generated by runoff transcription-translation.
Full-length
2 subunit were transcribed from the pBluescript
SK+ T3 promotor, using a construct kindly provided by T.
Kirchhausen (Harvard University)(16) . Luciferase was
translated with the sp6 T
T control construct (Promega).
-Galactosidase was produced using a pSP65-based construct provided
by V. Gurevich (Thomas Jefferson University). Transcription-translation
reactions were assembled according to the manufacturer's
instructions and incubated 90-120 min. Proteins were translated
in the presence of [
S]Translabel: although the
reagent contained both [
S]L-methionine
and -cysteine, a large excess of unlabeled cysteine in the translation
kit blocked the latter's incorporation. Prior to use, translation
reactions were centrifuged at 100,000 revolutions/min for 20 min at 2
°C in a Beckman TLA 100 rotor to clear ribosomes and aggregates.
All experiments utilized freshly translated subunits.
Digests of bound to cages were carried out in Buffer A. Cage high speed
pellets (see below) with bound
were resuspended in
Buffer A and an aliquot of this suspension combined with trypsin stock
freshly diluted in buffer A. Reactions were incubated 15 min at room
temperature and quenched with a 3-fold molar excess of soybean trypsin
inhibitor.
Figure 1:
In
vitro translated proteins. Translated reaction mixtures were analyzed
by SDS, 7.5% PAGE and autoradiography as described under
``Materials and Methods.'' Lane 1, full-length AP-2
expressed from the pSP65
plasmid;
the 107.7-kDa polypeptide migrates as M
=
114,000 band. Lane 2,
(C-terminal deletion mutant) expressed from the AvaI-restricted pSP65
plasmid; 67.5-kDa
polypeptide migrates as M
= 65,000 band. Lane 3, cotranscription-translation of pSP65
sense and pSP65
antisense plasmids. Lane
4, full-length AP-2
2 polypeptide translated using the
pBluescriptSK
2 plasmid; 105.1-kDa polypeptide migrates as M
= 105,000 band. Lane 5,
luciferase (M
= 62,000). Lane 6,
-galactosidase (M
=
116,000).
Similarly, proteolysis of the in vitro translated
or
2 subunits alone yielded fragments of
58-66 kDa and
40 kDa (Fig. 2). Identical results
were obtained when translated proteins were cleaved in the presence of
carrier AP-2, as assessed by comigration of Coomassie Blue-stained and
radiolabeled bands on SDS-PAGE (data not shown). Quantitative analysis
of the changes in the full-length
product and the
appendage domain confirm a precursor-product relationship (Fig. 3). The coincidence of the two curves in Fig. 3demonstrates that at all trypsin concentrations the
fraction of full-length
cleaved is virtually
identical to that of appendage generated. Furthermore, from the
published sequence of the
cDNA(6) , the
C-terminal 40 kDa portion of
(i.e. amino
acids 610-977) is predicted to contain five of the 17 methionine
residues of
, or 27% of the total
radiolabel. In close agreement with this prediction, 24% of the
undigested full-length counts were found in the 40-kDa proteolytic
product upon complete digestion of full-length
.
Hence, we conclude that the initial cleavage of translated
by trypsin occurs in a region corresponding to the relatively
exposed linker of an
subunit in the intact AP-2 complex.
Figure 2:
Limited proteolysis of translated
and
polypeptides. The formation of labile core (C) and
stable appendage (A) domains from the full-length subunit (F) are indicated (see text for details). Two-µl aliquots
of
(lanes 1-6) and
2 (lanes
7-12) translation reactions were digested for 15 min at 22
°C with the following trypsin concentrations and analyzed by
SDS-PAGE: lanes 1 and 7, no trypsin; lanes 2 and 8, 75 ng/ml; lanes 3 and 9, 150
ng/ml; lanes 4 and 10, 300 ng/ml; lanes 5 and 11, 600 ng/ml; lanes 6 and 12, 9600
ng/ml.
Figure 3:
Quantitative analysis of in vitro translated polypeptide cleavage. Comparison of progress of
cleavage of the full-length polypeptide (plotted as percent of maximal
cleavage: closed circles, solid line) with appearance
of the appendage domain (plotted as percent of maximal change: open
diamonds, dashed line) on limited trypsin digestion of in vitro translated
polypeptide. Coincidence
of the two curves supports a precursor-product relationship (see text
for details).
