From the
We separately expressed the 58-kDa C-terminal, 42-kDa middle,
16-kDa C-terminal, and 33-kDa N-terminal regions of AP-3 (also called
F1-20, AP180, NP185, and pp155), and determined their clathrin binding
and assembly properties. The 58-kDa C-terminal region of AP-3 is able
to bind to clathrin triskelia and assemble them into a homogeneous
population of clathrin cages and will also bind to preassembled
clathrin cages. The 42-kDa central region of AP-3 can bind to both
clathrin triskelia and to clathrin cages, but cannot assemble clathrin
triskelia into clathrin cages. The 16-kDa C-terminal region of AP-3 can
bind to clathrin cages, but cannot bind to clathrin triskelia or
assemble clathrin triskelia into clathrin cages. The clathrin binding
activities of the 42-kDa central region and 16-kDa C-terminal region
are weaker than the corresponding activity of either the 58-kDa
C-terminal region or full-length AP-3. Previous efforts had mapped a
clathrin binding site within the N-terminal 33 kDa of AP-3 (Murphy, J.
E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991)
J. Biol. Chem. 266, 4401-4408; Morris, S. A., Schroder,
S., Plessmann, U., Weber, K., and Ungewickell, E. (1993) EMBO J. 12, 667-675). However, although the N-terminal 33 kDa of
AP-3 is able to bind to clathrin triskelia (Murphy, J. E., Pleasure, I.
T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995)
[Abstract]
J.
Neurosci. Res. 41, 15-26), it does not promote their
assembly into clathrin cages (Murphy, J. E., Pleasure, I. T., Puszkin,
S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266,
4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26) or bind to preassembled clathrin cages (Ye, W.,
and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26). It
appears that the smallest functional unit that carries out all of the
reported clathrin binding and assembly properties of AP-3, essentially
as well as the full-length protein, is the 58-kDa C-terminal region.
Clathrin-coated vesicles are involved in pathways of
receptor-mediated intracellular transport
(4) . These include
the movement of proteins from the trans-Golgi network to secretory
vesicles for regulated secretion, the transfer of lysosomal hydrolases
from the trans-Golgi network to lysosomes, receptor-mediated
endocytosis, and the biogenesis and recycling of synaptic vesicles. The
protein coats of clathrin-coated vesicles have been well characterized
(5) . The major coat protein is clathrin
(6) . The
solution form of clathrin is the triskelion, which consists of three
identical clathrin heavy chains (192 kDa) and three clathrin light
chains (22-28 kDa)
(7, 8, 9, 10, 11) . Triskelions
can associate with one another to form icosahedral cages. When these
structures form at the surface of a cell membrane, the membrane
invaginates into a coated pit, followed by its separation from the
parent membrane as a coated vesicle. Coated vesicles also contain one
or more of the assembly proteins: AP-1, AP-2, AP-3, or auxilin
(12, 13, 14, 15) . The assembly proteins
all share the property that they promote the assembly of clathrin
triskelia into a homogeneous population of clathrin cages in solution
(12, 14, 16, 17) . In vivo, the
assembly proteins are believed to sit between the cell membrane and the
clathrin cage
(18) . The assembly proteins are thought to be
involved in directing clathrin cage assembly to a particular cell
membrane through an interaction with a membrane receptor
(19) .
Much remains to be elucidated concerning the mechanism by which
clathrin assembly is promoted by a clathrin assembly protein and how
this assembly might be regulated. The assembly proteins AP-1 and AP-2
are tetramers, whereas the assembly proteins AP-3 and auxilin are
monomers. AP-1, AP-2, and AP-3 have been cloned and sequenced
(20, 21, 22, 23) . As a monomer, AP-3
represents a simple system in which to investigate how a clathrin
assembly protein promotes clathrin assembly. AP-3 is the only clathrin
assembly protein shown to be synapse-specific
(24, 25, 26) , suggesting that it plays a
specialized role in the life cycle of the synaptic vesicle
(27) . Therefore, understanding how AP-3 functions will also
make a contribution toward elucidating the molecular mechanism
underlying neurotransmission.
