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(Received for publication, May 9, 1994; and in revised form, November 7, 1994) From the
The binding of clathrin-coated vesicles, clathrin triskelions,
and free clathrin light chains to calmodulin-Sepharose was compared.
When isolated from bovine brain, all three components bound to
calmodulin-Sepharose in the presence of calcium and could be eluted by
its removal. In contrast, coated vesicles and triskelions isolated from
bovine adrenal gland did not bind to calmodulin-Sepharose, although the
free light chains from adrenal gland bound as effectively as those from
brain. As distinct isoforms of the clathrin light chains are expressed
by brain and adrenal gland, these results implicate the clathrin light
chains as the calmodulin-binding component of coated vesicles and
triskelions. Furthermore, the insertion sequences found in the
neuron-specific isoforms, although not necessary for the binding of
free clathrin light chains to calmodulin, must facilitate the
interaction of heavy chain-associated light chains with calmodulin.
Recombinant mutants of LCa, with deletions spanning the entire
sequence, were tested for binding to calmodulin-Sepharose. Those
mutants retaining structural integrity, as assessed by the binding of a
panel of monoclonal antibodies, exhibited varying amounts of calmodulin
binding activity. However, deletion of the carboxyl-terminal 20
residues abolished calmodulin interaction. Thus, the carboxyl terminus
of LCa appears to constitute a calmodulin-binding site. Peptides
corresponding to the carboxyl terminus of LCa or LCb inhibited the
interaction of the light chains with calmodulin, suggesting that this
region forms the calmodulin-binding site of both LCa and LCb. The
carboxyl-terminal peptides of LCa and LCb inhibited the interaction of
light chains with calmodulin In eucaryotic cells, clathrin-coated vesicles are the agents for
receptor-mediated transport of macromolecules between membrane-bound
compartments. Formation of a coated vesicle is initiated by the binding
of adaptors to receptors, thereby forming the inner layer of the
protein coat. The outer layer is formed by the polymerization of
clathrin upon the clustered adaptors, giving the coat its
characteristic appearance of a polygonal lattice. Vesiculation, driven
by the polymerization of clathrin, is followed by disassembly of the
clathrin coat, allowing fusion of the vesicle with its target membrane
(reviewed in (1) and (2) ). The monomeric form of
clathrin is the triskelion, a three-legged structure that can
participate in many cycles of assembly and disassembly. Each leg of the
triskelion consists of a heavy chain bound to a light
chain(3, 4) . Mammalian cells express two homologous
light chains, LCa and LCb, in contrast to the single light chain of
yeast (5, 6, 7, 8, 9) .
Additional heterogeneity in mammalian light chains arises from
alternate mRNA splicing, which in neurons produces isoforms of LCa and
LCb that have additional insertion sequences of 30 and 18 amino acids,
respectively(5, 6) . Central to coated vesicle
function is the regulation of clathrin disassembly. Studies in
vitro show that disassembly is effected by the cytosolic heat
shock protein Hsc70 and is enhanced by the presence of calcium
ions(10, 11, 12, 13) . Hsc70 appears
to act through interaction with a binding site on the clathrin light
chains that lies adjacent to a site for binding
calcium(11, 14, 15) . Further evidence for
the involvement of calcium in clathrin function is the observation that
calmodulin, a cytoplasmic mediator of calcium-regulated processes,
binds to bovine brain clathrin-coated vesicles and to clathrin light
chains in a calcium-dependent
manner(9, 16, 17, 18, 19) .
The binding of calmodulin to clathrin triskelions has been visualized
by electron microscopy, but not by affinity chromatography, and when
clathrin heavy chains are immobilized on nitrocellulose, they, too,
bind calmodulin(17, 20, 21) . The
experiments reported here compare the binding of calmodulin to
clathrin-coated vesicles, triskelions, and light chains from tissues
expressing the different light chain isoforms and define a binding site
for calmodulin in the carboxyl-terminal region of the clathrin light
chains.