The
translated 2 subunit was slightly more resistant to proteolysis
initially than the
subunit, as has been observed by
others(17) . The initial proteolytic susceptibility of
translated
, resulting in the generation of cores and
appendages (Fig. 2, lanes 2 and 3) was
comparable to that of bovine brain
in an AP-2 complex
as assessed by digestion of translated
in the
presence of AP-2 (data not shown). However, further digestion of both
translated subunits revealed significant differences from those in
brain AP-2. The 58-66-kDa core domains of translated
, and especially of
2, were much more labile than
those of AP-2. While virtually no 60-66-kDa fragments of the
translated subunits remained at trypsin concentrations greater than 170
ng/ml (Fig. 2, lanes 4-6 and 10-12), AP-2 derived N-terminal products were stable at
much higher trypsin concentrations, in excess of 840 ng/ml (21 and data
not shown). In contrast, the 40-kDa C-terminal appendages of both the
translated
and
polypeptides were similar to AP-2 in their
relative stability, resistant even at trypsin concentrations of 9,600
ng/ml (Fig. 2, lanes 6 and 12).
Elsewhere,
we have reported that a high affinity inositol polyphosphate-binding
site exists near the N terminus of the subunit. (
)Inclusion of 1 mM phytic acid (1,2,3,4
5,6-IP
) did not alter the proteolytic pattern of
cleavage described above (data not shown).
Figure 4:
In vitro translated
, but not luciferase or
-galactosidase, binds to
assembled clathrin cages. Aliquots (5 µl) of translation mixes
containing controls, luciferase (lanes 1-4), or
-galactosidase (lanes 5-8), or
(lanes 9-14) were incubated in the absence (lanes 1, 3, 5, 7, and 9-11) or presence (lanes 2, 4, 6, 8, and 12-14) of 60 µg of
clathrin cages for 30 min at 4 °C. The samples were then
centrifuged and equal proportions of high speed pellets (lanes 1 and 2, 5 and 6, 10, and 13) and supernatants (lanes 3, and 4, 7 and 8, 11, and 14) were
electrophoresed; for
, low speed pellets (lanes 9 and 12) were also analyzed.
By incubating the in vitro translation
mixture with increasing concentrations of clathrin cages we obtained a
dissociation constant of 1.1 10
M (Fig. 5). From the asymptote of the binding curve, the
maximal
fraction bound was 0.71, implying that not all of the
translated protein was capable of binding clathrin. The binding was not
inhibited by 1 mM phytic acid, a potent inhibitor of AP-2
self-association(9) , further evidence that binding was
specific and not due to aggregation or self-association (data not
shown).
Figure 5:
Binding of translated full-length
and
as a function of
clathrin cage concentration. Aliquots of translation reaction
containing in vitro translated full-length
(filled squares, solid line) or
(open squares, dashed
line) were incubated with clathrin cages at the indicated
concentrations for 30 min and fractionated by centrifugation. The
fraction bound was determined by excision and counting of the high
speed pellet and supernatant bands and was corrected for nonspecific
sedimentation. The data were fit to rectangular hyperbolas
(Kaleidagraph) yielding values of K
1.1
10
M and 71% maximal binding (r = 0.999) for the full-length
, and K
3.1
10
M and 36% maximal binding (r = 0.955)
for
.
Clathrin cage binding by translated subunit was inhibited by saturating quantities of AP-2,
confirming that the interaction was specific (Fig. 6). From the
apparent IC
and the published K
for
the AP-2-clathrin interaction of 10
M(17, 20) , we calculate a K
for
the
-clathrin interaction of 0.7
10
M, in reasonable agreement with the
value estimated by direct binding.
Figure 6:
Binding of translated to
clathrin cages is blocked by brain AP. Cages (20 µg) were incubated
with a bovine brain AP preparation containing the indicated
concentration of AP-2 in Buffer T for 90 min, followed by incubation
with 4 µl of translated
for an additional 30 min.
Cage-associated
was determined after centrifugation.
The data were corrected for nonspecific sedimentation and are expressed
relative to binding in the absence of exogenous brain
AP.
Both clathrin-clathrin and
AP-clathrin interactions are readily reversed by high concentrations of
protonated amines such as Tris-HCl (1, 6, 15) . The -clathrin interaction
was also reversible. Brief (5 min) treatment of the sedimented cages,
to which translated
was bound, with 500 mM Tris-HCl, pH 7, followed by a high speed spin released most
(>80%) of the
into the supernatant. Furthermore,
the solubilized
again cosedimented with the clathrin
cages reformed by stepwise dialysis of the dissociated preparation into
Buffer C and then into Buffer A (data not shown).