AP-3 was independently discovered in a
number of laboratories, and has been known as pp155
(28) , AP180
(16) , NP185
(29) , and F1-20
(23, 30) .
pp155, AP180, and NP185 were shown to be the same protein, and renamed
AP-3
(1) . F1-20 and AP-3 were then shown to be identical
(2, 27) . AP-3 is a neuronal-specific
(2, 26, 29) phosphoprotein
(23, 28, 31, 32) and glycoprotein
(32) , which is unusually acidic
(16, 23, 28) , and migrates anomalously on
SDS-PAGE
In order to begin to understand the structure-function
relationships of AP-3, clathrin binding properties of proteolytic
fragments of AP-3 were studied
(1, 2) . It was found
that the 33-kDa N-terminal region could bind to clathrin triskelia,
leading to the conclusion that the 33-kDa N-terminal region was a
clathrin binding domain
(1) . In this same study it was found
that the 33-kDa N-terminal region was insufficient for clathrin
assembly. We confirmed these findings utilizing the bacterially
expressed N-terminal 33-kDa region of AP-3 and extended them by showing
that the 33-kDa N-terminal region could bind to clathrin triskelia, but
not to preassembled clathrin cages
(3) . This suggested that
clathrin triskelion binding and clathrin cage binding are not
functionally equivalent. It was also shown that the 33-kDa N-terminal
region contained a high affinity binding site for specific inositol
polyphosphates
(33) . In the current study, we undertook an
analysis of the clathrin binding and assembly properties of additional
individually expressed regions of AP-3. We found that the 58-kDa
C-terminal region of AP-3 had all of the clathrin binding and assembly
properties of full-length AP-3. Previous proteolysis studies
(1) and sequence analyses
(2) led to the suggestion
(2) that the 58-kDa C-terminal region could be further divided
into a 42-kDa central domain and a 16-kDa C-terminal domain. We found
that the 42-kDa central region could bind to clathrin cages and
clathrin triskelia and that the 16-kDa C-terminal region could bind to
clathrin cages. However, neither of the latter two regions alone could
assemble clathrin triskelia into clathrin cages.
Materials
A construct
expressing GST-C58 kDa was constructed as described previously
(33) . This construct (pGEX3X-F1-20-COOH58 kDa) was introduced
into Escherichia coli BL21 for protein expression. GST-C58 kDa
was expressed and purified as described previously
(33) . A
construct expressing GST-C16 kDa was constructed as follows. Plasmid
pGEX3X-F1-20-COOH58 kDa was digested with restriction endonucleases
BamHI and StuI, followed by mung bean nuclease
treatment and agarose gel electrophoresis. The largest fragment was
purified and ligated to form closed circular DNA. The new construct
(pGEX3X-F1-20-COOH16 kDa) was introduced into E. coli DH5
When these
experiments were carried out over a wider range of protein
concentrations, and subjected to a quantitative analysis
(Fig. 4), it was found that the 58-kDa C-terminal region of AP-3
assembles clathrin as well as the full-length protein. No significant
clathrin assembly was found by GST-M42 kDa, GST-C16 kDa, GST-N33 kDa,
or GST. A hallmark of factor-dependent clathrin assembly is the
homogeneity of the cages formed. Analysis of the morphology of the
cages assembled in the experiment shown in Fig. 3is presented in
Fig. 5
. It is apparent that no cages were formed with GST
(Fig. 5 A) and that both GST-AP-3
(Fig. 5 B) and GST-C58 kDa (Fig. 5 C)
assembled clathrin triskelia into a homogeneous population of clathrin
cages. We conclude that the 58-kDa C-terminal region of AP-3 is
sufficient to assemble clathrin triskelia into clathrin cages.
The results of the study presented here were integrated with
the previously published studies, to prepare a summary of
structure-function relationships for AP-3 (Fig. 6). These
relationships are diagrammed against the distribution of acidic and
basic residues in the primary structure of AP-3. Previously, it was
reported that the 33-kDa N-terminal region of AP-3 contained a high
affinity binding site for specific inositol polyphosphates, with
similar binding parameters as full-length AP-3
(33) . We show
that the 58-kDa C-terminal region of AP-3 is able to bind to clathrin
triskelia and assemble them into a homogeneous population of clathrin
cages and will also bind to preassembled clathrin cages. We also show
that the clathrin triskelia binding, clathrin cage binding, and
clathrin assembly activities of the 58-kDa C-terminal region of AP-3
are essentially indistinguishable from that of the full-length protein.