This experiment
shows that bovine brain clathrin-coated vesicles bind to
calmodulin-Sepharose and can be eluted in a form that still sediments
at 100,000
Figure 1:
Binding of brain clathrin-coated
vesicles (A) and triskelions (B) to calmodulin. A, the leftpanel shows binding to
calmodulin-Sepharose, whereas the rightpanel shows
the negative control binding to Sepharose alone. Lanes1 and 2 show 124 µg of pelleted and nonpelleted brain
clathrin-coated vesicles, respectively. An equivalent amount of
pelleted vesicles was incubated with each resin. The unbound material
was centrifuged, and the pellet is shown in lane3 and the supernatant in lane4. The resin was
washed 10 times in the presence of Ca
A similar
analysis of clathrin triskelions derived from bovine brain
clathrin-coated vesicles was performed. Buffer conditions were those
that stabilize triskelions, and the samples for chromatography were
centrifuged at 100,000 When similar experiments were performed
using clathrin-coated vesicles and triskelions isolated from bovine
adrenal glands, no binding to calmodulin-Sepharose was observed (Fig. 2). For triskelions, the known differences between the
preparations isolated from brain and adrenal gland are the presence of
insertion sequences in the neuronal isoforms of LCa and LCb. These
results suggested that the light chains could be involved in the
interaction of calmodulin with brain clathrin-coated vesicles and
triskelions and that the neuronal insertion sequences may play a role
in these interactions.
Figure 2:
Binding of adrenal gland clathrin-coated
vesicles (A) and triskelions (B) to calmodulin. A, the left panel shows binding to
calmodulin-Sepharose, whereas the rightpanel shows
the negative control binding to Sepharose alone. Lanes1 and 2 show 100 µg of pelleted and nonpelleted adrenal
clathrin-coated vesicles, respectively. An equivalent amount of
pelleted vesicles was incubated with each resin. The unbound material
was centrifuged, and the pellet is shown in lane3 and the supernatant in lane4. The resin was
washed 10 times in the presence of Ca
Figure 3:
Interaction of brain (A) and
adrenal gland (B) clathrin light chains with calmodulin. In
both A and B, the upperpanel shows
the binding of LCa, and the lowerpanel shows the
binding of LCb. A, 20-µg aliquots of the brain LCa and LCb
preparations (lane 1) were used as input. Light chains were
incubated with calmodulin-Sepharose, and the unbound material (lane
2) was removed. The calmodulin-Sepharose was then washed 10 times
in the presence of Ca
Figure 4:
Cartoon showing linear organization of
neuronal form of clathrin light chain LCa into structural and
functional domains. Beneath the cartoon are line diagrams of the two
LCa isoforms and the deletion mutants (mt) used in this study. Light gray box, conserved region; white wide striped
box, Hcs70-binding site; dark gray box, calcium-binding
site; white narrow striped box, heavy chain-binding site; black striped box, neuronal insert; black box,
calmodulin-binding site.
To
identify a region of LCa important for calmodulin binding, the deletion
mutants were analyzed for interaction with calmodulin-Sepharose (Fig. 5). The calmodulin binding activity was calculated by
dividing the radioactivity of the material eluted from the calmodulin
column by the radioactivity of the total material (bound and unbound)
recovered from the calmodulin column. The COOH-terminal truncation
mutants
Figure 5:
Interaction of recombinant LCa and mutant
LCa proteins with calmodulin-Sepharose (triplicate determinations are
shown by filled bars) and Sepharose (clear bars).