Figure 7:
pH
dependence of the binding of in vitro translated
and
2 to clathrin-Sepharose. A, aliquots (3 µl)
of translation reactions of
(lanes
1-6) or
2 (lanes 7-12) were incubated
for 30 min with 300 µl of clathrin-Sepharose (lanes 2-5 and 8-11) or underivatized Sepharose (lanes
1, 6, 7, and 12) which had been
pre-equilibrated with buffer A adjusted to the indicated pH (see
below). Columns were washed with 1200 µl of the equilibration
buffer (panel W) and the remaining bound protein eluted with
1200 µl of Buffer B supplemented with 0.05 mg/ml bovine serum
albumin (panel E). All fractions were precipitated with 10%
trichloroacetic acid, and equal proportions of washes and eluants were
analyzed by electrophoresis and autoradiography. Lanes 1, 2, 7, and 8, pH 6.5; lanes 3 and 9, pH 6.8; lanes 4 and 10, pH 7.2; lanes
5, 6, 11, and 12, pH 7.6. B,
data from panel A for each polypeptide are quantified and
plotted as the percentage of maximal binding. Maximal binding for each
polypeptide was observed at pH 6.5 and was 80% for
and essentially 100% for
2.
Alternatively, to assess the
ability of the isolated N-terminal region of translated to bind to preformed clathrin cages, we also produced a truncated
polypeptide, designated
, by runoff
transcription-translation (Fig. 1). This protein did bind in a
saturable manner to clathrin cages, although the apparent binding
affinity (K
= 3
10
M) was somewhat lower that of the full-length protein (Fig. 5). Only 36% of the total protein was capable of binding,
suggesting either that a greater proportion of the translated protein
was misfolded or that a binding equilibrium had not been established.
In contrast to the failure of either core or appendage domain of
translated and digested to bind, when cages with
bound
were incubated with trypsin the core fragments
were preferentially retained by the cages while the appendage domain
was released into the supernatant (Fig. 8). A similar result has
been obtained with native AP-2(12) . Interestingly, on more
vigorous proteolysis of the cage-bound translated
a
discrete 29-kDa cage-associated fragment became evident and was
prominent only in the presence of clathrin cages (compare Fig. 8, lane 2, with Fig. 2). This fragment
likely corresponds to the clathrin-binding domain of the
subunit.
Figure 8:
Proteolysis of in vitro translated
subunit bound to clathrin cages reveals a unique
29-kDa fragment. Clathrin cages containing bound in vitro translated
in Buffer A were incubated for 15 min
at 22 °C in the presence (lanes 2 and 4) or
absence (lanes 1 and 3) of trypsin (100 ng/ml).
Following sedimentation, cage-associated and released
fragments were analyzed by electrophoresis and autoradiography. Lanes 1 and 2, high speed pellets; lanes 3 and 4, supernatants.
AP-2 is capable of binding clathrin trimers with high
affinity, an interaction representing an initial step in the coat
assembly process. Following treatment with urea or guanidinium
chloride, the AP-2 complex has been fractionated by gel filtration or
hydroxylapatite chromatography, yielding partially purified ,
, and 50 kDa/17 kDa subunits. Of these, only the large
and
2 subunits of AP-2 were required for clathrin coat assembly in
vitro(13) . Dissociated
2 subunits from such
preparations were shown to bind to clathrin cages but could not alone
sponsor clathrin assembly(7) . Recent studies using recombinant
protein have reported that
2 alone is capable of inducing clathrin
assembly(14) , and
1 has been implicated in clathrin
recruitment in the trans-Golgi network(22) . Previous
work from this laboratory suggested that
subunits could bind to
clathrin trimers and cages(23) . However, these experiments
suffered from potential limitations in that the
fractions
contained small quantities of
and 50-kDa/17-kDa polypeptides,
preventing an unambiguous assignment of clathrin binding activity to
the
subunit.
To further examine the issue of clathrin-binding
subunits, we have translated the and
2 subunits
of AP-2 in vitro in a rabbit reticulocyte lysate system to
assess their respective clathrin binding capabilities de novo.