We found that the 42-kDa central region of AP-3 can bind to both
clathrin triskelia and to clathrin cages, but cannot assemble clathrin
triskelia into clathrin cages. We found that the 16-kDa C-terminal
region of AP-3 can bind to clathrin cages, but cannot bind to clathrin
triskelia or assemble clathrin triskelia into clathrin cages. We noted
that the clathrin binding activities of the 42-kDa central region and
16-kDa C-terminal region are weaker than the corresponding activity of
either the 58-kDa C-terminal region or full-length AP-3. Previous
efforts had mapped a clathrin binding site within the N-terminal 33 kDa
of AP-3
(1, 2) . However, although the N-terminal 33 kDa
of AP-3 is able to bind to clathrin triskelia
(1, 3) ,
it does not promote their assembly into clathrin cages
(1, 3) or bind to preassembled clathrin cages
(3) . It
appears that the smallest functional unit that carries out all of the
reported clathrin binding and assembly properties of AP-3, essentially
as well as the full-length protein, is the 58-kDa C-terminal region.
Analysis
(27, 36) of the amino acid sequence of AP-3
(23) revealed clear divisions between the characteristics of the
The results of this study lend additional
support to the assignment of a 33-kDa N-terminal domain and a 58-kDa
C-terminal domain. The 33-kDa N-terminal domain is a high affinity
receptor for inositol polyphosphates
(33) and also binds to
clathrin triskelia
(1, 3) . The 58-kDa C-terminal domain
is a fully functional clathrin binding and assembly domain. Evidence
supporting the further subdivision of the 58-kDa C-terminal domain into
a 42-kDa central domain, and a 16-kDa C-terminal domain, is more
tenuous at this time. We found that each of these proteins is less
stable than the 33-kDa N-terminal or 58-kDa C-terminal regions. For
example, we were able to identify conditions under which we could
stably prepare both the 33-kDa N-terminal region and the 58-kDa
C-terminal region free of GST. However, we were not able to identify
conditions, even with extensive testing, under which we could prepare
stable 42-kDa central region and 16-kDa C-terminal region free of GST
(data not shown). The reported binding properties of these fragments
are interesting, but their meaning is subject to several possible
interpretations. For example, it is possible that the 42-kDa central
region, and the 16-kDa C-terminal region, each contain a distinct
binding site for clathrin cages. Alternatively, it is possible that in
the 58-kDa C-terminal region, residues from both the 42-kDa central
region and 16-kDa C-terminal region contribute to a single clathrin
binding and assembly site. When expressed and studied separately, the
residues can make sufficient contacts to account for the clathrin
binding observed in this study, but not to carry out clathrin assembly.
Distinguishing between these various possibilities may require the
elucidation of the three-dimensional structure of AP-3.
This study
raises two intriguing and important questions. Why is there is a
binding site for clathrin triskelia in the 33-kDa N-terminal domain,
when the 58-kDa C-terminal domain binds and assembles clathrin as well
as the full-length protein? How does an inositol polyphosphate binding
site in the 33-kDa N-terminal domain modulate the clathrin assembly
activity which we show here is present in the 58-kDa C-terminal domain?
While at this point in time we can only speculate, it is interesting to
consider that the 33-kDa N-terminal domain may function as a regulatory
domain, and the 58-kDa C-terminal domain may function as a clathrin
assembly domain. In this light, it is provocative that the 33-kDa
N-terminal domain binds to a site on clathrin triskelia that either
becomes inaccessible or changes conformation upon assembly of the
triskelia into cages. Answers to these interesting questions await
future studies.
(
)(1, 16, 17, 23) . Two
primary functional activities have been found for AP-3. AP-3 is a
clathrin assembly protein
(16) , as well as a high affinity
receptor for specific inositol polyphosphates
(33) . These two
functions are intimately related, since binding of specific inositol
polyphosphates has been shown to inhibit clathrin assembly
(33) .
Proteins
All buffers used in the protein work, even if
not explicitly indicated in the citations, contained 0.1 mM
PMSF. The proteins GST-AP-3
(AS15AS108
)
(27) , GST-N33
kDa
(3) and GST were all expressed and purified exactly as
described previously
(3) . Bovine brain clathrin was purified
from bovine brain clathrin-coated vesicles as described previously
(3) , based on subtle modifications of
(16) . SDS-PAGE
analysis of the purified clathrin revealed three silver-stained bands,
corresponding in apparent molecular weight to clathrin heavy chain and
to the two clathrin light chains. Clathrin was determined to be free of
detectable contaminating AP-3 by Western blot analysis with the F1-20
monoclonal antibody utilizing the ECL detection system
(27) .