After incubation of mutant LCa proteins with resin, unbound material
was removed, and the resin was washed 10 times with buffer containing
Ca
Four peptides spanning residues 224-243
of either LCa (LCa20 and LCa23) or LCb (LCb20 or LCb23) inhibit the
calcium-dependent binding of
Figure 6:
Competition for binding of
As a further
test of the specificity of the inhibitory sequence, peptides
corresponding to the amino-terminal (LCa12) and carboxyl-terminal
(LCa11) halves of peptide LCa23 were synthesized (Table 1). The
LCa11 peptide had no effect, whereas the LCa12 peptide gave a slight
inhibition of These studies with synthetic peptides support the assignment of
residues 224-243 of LCa and LCb as a site of direct interaction
with calmodulin. This site has a similar sequence in the two light
chains: 15 of the residues are identical, and conservative
substitutions are found at the remaining 5 (Table 1). Comparison
of light chain peptide inhibition with well characterized
calmodulin-binding peptides suggests that the affinity for the clathrin
light chain-calmodulin interaction is within the range of other
calmodulin-binding proteins and thus of potential physiological
importance(37) . Previous investigations have created a picture in which
clathrin heavy and light chains are seen to play highly complementary
roles. The characteristic structures of the triskelion and the clathrin
coat are a function of the heavy chain. In contrast, the light chains
contribute little to the appearance of clathrin structures, but provide
an array of regulatory motifs implicated in the control of clathrin
function (38) . Light chains are the target for
phosphorylation, Hsc70 binding, and calcium binding. Further
evidence supporting this image of the light chains is contributed by
the study described here; the carboxyl-terminal region of the light
chains is demonstrated to be critical for interaction with calmodulin
and in all likelihood forms the site of calmodulin binding. Calmodulin
binding is the first activity mapped to the carboxyl-terminal region of
the light chains that has been noted for its conservation between LCa
and LCb(5, 6, 7) . Consistent with the
conservation in sequence, we find that all four forms of free light
chain, as represented by the neuronal and adrenal gland forms of LCa
and LCb, bind to calmodulin affinity columns in a calcium-dependent
fashion, confirming and extending the previous findings of Linden et al.(16) , Lisanti et al.(18) ,
Merisko et al.(21) , and Moskowitz et
al.(17) . Although assignment of the calmodulin-binding
site to residues 224-243 is primarily based upon the analysis of
deletion mutants of LCa, the circumstantial evidence derived from
binding studies with intact light chains, inhibition studies with
synthetic peptides, and the conservation of sequence indicate that the
homologous region of LCb is also the calmodulin-binding site of that
light chain. Many cytoplasmic proteins bind calmodulin. A common
motif in their calmodulin-binding sites is a basic amphiphilic
Free LCa and LCb are not the physiologically
active forms of clathrin light chains, and assessment of the functional
significance of their binding to calmodulin therefore requires an
understanding of calmodulin interaction with triskelions and
clathrin-coated vesicles. We find that brain triskelions and coated
vesicles bind to calmodulin, whereas those isolated from adrenal gland
do not. The clathrin heavy chain isolated from brain can bind
calmodulin, whereas the heavy chain isolated from adrenal glands has
not been analyzed(20, 21) . Therefore, it is unknown
whether tissue-specific differences between the heavy chains exist.
However, the known structural difference between the triskelions from
these two tissues is the presence of neuron-specific insertion
sequences in the light chains, suggesting that these sequences, which
do not affect calmodulin binding of the free light chain, affect the
conformation or accessibility of the calmodulin-binding sites on the
light chains and possibly the heavy chain in triskelions and
clathrin-coated vesicles. The insertion sequences in the light chains
are close to the proposed calmodulin-binding site and are suitably
situated to perform such modification. Alternatively, if differences
between the heavy chains from the two tissues exist, unique features of
the adrenal gland heavy chain might prevent adrenal gland triskelions
from binding to calmodulin. Differential interaction with calmodulin
is the first assay of potential functional importance to distinguish
the tissue-specific isoforms of the clathrin light chain. That
calmodulin binds to neuronal clathrin but not to the clathrin of other
cell types points to the calmodulin interaction serving a
neuron-specific function. Potential candidates are the recycling of
synaptic vesicle membrane proteins and the transport of triskelions
along axons to the nerve terminals as both triskelions and calmodulin
are transported to the synapse in the slow component
SCb(42, 43) .
Volume 270,
Number 5,
Issue of February 3, 1995 pp. 2395-2402
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
LOCALIZATION OF A BINDING SITE (*)
10-fold less effectively than a
calmodulin-binding peptide derived from smooth muscle myosin light
chain kinase, but much more effectively than a calmodulin-binding
peptide derived from adenylate cyclase. This comparison places the
clathrin light chain-calmodulin interaction within the physiological
range seen for other calmodulin-binding proteins.
