(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 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.
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 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).
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. 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.
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 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).
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. 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.
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. 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).
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 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.
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. 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.
Four peptides spanning residues 224-243
of either LCa (LCa20 and LCa23) or LCb (LCb20 or LCb23) inhibit the
calcium-dependent binding of 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.
Figure 6:
Competition for binding of 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.
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 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).
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
-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.
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) .