Three cycles of assembly-disassembly was carried out
(3) .
Protein concentrations were determined spectrophotometrically using
extinction coefficients which were calculated from the reported amino
acid sequences of clathrin
(8, 9, 10) and AP-3
(23) , according to the relation
=
number of tryptophan residues (5690) + number of tyrosine residues
(1280)
(34) , and found to be as follows: GST-AP-3
(80, 650) , GST-C58 kDa
(63, 440) ,
GST-M42 kDa
(46, 370) , GST-C16 kDa
(57, 750) , GST-N33 kDa
(57, 890) , GST
(40, 680) , and clathrin triskelion
(676, 770) . Monoclonal antibody F21-5 was generated by
fusion of spleens from mice immunized with clathrin which was free of
AP-3 as determined by Western blot analysis with monoclonal antibody
F1-20. F21-5 was found to be specific for clathrin heavy chain and did
not display cross-reactivity with bacterially expressed or bovine AP-3,
clathrin light chains, AP-1 or AP-2.
(
)
Monoclonal
antibody F1-20 has been shown to be specific for AP-3
(23, 26, 27, 30) .
for protein expression. GST-C16 kDa was expressed and purified under
the same conditions as GST-AP-3
(3) , except that the cells were
grown for 2 h following
isopropyl-1-thio-
-D-galactopyranoside induction. A
construct expressing GST-M42 kDa was constructed as follows. Plasmid
pGEX3X-F1-20-COOH58 kDa was digested with restriction endonucleases
StuI and EcoR I, followed by mung bean nuclease
treatment and agarose gel electrophoresis. The largest fragment was
purified and ligated to form closed circular DNA. The new construct
(pGEX3X-F1-20-middle 42 kDa) was introduced into E. coli DH5
for protein expression. GST-M42 kDa was expressed and
purified under the same conditions as GST-AP-3
(3) , except that
the cells were grown for 4 h following
isopropyl-1-thio-
-D-galactopyranoside induction. Methods
Clathrin Cage Binding Assay
Clathrin cage binding
assays were carried out as described
(3, 35) , except
that all the GST-fusion proteins were used at 20 µM,
clathrin was used at 1.8 µM (corresponding to molarity of
triskelia; 1.2 mg/ml), and all of the binding reactions were carried
out in isolation buffer (0.1 M MES, 1 mM EGTA, 0.5
mM MgCl, 0.1 mM PMSF, pH 6.7). The
samples were analyzed by SDS-PAGE followed by Coomassie Blue staining
and Western blot analysis. The Western blots were stained with a
monoclonal antibody against GST (``GST
(12) '' from
Santa Cruz Biotechnology, Inc.) and visualized utilizing the ECL
detection method, as described
(27) . The distribution of the
GST-fusion proteins between the pellet and supernatant fractions was
quantitated using the Millipore BioImage system with 3cx scanner.
Values given in the text for the percent fusion protein bound
represent: the percent fusion protein in the pellet in the sample
containing clathrin - the percent fusion protein in the pellet in
the parallel reaction not containing clathrin.
Clathrin Triskelia Binding Assay
GST-AP-3, GST-C58
kDa, GST-M42 kDa, GST-C16 kDa, and GST were each bound to
glutathione-Sepharose (Pharmacia Biotech Inc.)
(3) . Rather then
eluting the bound proteins with glutathione, each resin was then
equilibrated with ice-cold isolation buffer (0.1 M MES, 1
mM EGTA, 0.5 mM MgCl, pH 6.7) containing
0.1 mM PMSF and 0.01% gelatin. 0.4 mg/ml clathrin triskelia in
isolation buffer were incubated with an equal volume of each resin at 4
°C for 2 h with rocking. The resins were pelleted by a 5-s
microcentrifugation, and the supernatants were removed. The
supernatants were labeled F for flow-through in
Fig. 2
and correspond to the unbound material. The resins were
washed six times with an equal volume of ice-cold isolation buffer
containing 0.1 mM PMSF and 0.01% gelatin. The last wash was
found to be free of clathrin by Western blot analysis using the
clathrin heavy chain-specific monoclonal antibody F21-5, indicating
that all nonspecifically bound material was removed. The beads were
then eluted by boiling for 5 min in an equal volume of 1
SDS
sample buffer, followed by a 5-s microcentrifugation, and the
supernatants were removed. These supernatants were labeled B for bound material in Fig. 2. All of the samples were
analyzed by SDS-PAGE, followed by Coomassie Blue staining and Western
blot analysis using the clathrin heavy chain-specific monoclonal
antibody F21-5 with the ECL detection system. A parallel set of
experiments was carried out using 0.4 mg/ml BSA instead of clathrin
triskelia in the incubation mix to ensure that the resins did not
nonspecifically bind to proteins in the solution. These blots were
blocked with 0.10% gelatin instead of BSA and stained with a monoclonal
anti-BSA antibody (Sigma B2901).