Coated Vesicle Preparation and Clathrin
Purification
Clathrin-coated vesicles were prepared from six
bovine brains or 2 pounds of adrenal glands as described previously (22) using a modified isolation buffer in which magnesium
chloride and dithiothreitol were omitted. For the preparation of
adrenal gland clathrin-coated vesicles, the protease inhibitors
aprotinin (5 µg/ml), leupeptin (5 µg/ml), and pepstatin (7
µg/ml) were included, in addition to phenylmethylsulfonyl fluoride,
in the isolation buffer. Triskelions were extracted from
clathrin-coated vesicles and purified according to
Näthke et al.(15) . Triskelions
were boiled for 10 min to release the clathrin light chains (23, 24) . LCa and LCb were separated on a
DEAE-cellulose column as described by Parham et
al.(25) .Light Chain Mutagenesis
Construction of the LCa
mutants 
26-56, 22-96, and 162-243 was
reported previously(15) . LCa mutant 98-161 was
produced by removing the ClaI-HindIII fragment from a
full-length cDNA clone of bovine brain LCa(6) . Filling in of
the restriction site overhangs and blunt-ended ligation then eliminated
the codons for Leu-Glu
(numbers refer
to those of the original bovine LCa sequence(6) ). LCa mutant
2-21 was generated by deleting the NcoI-NaeI fragment from a full-length cDNA clone of
bovine brain LCa(6) , followed by filling in and blunt-ended
ligation. This resulted in a mutant LCa lacking residues
Ala
-Ala. LCa mutant
2-21,26-56 is a combination of LCa mutant
2-21 and 26-56. LCa mutants 188-243,
196-243, 204-243, and 224-243 were
constructed from a bovine LCa cDNA clone in pUC18 containing the entire
coding sequence of the major neuron-specific isoform. Stop codons were
introduced at the desired locations through site-directed mutagenesis
by overlap extension using the polymerase chain reaction(26) .
Mutations were verified by DNA sequencing.Preparation of Recombinant Mutant Light
Chain
Mutant and wild-type LCa were cloned into either the
pET11d or pET21d vector (Novagen) and expressed in Escherichia coli strain BL21(DE3) by
isopropyl-
-D-thiogalactopyranoside induction. Cell
extracts containing recombinant LCa proteins were prepared by lysing
cells in 50 mM Tris, pH 8.0, 70 mM EDTA, 9.5%
sucrose, 0.06% Triton X-100, and 0.16 mg/ml lysozyme in the presence of
the protease inhibitors aprotinin, leupeptin, pepstatin, and
phenylmethylsulfonyl fluoride as described under ``Coated Vesicle
Preparation and Clathrin Purification.'' Debris was removed from
the cell lysates by a 10-min centrifugation at 50,000 
g. The supernatants were boiled and centrifuged to give a
light chain-containing supernatant.Radiolabeling of Rabbit Anti-mouse IgG F(ab`)
50 µg of rabbit anti-mouse IgG F(ab`) Fragment fragment was iodinated using 1 mCi of NaI (100
mCi/ml; Amersham Corp.) and one bead of IODO-BEADS (Pierce) to a
specific activity of 14 µCi/µg.
Calmodulin-Sepharose Affinity Chromatography
To
assay the interaction of light chains and mutant LCa proteins with
calmodulin, 0.2 ml of settled calmodulin-Sepharose 4B resin (400 µg
of calmodulin) was pre-eluted with 20 mM Tris, pH 7.8, 100
mM NaCl, 20 mM 2-mercaptoethanol, and 2 mM EGTA (buffer A) and re-equilibrated in 20 mM Tris, pH
7.8, 100 mM NaCl, 20 mM 2-mercaptoethanol, and 1
mM CaCl (buffer B). Light chains (10-20
µg) in 0.5 ml of buffer B were incubated with the
calmodulin-Sepharose resin for 1 h at 4 °C under moderate agitation
on a rotating platform. The resin was then poured into a 1-ml
tuberculin syringe to make a column from which the following fractions
were collected: 0.5 ml of supernatant from the incubation mixture, 10
washes of 1 ml each of buffer B, and four elutions of 0.5 ml each of
buffer A. The protein in each fraction was precipitated by 5%
trichloroacetic acid and then analyzed by 12.5% SDS-PAGE (
)(27) and Western blotting(28) . LCa was
detected using monoclonal antibody CVC.7 (29) or
X16(30) , and LCb was detected using monoclonal antibody LCB.1
or LCB.2(31) . The secondary antibodies were anti-mouse