The approach of in vitro translation has several important
advantages. The individual subunits are generated without resort to the
strong denaturants that make it extremely difficult to be certain that
the native state has been reattained. In contrast, the translation
system produces polypeptides in a physiological environment with the
appropriate folding factors, more closely resembling the intracellular
milieu. Further, readily detectable radioactive polypeptides are
generated that can be used at tracer levels (
10
M) in functional assays. This is a major advantage in
the study of AP-2 structure and function because the protein and its
subunits are prone to aggregation and self-association (8) even at relatively low protein concentrations
(
µg/ml or 10
M). Finally, the study
of individual polypeptides of multisubunit proteins by in vitro transcription-translation (24) may be particularly
appropriate for the APs. Although these proteins have been assumed to
function only as intact tetrameric complexes, there is recent evidence
that the AP50 functions independently of the AP-2 complex as an
activator of the vacuolar proton pump(25, 26) . The
structural and functional attributes of the isolated
and
polypeptides reported here and previously (7, 14) suggest that they, too, could have independent
roles.
Our results show that readily detectable amounts of AP-2
and
2 polypeptides can be expressed in a
functional form in vitro. Both appear to assume the proper
secondary and tertiary conformation by folding into the core and
appendage domains that are characteristic of the intact AP-2 protein.
The fragments obtained on limited proteolysis correspond well to those
expected from the large subunits of bovine brain AP-2, though there are
differences. While the C-terminal appendage fragment is resistant to
further proteolysis, the core fragments appear to be more heterogenous
and extremely labile. This is consistent with the proposed quaternary
structure of isolated AP-2(10, 21) . The
and
2 C-terminal appendages do not display stable intermolecular
contacts with other subunits of AP-2 and likely function as
independently folded and stable domains. In contrast, in native AP-2
protein the N-terminal core domains of
and
are in proximity
to each other and to the AP50 and AP17 polypeptides. These interactions
do not occur with the translated polypeptides. Consequently, the in
vitro translated subunits may be relatively unprotected and more
prone to proteolysis, yielding the results seen in Fig. 2.
Upon proteolysis of cage-bound , the appendage
fragments are preferentially released, while the core is almost
entirely retained. Of particular interest is the appearance of a novel
29-kDa fragment when cage-bound
is proteolyzed (Fig. 8). The appearance of this band correlates well with the
disappearance of the core 58-66-kDa fragments, suggesting that
further digestion is blocked by tight association and stabilization by
clathrin. Conversely, if dissociated this fragment may be more rapidly
degraded: in the absence of clathrin, heterogeneous bands of this size
are barely detectable (Fig. 2). It seems likely that this
fragment comprises part of a discrete clathrin-binding domain within
the
subunit.
In contrast to the tight retention of the core
and 29-kDa fragment when the full-length protein is proteolyzed,
and proteolytic core fragments generated
prior to cage binding interact with much lower affinity. This may
indicate that the appendage and/or C-terminal linker regions of
are required to maintain the free core domain in a
conformation in which it is able to interact with clathrin.
Alternatively, the appendage or linker regions may interact with
clathrin directly.
The observation that both and
2 subunits of AP-2 have clathrin-binding sites supports the
concept that coat assembly proceeds by bivalent binding and
stabilization of overlapping clathrin triskelia in a conformation that
leads to polygon formation, essentially the cross-linking model
proposed earlier(1, 2) . Whether this hypothetical
mechanism extends to other proteins such as AP-3/AP180, auxillin (27, 28) and a novel AP-20 (29) that have been
reported to promote clathrin assembly in vitro remains to be
determined. In any case, the expanding group of proteins capable of
promoting polymerization suggests that coat assembly may be invoked by
different effectors under a variety of different circumstances in
vivo.
Though both and
2 subunits bind
strongly to assembled clathrin lattices,
subunit
binding to clathrin trimers is much more sensitive to pH in the
physiological range than is the
subunit. This seems unlikely to
be a consequence of lability of the isolated
conformation in
solution, as we detect no change in either the proteolysis pattern or
susceptibility of translated
subunit over this pH range (data not
shown). The increased affinity of the
subunit for clathrin with
decreasing pH correlates with the increased ability of AP-2 to drive
coat formation with decreasing pH(21) , arguing for a role of
the
subunit in lattice assembly. In addition, cytoplasmic
acidification to pH 6.3-6.5 also results in
``freezing'' of clathrin lattices with increased curvature (32) thereby arresting receptor-mediated
endocytosis(19, 21, 30, 31) . These
observations suggest that the
-clathrin interaction may also be
involved in lattice shape changes during vesiculation and endocytosis,
or conceivably, that through this binding clathrin may affect other
functions.
Note Added in
Proof-We have recently found that the translated mouse
subunit of the AP-1 complex binds to clathrin cages with K
0.15 µM, comparable
to the
results shown in Fig. 5. In view of
published results(22) , this suggests that AP-1, like AP-2, can
bind bivalently to clathrin.