Figure 2:
The 58-kDa C-terminal and 42-kDa central
region of AP-3 bind to clathrin triskelia. Clathrin triskelia binding
assays were carried out as described under ``Experimental
Procedures.'' Glutathione-Sepharose coupled to either GST-AP-3
( lanes 1, 2, 11, and 12), GST-C58 kDa ( lanes 3,
4, 13, and 14), GST-M42 kDa ( lanes 5, 6, 15, and
16), GST-C16 kDa ( lanes 7, 8, 17, and 18),
or GST ( lanes 9, 10, 19, and 20) was incubated with
an equal volume of either 0.4 mg/ml clathrin triskelia ( lanes
1-10) or 0.4 mg/ml BSA ( lanes 11-20).
Following a 2-h incubation at 4 °C with agitation, the beads were
pelleted by a 5-s microcentrifugation, and the supernatant was removed
(this is the flow-through, corresponding to the unbound material,
labeled as F in all odd lanes). The beads were washed six
times with an equal volume of ice-cold isolation buffer. The beads were
then eluted by boiling for 5 min in an equal volume of 1 SDS
sample buffer (this is the eluate, corresponding to the bound material,
labeled as B in all even lanes). All of the samples were
analyzed by SDS-PAGE followed by Coomassie Blue staining ( upper
panel) and by Western blot ( lower panel) using either a
clathrin heavy chain-specific monoclonal antibody ( lanes
1-10) or a BSA-specific monoclonal antibody ( lanes
11-20), visualized by the ECL method. Positions of all
proteins are indicated using the abbreviations defined in the legend to
Fig. 1. Essentially identical results were obtained in three
independent experiments.
Clathrin Assembly Assays
3 µM
clathrin triskelia were dialyzed overnight at 4 °C against
isolation buffer with the addition of 20 µM of either GST,
GST-AP-3, GST-C58 kDa, GST-M42 kDa, GST-C16 kDa, GST-N33 kDa, or GST,
as indicated in the figure legend to Fig. 3. Following a 3-min
centrifugation at 13,600 g to remove nonspecific
aggregates, newly assembled clathrin cages were pelleted by
ultracentrifugation for 20 min at 100,000
g. The
pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE,
followed by Coomassie Blue staining and Western blot analysis using the
anti-clathrin heavy chain-specific monoclonal antibody F21-5. The
distribution of clathrin heavy chain between the pellet and supernatant
fractions was quantitated using the Millipore BioImage system with 3cx
scanner. For the quantitative assembly plot (Fig. 4), assays were
carried out as described above, except that the concentration of test
assembly protein was varied, as indicated on the plot. Each point on
the plot represents the average of triplicate determinations. The
morphology of the products of the assembly assays using either 20
µM GST-AP-3, 20 µM GST-C58 kDa, or 20
µM GST were evaluated by negative staining electron
microscopy, as described
(3) .
Figure 3:
The 58-kDa C-terminal region of AP-3
assembles clathrin. Clathrin assembly assays were performed as
described under ``Experimental Procedures.'' 3
µM clathrin triskelia were dialyzed overnight at 4 °C
against isolation buffer with the addition of 20 µM
concentrations of either GST-AP-3 ( lanes 1 and 2),
GST-C58 kDa ( lanes 3 and 4), GST-M42 kDa ( lanes 5 and 6), GST-C16 kDa ( lanes 7 and 8),
GST-N33 kDa ( lanes 9 and 10), or GST ( lanes 11 and 12). Following a low speed spin to remove nonspecific
aggregates, newly assembled clathrin cages were pelleted by
ultracentrifugation at 100,000 g. The pellet
( P) and supernatant ( S) fractions were analyzed by
SDS-PAGE, followed by Coomassie Blue staining ( upper panel)
and Western blotting ( lower panel) using an anti-clathrin
heavy chain-specific monoclonal antibody. Positions of all the proteins
are indicated in the same way as in Fig. 1. Essentially identical
results were obtained in three independent
experiments.