IgG-alkaline phosphatase for LCa and LCb proteins (Boehringer Mannheim)
and radiolabeled rabbit anti-mouse IgG F(ab`) fragments for
recombinant LCa and LCa mutant proteins. As the negative control, a
resin of Sepharose CL-4B (Pharmacia Biotech Inc.) with no calmodulin
conjugated was used. For quantitation of recombinant LCa and LCa mutant
proteins binding to calmodulin-Sepharose, Western blots were exposed to
Phosphor Imager screens (Molecular Dynamics, Inc.) and read on a
PhosphorImager SF (Molecular Dynamics, Inc.), and protein band
intensities were determined using the ImageQuant software
package (Molecular Dynamics, Inc.). The supernatant from the incubation
mixture and the first and the tenth wash fractions were counted as
material not bound to calmodulin-Sepharose. The eluted fractions were
counted as material bound to calmodulin-Sepharose. The percentage of
mutant light chain bound to calmodulin was calculated by dividing the
radioactivity of the material eluted from the calmodulin columns by the
total radioactivity of the bound and the unbound material from the
calmodulin columns. Assays of clathrin-coated vesicle binding to
calmodulin-Sepharose were done in 10 mM MES, pH 6.5, 100
mM KCl, 0.1% bovine serum albumin, 0.02% NaN
, and
either 2 mM EGTA or 1 mM CaCl. The buffer
originally used for the binding of triskelions to calmodulin-Sepharose
was 0.5 M Tris, 50 mM MES, pH 6.2, 0.25 mM MgCl, 0.5 mM EGTA, 1.5 mM CaCl, and 0.02% NaN. Triskelions were
eluted from calmodulin-Sepharose with 0.5 M Tris, 50 mM MES, pH 6.2, 0.25 mM MgCl, 2 mM EGTA, and 0.02% NaN. Upon review of this report, the
binding of brain triskelions to calmodulin-Sepharose was also performed
in buffers containing 100 mM NaCl in addition to all other
components listed above, and the results were identical.Radiolabeling of LCa and LCb
200 µg of
purified bovine brain LCa or LCb was radioiodinated using 1 mCi of
NaI (100 mCi/ml) and one bead of IODO-BEADS per reaction
for 15 min in buffer containing 25 mM Tris, 0.1 M NaCl, pH 7.0.
I-Labeled light chain and
unincorporated Na
I were separated by gel filtration on a
0.8
16-cm Sephadex G-25 column equilibrated in 25 mM Tris, 0.1 M NaCl, and 1 mg/ml bovine serum albumin, pH
7.0. LCa and LCb were radiolabeled to specific activities of 12.9 and
2.89 µCi/µg, respectively.Light Chain-Calmodulin Interaction in Presence of
Peptides
Peptides (see Table 1) were synthesized by
Research Genetics (Huntsville, AL) and desalted using Sep-Pak C
cartridges (Waters Associates) using published
procedures(32) . After purification, the concentrations of the
peptide preparations were determined using the Pierce
micro-bicinchoninic acid assay according to the manufacturer's
instructions. Assays were performed using 1.5 µg of I-labeled LCb or 0.5 µg of
I-labeled
LCa and 100 µl of settled resin of a mixture of
calmodulin-Sepharose (Pharmacia Biotech Inc.) and Sepharose CL-4B.
Assays contained 15 µg of calmodulin. Peptides at 1-, 10-, 100-,
and 1000-fold molar excesses over light chain were coincubated with the
radiolabeled light chains and the calmodulin-Sepharose/Sepharose CL-4B
mixture for 1 h. Fractions were obtained as described above and
analyzed by counting aliquots in triplicate on a Pharmacia Biotech 1260
Multigamma II counter.
Brain, but Not Adrenal Gland, Clathrin-coated Vesicles
and Triskelions Bind to Calmodulin-Sepharose
The interaction of
calmodulin with clathrin-coated vesicles (assembled clathrin) and
purified triskelions (unassembled clathrin) was assessed in separate
experiments. Clathrin-coated vesicles were isolated from bovine brains
and tested for binding to calmodulin conjugated to Sepharose 4B. In the
experiments using clathrin-coated vesicles, the presence of
disassembled clathrin in the vesicle preparations was reduced by
centrifugation at 100,000 g. After discarding the
supernatant that contained unassembled clathrin (triskelions), the
pelleted clathrin-coated vesicles were resuspended just prior to use.