Figure 4:
The 58-kDa C-terminal region of AP-3
assembles clathrin as efficiently as full-length AP-3. Quantitative
assembly assays were carried out as described under ``Experimental
Procedures.'' 3 µM clathrin triskelia were dialyzed
overnight at 4 °C against isolation buffer with the addition of one
of the following test assembly proteins at the concentrations indicated
in the figure: GST-AP-3 ( closed circle), GST-C58 kDa ( open
circle), GST-C16 kDa ( closed square), GST-M42 kDa
( closed triangle), or GST (). Each data point
represents the mean from three experiments with the error bars representing the standard deviations derived from three
experiments.
The 58-kDa C-terminal Region, the 16-kDa C-terminal
Region, and the 42-kDa Central Region of AP-3 Bind to Clathrin
Cages
Previous studies had demonstrated that bacterially
expressed AP-3 could bind to clathrin cages, whereas the 33-kDa
N-terminal region of AP-3 (amino acid residues 1-304) could not
bind to clathrin cages
(3) . Consequently, we examined the
ability of the 58 kDa C-terminal region (amino acid residues
305-901), the 42 kDa central region (amino acid residues
305-744), and the 16 kDa C-terminal region (amino acid residues
745-901) of AP-3 to bind to preassembled clathrin cages
(Fig. 1). In this assay, the ability of the indicated GST fusion
proteins to co-sediment with the pre-assembled clathrin cages was
monitored by Western blot analysis with an anti-GST monoclonal
antibody, and subjected to a quantitative analysis, as described under
``Experimental Procedures.'' Consistent with previous
observations
(3) we found 88% binding of GST-AP-3 ( lanes
1-4) (positive control) and 0.2% binding of GST-N33 kDa
( lanes 5-8) (negative control) to the clathrin cages. In
experiments utilizing the newly expressed domains, we found 86% GST-C58
kDa ( lanes 9-12), 42% GST-M42 kDa ( lanes
13-16), and 51% GST-C16 kDa ( lanes 17-20)
bound to the clathrin cages. The GST negative control showed 0% binding
to the clathrin cages ( lanes 21-24). We conclude that
the 58-kDa C-terminal region, the 42-kDa central region, and 16-kDa
C-terminal region of AP-3 all bind to clathrin cages.
Figure 1:
The 58-kDa
C-terminal, 16-kDa C-terminal, and 42-kDa central region of AP-3 all
bind to clathrin cages. Clathrin cage binding assays were carried out
as described under ``Experimental Procedures.'' 20
µM concentrations of either GST-AP-3 ( lanes
1-4), GST-N33 kDa ( lanes 5-8), GST-C58 kDa
( lanes 9-12), GST-M42 kDa ( lanes 13-16),
GST-C16 kDa ( lanes 17-20), or GST ( lanes
21-24) was incubated in the absence (-) or presence
(+) of 1.8 µM (corresponding to molarity of
triskelia, 1.2 mg/ml) preassembled clathrin cages. Following a low
speed spin to remove nonspecific aggregates, all samples were pelleted
by ultracentrifugation at 100,000 g. The pellet
( P) and supernatant ( S) fractions were analyzed by
SDS-PAGE, followed by Coomassie Blue staining ( upper panel)
and Western blotting ( lower panel) using an anti-GST-specific
monoclonal antibody visualized by the ECL detection method. Positions
of clathrin heavy chain ( Cla HC), GST-AP-3, GST-C58 kDa,
GST-M42 kDa, GST-N33 kDa, GST-C16 kDa, clathrin light chains ( Cla
LCs), GST, and molecular mass markers ( MW) are indicated.
Essentially identical results were obtained in three independent
experiments.
The 58-kDa C-terminal Region and the 42-kDa Central
Region of AP-3 Bind to Clathrin Triskelia
Previously, we showed
that the bacterially expressed 33-kDa N-terminal region of AP-3 could
bind to clathrin triskelia, but could not assemble clathrin triskelia
into cages or bind to preassembled clathrin cages
(3) . This
suggested that binding to clathrin triskelia and clathrin cages were
not functionally equivalent. In the present study, we examined the
ability of the 58-kDa C-terminal region, the 42-kDa central region, and
the 16-kDa C-terminal region of AP-3 to bind to clathrin triskelia. For
these experiments, we coupled proteins GST-AP-3, GST-C58 kDa, GST-M42
kDa, GST-C16 kDa, and GST to glutathione-Sepharose. A solution of
clathrin triskelia, or as a control a solution of BSA, was incubated
with each resin. The resins were washed to remove nonspecifically bound
protein, followed by solubilization of the bound material by boiling
each resin in SDS-sample buffer. The flow-through (labeled F)
and bound (labeled B) fractions were analyzed by SDS-PAGE
(Fig. 2). The gels in the top panel were stained for
total protein with Coomassie Blue, and the gels in the bottom panel were transferred to nitrocellulose and stained with either an
anti-clathrin heavy chain-specific monoclonal antibody ( lanes
1-10) or anti-BSA monoclonal antibody ( lanes
11-20). Note that the GST fusion proteins also eluted from
the resins by boiling in SDS-sample buffer and showed up in the
Coomassie Blue-stained gels. None of the resins retained the BSA
( lanes 11-20), indicating that the resins did not
nonspecifically bind to proteins. In experiments utilizing GST-AP-3 and
GST-C58 kDa resins, all the clathrin triskelia were found in the bound
fractions ( lanes 1-4). This confirmed that GST-AP-3 can
bind to clathrin triskelia and demonstrated that GST-C58 kDa can bind
to clathrin triskelia. In experiments utilizing GST-M42 kDa resin,
clathrin triskelia were found both in the flow-through fraction
( lane 5) and bound fraction ( lane 6). This indicated
binding between clathrin triskelia and GST-M42 kDa. In experiments
utilizing either GST-C16 kDa, or GST (negative control), essentially
all of the clathrin triskelia were found in the flow-through fractions
( lanes 7-10), indicating that clathrin triskelia did not
bind to either of these proteins under these assay conditions. We also
increased the concentration of clathrin in the assay by a factor of 3
(1.2 mg/ml) and were still unable to detect any binding of clathrin
triskelia to GST-C16 kDa.
The 58-kDa C-terminal Region of AP-3 Is Sufficient for
Clathrin Assembly
It had been shown previously that bacterially
expressed GST-AP-3 could assemble clathrin triskelia into cages,
whereas the bacterially expressed 33-kDa N-terminal region of AP-3 was
insufficient for clathrin assembly
(3) . In order to further
localize the regions of AP-3 required for clathrin assembly, we
compared the ability of GST-AP-3, GST-C58 kDa, GST-M42 kDa, GST-C16
kDa, GST-N33 kDa, and GST to assemble clathrin triskelia into clathrin
cages. In this assay, we monitored clathrin assembly as the movement of
clathrin from a 100,000 g soluble form (triskelia) to
a 100,000
g insoluble form (cages) following dialysis
with a test assembly protein (Fig. 3). In the gel displayed, it
is clear that clathrin moves from the supernatant to the pellet in the
presence of GST-AP-3 ( lanes 1 and 2) or GST-C58 kDa
( lanes 3 and 4), indicative of clathrin assembly.
However, no movement of clathrin is seen from the supernatant to the
pellet in the presence of GST-M42 kDa ( lanes 5 and
6), GST-C16 kDa ( lanes 7 and 8), or GST-N33
kDa ( lanes 9 and 10), compared with the negative
control GST ( lanes 11 and 12).
Figure 5:
Ultrastructural analyses of the cages
assembled by the 58-kDa C-terminal region of AP-3 reveals a homogeneous
population of clathrin cages. The morphology of the products of the
clathrin assembly assays displayed in Fig. 3 were evaluated by negative
staining electron microscopy. Electron micrographs are shown of
clathrin assembly reactions promoted by either GST (negative control)
( A), GST-AP-3 (positive control) ( B), or GST-C58 kDa
(test assembly protein) ( C). The distribution of cage
diameters assembled by GST-AP-3 and GST-C58 kDa was
indistinguishable.