The resin was incubated with clathrin-coated vesicles in the presence
of Ca, extensively washed in the presence of
Ca
, and then eluted with buffer containing EGTA and
no Ca
. Fractions obtained from the chromatography
were first centrifuged at 100,000
g to separate
assembled (clathrin-coated vesicles) from disassembled (triskelion)
clathrin and were then analyzed separately for the presence of clathrin
polypeptides using Western blots and a mixture of monoclonal antibodies
specific for LCa, LCb, and the clathrin heavy chain. g (Fig. 1A, leftpanel). 50% of the input material bound to the
calmodulin column, and
10% of that was sedimentable following
elution. The binding was dependent upon calmodulin as shown by the
control experiment using unconjugated Sepharose (Fig. 1A, rightpanel).
. In lanes5 and 6 are the pellet and the supernatant,
respectively, obtained by centrifugation of the combined first and
second washes. Similarly, lanes7 and 8 are
the pellets and supernatants, respectively, obtained from the combined
ninth and tenth washes. After washing, the resins were eluted with
EGTA. The first two fractions eluted were combined and centrifuged; lanes9 and 10 show the pellet and the
supernatant, respectively. Samples were analyzed by SDS-PAGE and
Western blotting using a mixture of monoclonal antibodies against the
clathrin heavy chain (TD.1), LCa (CVC.7), and LCb (LCB.2). The ratio of
clathrin heavy chain to clathrin light chains may not seem constant in
all lanes for several reasons. First, the Western blotting method
employed was not intended to be quantitative. Second, the experiment
analyzed intact clathrin-coated vesicles (in the lanes showing pelleted
material) as well as clathrin-coated vesicles that disassembled and
perhaps partially degraded during the duration of the experiment (in
the lanes showing material of the supernatants). The positions of
prestained molecular mass markers are indicated by the horizontallines to the left of the panels (from top to bottom):
phosphorylase b (106 kDa), bovine serum albumin (80 kDa),
ovalbumin (49.5 kDa), carbonic anhydrase (32.5 kDa), soybean trypsin
inhibitor (27.5 kDa), and lysozyme (18.5 kDa). B, equivalent
amounts of clathrin triskelions (lane6) were used to
assess binding to calmodulin-Sepharose (lanes 1-5) and
to Sepharose (lanes 7-11). After removal of unbound
triskelions (lanes1 and 7), the resin was
washed 10 times in the presence of Ca
. Lanes2 and 8 show the first wash fraction, and lanes3 and 9 show the last wash fraction.
Then, the resin was eluted with EGTA. Lanes4 and 10 show the combined first and second eluted fractions, and lanes5 and 11 show the combined third and
fourth eluted fractions. The clathrin subunits were detected by silver
staining of the SDS-polyacrylamide gel. Molecular mass markers were as
described for A.
g to remove aggregates prior to
the incubation with the calmodulin-Sepharose. As observed for
clathrin-coated vesicles, triskelions bound to calmodulin-Sepharose,
but not Sepharose, in a Ca-dependent fashion (Fig. 1B).
. In lanes 5 and 6 are the pellet and the supernatant, respectively,
obtained by centrifugation of the first wash. Similarly, lanes7 and 8 are the pellets and supernatants,
respectively, obtained from the tenth wash. After washing, the resins
were eluted with EGTA. The first two fractions eluted were combined and
centrifuged; lanes9 and 10 show the pellet
and the supernatant, respectively. The observation of a small amount of
heavy chain in lane9 was not reproducible. Samples
were analyzed by SDS-PAGE and Western blotting using a mixture of
monoclonal antibodies against the clathrin heavy chain (TD.1), LCa
(CVC.7), and LCb (LCB.1). Molecular mass markers were as described for Fig. 1A. B, equivalent amounts of clathrin
triskelions (lane6) were used to assess binding to
calmodulin-Sepharose (lanes 1-5) and to Sepharose (lanes 7-11). After removal of unbound triskelions (lanes1 and 7), the resin was washed 10
times in the presence of Ca
. Lanes 2 and 8 show the first wash fraction, and lanes3 and 9 show the last wash fraction. Then, the resin was
eluted with EGTA. Lanes4 and 10 show the
combined first and second eluted fraction, and lanes5 and 11 show the combined third and fourth eluted
fractions. Samples were analyzed as described for A. Molecular
mass markers were as described for Fig. 1A.