Figure 6:
Summary of the structure-function studies
of AP-3. A clathrin triskelia binding site had been found in the 33-kDa
N-terminal region (1, 3). A high affinity inositol polyphosphate
binding site had also been found in the 33-kDa N-terminal region (33).
The 33-kDa N-terminal region had been found to not bind to clathrin
cages (3) or assemble clathrin triskelia into cages (1, 3). All data
concerning the 58-kDa C-terminal region, the 42-kDa central region, and
the 16-kDa C-terminal region are presented in this paper. For
reference, all of the binding data are shown along with the
distribution of acidic ( A) and basic ( B) residues
across the mouse AP-3 sequence (23, 27).
The data presented in this study allow us to consider alternative
interpretations to previously published studies. In clathrin cage
binding assays utilizing a partial trypsin digestion of AP-3, bands
with apparent molecular masses of 148, 107, and 33 kDa were found to
co-sediment with preassembled clathrin cages
(2) . Sequencing of
the digestion products revealed that the 33-kDa fragment corresponded
to the 33-kDa N-terminal region of AP-3 and that the 107-kDa apparent
molecular mass fragment began at amino acid 304. The interpretation
presented was that the 33-kDa N-terminal region bound specifically to
the cages and that the 107-kDa apparent molecular mass fragment was
only retained by the cages due to a noncovalent association with the
33-kDa N-terminal fragment. Our demonstration of clathrin cage binding
by the 58-kDa C-terminal region, the 42-kDa central region, and the
16-kDa C-terminal region, coupled with the lack of clathrin cage
binding by the 33-kDa N-terminal region
(3) , suggest an
alternative interpretation of these proteolysis studies. Possibly it
was the 107-kDa apparent molecular mass fragment that was bound
specifically to the cages, and the 33-kDa N-terminal fragment was only
retained by the cages due to a noncovalent association with the 107-kDa
apparent molecular mass fragment. Similarly, in the clathrin triskelia
binding assays in which it was shown that the 33-kDa N-terminal region
could bind to clathrin triskelia, some retention by the
clathrin-Sepharose of a 135-kDa apparent molecular mass fragment and a
100-kDa apparent molecular mass fragment were also observed
(1) . It was suggested that the 100-kDa apparent molecular mass
fragment was not bound directly to the clathrin triskelia, but was
retained by a noncovalent association with the 33-kDa N-terminal
fragment. Our data allow one to consider the possibility that both the
33-kDa N-terminal fragment and 100-kDa apparent molecular mass fragment
were specifically retained by the clathrin-Sepharose.
30-kDa N-terminal region and the
60-kDa C-terminal region.
The
60-kDa C-terminal region has an unusual self-repeating primary
structure, a preponderance of acidic residues, an unusually high
proline, serine, threonine, and alanine content, and is predicted to
have a high content of irregular secondary structure. The
30-kDa
N-terminal region of AP-3 has an amino acid composition typical of a
globular protein, with a roughly equal balance of acidic and basic
residues, and is predicted to have a high alpha helix content. It was
suggested that AP-3 has an essentially neutral
30-kDa N-terminal
domain with an amino acid composition typical of a globular structure
and an acidic
60-kDa C-terminal domain with a highly repetitive
and redundant amino acid composition
(23, 27) . These
data were consistent with published proteolysis studies
(1) ,
which had shown that limited digestion with a number of different
proteases generated a stable
30-kDa fragment, which was later
shown
(2) to correspond to the 33-kDa N-terminal region. Other
investigators
(2) proposed a further division within the 58-kDa
C-terminal region into a 42-kDa central acidic domain and a 16-kDa
C-terminal domain. These assignments were based on the identification
of these regions as products of partial proteolysis reactions
(1, 2) and the finding that the 16-kDa C-terminal
region was more basic, and had a higher proline content, than any other
part of the protein.
AS108
); GST-C58 kDa, GST, fused
with amino acids 305-744 of AP-3 (isoform
AS15
AS108
); GST-N33 kDa, GST, fused
with amino acids 1-304 of AP-3 (isoform
AS15
AS108
); GST-C16 kDa, GST, fused
with amino acids 745-901 of AP-3 (isoform
AS15
AS108
); GST-M42 kDa, GST, fused
with amino acids 305-744 of AP-3 (isoform
AS15
AS108
); PMSF,
phenymethylsulfonyl fluoride; MES, 4-morpholineethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.