Isolated Brain and Adrenal Gland Forms of LCa and LCb
Bind Calmodulin
Free light chains obtained from brain and
adrenal gland triskelions were assessed for their binding to
calmodulin-Sepharose. As previously shown by Lisanti et al.(18) , brain LCa and LCb bind well to
calmodulin-Sepharose, and in our experiments, the binding was nearly
complete (Fig. 3). Adrenal gland LCa and LCb also bound to
calmodulin-Sepharose (Fig. 3), a result contrasting with that
obtained for adrenal gland clathrin-coated vesicles and triskelions. As
adrenal gland light chains lack neuronal insertion sequences, their
binding cannot involve these sequences. The results from the binding of
clathrin-coated vesicles, triskelions, and free light chain suggest the
hypothesis that the calmodulin-binding site is formed by another part
of the light chain molecule, but is modified by the presence of a
neuronal insertion sequence.
. Lanes 3-7 show
successive pairs of the wash fractions. The resin was eluted four times
with buffer containing EGTA (lanes 8-11). Samples were
analyzed by SDS-PAGE and Western blotting with monoclonal antibodies
against LCa (CVC.7) and LCb (LCB.2). B, input aliquots of
adrenal gland LCa and LCb (lane1) were used for
binding to Sepharose (lanes 2-6) and
calmodulin-Sepharose (lanes 7-11). After coincubation of
light chain and resin, material that was not bound to the resins was
removed (lanes2 and 7), and the resins were
then washed 10 times with Ca
-containing buffer. Lanes 3 and 8 show the first wash fractions, and lanes4 and 9 show the tenth wash fractions.
Then, the resins were eluted with buffer containing EGTA. Lanes5 and 10 show the combined first and second
eluted fractions, and lanes6 and 11 show
the combined third and fourth eluted fractions. Samples were analyzed
by SDS-PAGE and Western blotting with monoclonal antibodies against LCa
(CVC.7) and LCb (LCB.1).
COOH Terminus of LCa (Amino Acid Residues 224-243)
Is Important for Interaction with Calmodulin
Ten deletion
mutants of the major neuronal form of LCa were constructed. These
deletions cover the entire LCa sequence (Fig. 4). To assess the
structures of the LCa mutants, they were first compared with wild-type
LCa for their recognition by eight LCa-specific monoclonal antibodies
and polyclonal antisera on Western blots and by radioimmunoassay.
Previous analysis mapped the epitopes recognized by the antibodies
within the LCa sequence, and they are distributed throughout the length
of LCa(11, 31, 33) . Wild-type LCa and all
LCa mutants except mutant 162-243 reacted as predicted from
the presence of antibody epitopes in the amino acid sequences. In
contrast, LCa mutant 162-243 reacted weakly with five of the
six antibodies tested recognizing epitopes in the NH terminus and the heavy chain-binding domain, regions that are not
deleted in the mutant. Thus, the deletion in mutant 162-243
grossly affects the conformation of LCa, whereas the other deletion
mutants appear to fold sufficiently to retain antibody epitopes.
162-243, 188-243, 196-243,
204-243, and 224-243 did not bind calmodulin. In
contrast, the LCa mutants carrying deletions in the NH terminus or the heavy chain-binding domain exhibited calmodulin
binding activity comparable to or exceeding that of wild-type
recombinant LCa. These findings suggest that residues 224-243 at
the carboxyl terminus of LCa are responsible for the calmodulin binding
activity. The smallest COOH-terminal deletion that abolished calmodulin
binding was that of mutant 224-243. As the serological
analysis showed that the overall conformation of LCa mutant
224-243 was not disrupted, we hypothesize that residues
224-243 constitute a calmodulin-binding site.
. LCa protein in these fractions was counted as not
bound to the resin. Then, the resin was eluted with EGTA, and the
``bound'' protein was recovered. The yaxis shows the percentage of light chain bound as calculated from the
ratio of bound protein and total protein (bound plus unbound). LCa and
LCa mutant (mt) proteins were detected by Western blotting
using monoclonal antibodies against LCa (CVC.7 and X16). The amount of
protein in each band was quantitated as described under
``Materials and Methods.'' Black bar, calmodulin 1; light gray bar, calmodulin 2; dark gray bar,
calmodulin 3; white bar,
Sepharose.
Peptides Spanning Residues 224-243 of LCa and LCb
Inhibit Light Chain-Calmodulin Interaction
The properties of the
LCa deletion mutants identify residues 224-243 in the
carboxyl-terminal region as a site of interaction with calmodulin. We
therefore examined whether synthetic peptides derived from this LCa
sequence and the homologous region of LCb (Table 1) inhibited the
binding of I-labeled light chain to calmodulin-Sepharose.
To assess the relative affinities of the clathrin light chain peptides
for calmodulin, two peptides with known binding affinities for
calmodulin, peptide SMMLCK derived from smooth muscle myosin light
chain kinase (K
= 1 nM) (34, 35) and peptide AC derived from adenylate cyclase (K = 580
nM)(36, 37) , were tested for their capacity
to inhibit the binding of clathrin light chains to calmodulin in this
assay (Table 1).I-LCb to
calmodulin-Sepharose, whereas a peptide corresponding to residues
158-175 of the neuronal insertion sequence of LCa (nLCa) had no
effect (Fig. 6B, upperpanel).
Peptide SMMLCK was the most potent inhibitor of
I-LCb
binding to calmodulin, whereas peptide AC was ineffective. The light
chain-derived peptides LCa20, LCa23, LCb20, and LCb23 are within an
order of magnitude as effective inhibitors as peptide SMMLCK of the
binding of
I-LCb to calmodulin-Sepharose; we estimate
their affinity constants for calmodulin to be
10 nM. To
assess the importance of the sequence of the inhibitory peptides, as
opposed to their amino acid composition, a ``scrambled''
peptide (LCa23sc) having the same length and composition as LCa23 but a
different sequence was synthesized (Table 1). The LCa23sc peptide
inhibited
I-LCb binding to calmodulin-Sepharose, but with
a potency
10-fold less than that of the LCa23 peptide (Fig. 6B, upperpanel). Thus, there
appears to be contributions to calmodulin binding from both the
sequence and the composition of the peptide inhibitors.
I-labeled LCa (A) and
I-labeled
LCb (B) to calmodulin-Sepharose by light chain peptides. The upper and lowerpanels show the results for
different peptides.
I-Labeled LCa (0.5 µg) or
I-labeled LCb (1.5 µg) was incubated with peptides at
different molar ratios ranging between 1:1 and 1000:1 and
calmodulin-Sepharose. After incubation, material that did not bind to
the resin was removed, and the resin was washed six times with
Ca
-containing buffer. Radioactivity obtained in these
fractions was counted as protein ``not bound'' to
calmodulin-Sepharose. Then, the resin was eluted with EGTA, and the
radioactivity recovered in these fractions was counted as protein bound
to the calmodulin-Sepharose. The results are plotted as the percentage
of light chain bound in the presence of peptide relative to that bound
in the absence of peptide. Upper panels: black
circle, LCa20; white circle, LCa23; multiplication
sign, LCb20; black square, LCb23; black
triangle, LCa23sc; white square, nLCa; white
triangle, peptide SMMLCK; small dotted black square,
peptide AC. Lower panels: white circle, LCa23; black triangle,LCa23sc; dotted black square, LCa12; dotted black diamond, LCa11; dotted white square,
LCa12+11.
I-LCb binding to calmodulin-Sepharose (Fig. 6B, lowerpanel). An analogous
set of experiments assessed the capacity of the synthetic peptides to
inhibit the binding of
I-LCa to calmodulin-Sepharose, and
the results are comparable to those obtained for the binding of
I-LCb to calmodulin-Sepharose (Fig. 6A).
-helix(37) . The amino acid sequences of LCa and LCb are
not predicted, however, to form such structures; instead, the light
chains are, like calmodulin, characteristically acidic polypeptides.
Within the calmodulin-binding region, aspartate 225 and proline 240 are
predicted to perturb the basic nature of the site and -helix
formation, respectively. Although 4 basic and 7 hydrophobic residues
are found within the region, they are spaced differently than in
sequences known to bind calmodulin with high affinity(39) .
Thus, it is possible that LCa and LCb bind calmodulin with structures
distinct from a basic amphiphilic -helix, precedent for which has
been found in another calmodulin-binding protein, the -subunit of
skeletal muscle phosphorylase kinase(40) . Moreover,
amphiphilic peptides that are composed entirely of D-amino
acids are able to bind calmodulin, demonstrating another permissible
deviation from naturally occurring amphiphilic
-helices as a
calmodulin-binding motif(41) . Furthermore, the light chain
conformation is influenced by interaction with the heavy chain, which
may affect the accuracy of predictions based solely upon light chain
primary structure.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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