Mar 25, 1985 - uncoating, we have found that uncoating protein func- tions as an enzyme in ... two domains, each of which has a binding site for the uncoat-. * This work was .... equilibrated between free and bound forms to about the same extent as ..... dissociate rapidly at 37 "C. Therefore, the term stable refers only to the ...
Vol ,260, No.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.
Issue of August 25, PP,10050-10056,1985 Printed in U.S.A.
Two Classes of Binding Sites for Uncoating Protein in Clathrin Triskelions" (Received for publication, March 25, 1985)
Sandra L. SchmidS andJames E. Rothman From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305
Clathrinreleasedfromcoated vesicles orempty ing protein. Binding at one site requires the presence of cages by the ATP-dependent action of uncoating pro- clathrin light chains and is unstable to gel filtration. Light tein exists as a complex with the uncoating protein. chains are not a component, however, of the second site, Despite its apparent consumption during a round of which retains bound uncoating protein during gel filtration. uncoating, we have found that uncoating protein functions as an enzyme in that it rapidly and spontaneously EXPERIMENTALPROCEDURES recycles from its product (triskelions) to its substrate Materials-Trypsin (treated with ~-l-tosylamido-2-phenylethyl (cages). The binding of uncoating protein to clathrin chloromethyl ketone) and soybean trypsin inhibitor were from Wortriskelions is a complex equilibrium thatinvolves the thington. All other chemicals and buffers were as described in the interaction of uncoating protein with at least two dis- previous paper (1). tinct sites on the clathrin molecule. Limited proteolysisUncoating ATPase and clathrin were prepared from bovine brain dissectedclathrininto two domains,eachofwhich as previously described (2). In later preparations crude membranes, contained distinct binding sites. Binding to one of these from which clathrin is extracted, and cytosol, from which uncoating sites, located on the proximal leg of a triskelion, was enzyme is purified, were obtained from the same brain homogenate. dependent upon the presence of light chains and was In this case, bovine brains werehomogenized in 1.5 volumes of isotonic Hepes' Buffer containing 150 mM NaCl, 10 mM Hepes, pH unstable togel filtration. Binding to the second kind of site, located on the distal portion of a triskelion leg, 6.5,0.5 mM MgCl,, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride. Clathrin and uncoating protein were then purified from was stable togel filtration and was independent of the crude membrane and cytosol factors, exactly as described. Preparapresence of light chains. tion of brain cytosol in isotonic Hepes Buffer gave -50% higher yields of uncoating protein compared to standard preparations of cytosol using 25 mM Tris (pH 7.0),250 mM sucrose. Uncoating protein and clathrin were radiolabeled as before (1).Clathrin light chains were Uncoating protein utilizes the energy of ATP hydrolysis to prepared by boiling clathrin (3,4). Assays-ATPase assays were performed exactly as described in the actively disrupt clathrincages, transiently displacing portions previous paper and elsewhere (1, 5). Clathrin release assays were of the triskelion from the cage lattice (1).Then, uncoating performed using cages reassembled from [3H]clathrin(2). When protein rapidly captures the displaced triskelion by binding unlabeled clathrin cages or proteolyzed cages were used as substrate, to it, preventing its reassembly into the lattice (1).When all clathrin release was determined by electrophoresis of supernatants points of attachment of a triskelion to a cage have been and pellets on SDS-polyacrylamide minigels. Coomassie Blue-stained severed, the triskelion is released as a stoichiometric complex gelswere quantitated from densitometric tracings (2, 6). Binding assays were performed, using [3H]uncoating protein as described (1, with uncoating protein (1). It would thus appear that uncoating protein might be con- 7). Bulk Preparation of Uncoating Protein-Clathrin Complexessumed during asingle round of uncoating as itoccurs in uitro. Clathrin triskelions, 1mg, and 'H-labeled uncoating protein, 600 pg, Is this the case? How stable are these complexes? Are addi- were incubated at 37 "C for 45-60 min in 250 pl of Buffer B, pH 7.8, tional components or energy input required to recycle un- containing 50 p~ ATP. The incubation mixture was chilled to 4 "C and loaded onto a Bio-Gel A-1.5m column (22 X 0.8 cm) equilibrated coating protein and clathrin from complexes? Or, are the uncoatingprotein-clathrin complexes the products of a readily in Buffer B, pH 7.0, at 4 "C. The column was eluted at 1.5 ml/h, collecting 250-pl fractions. Fractions from the leading edgeof the reversible binding so that uncoatingproteincanspontavoid peak werepooled. Uncoating protein-clathrin complexes preneously recycle for additional rounds? pared in this way contain approximately 0.7 mol ofuncoating protein/ In thispaper, we report that uncoating protein in fact binds clathrin heavy chain. These complexes were stable to storage a t 4 "C to clathrin in areversible equilibrium, Recycling of uncoating for a t least 36 h and could be stored frozen at -70 "C for at least 48 protein is a facile and spontaneous process that occurs because h. Recovery of clathrin was 40-60%. Proteolysis of Clathrin Cages-Proteolysis of clathrin cages to yield uncoating protein has a markedly higher affinity for cages domains of clathrin was performed as described elsewhere (8,9). than for triskelions. The binding equilibrium is complex and two Briefly, clathrin cages (1-1.5 mg/ml) were incubated with L-1-tosreflects the distribution of uncoating protein between two ylamido-2-phenylethyl chloromethyl ketone-treated trypsin (1%by kinds of binding sites of similar affinity present on each weight of clathrin) for 30 min at 37 "C. Digestion was stopped by clathrin triskelion. Limited proteolysis dissects clathrin into addition of soybean trypsin inhibitor (twice the weight of trypsin two domains, each of which has a binding site for the uncoat- present). Trypsin-digested cages (containing a family of 110-kDa tryptic fragments) were collected by sedimentation in a Beckman * This work was supported by National Institutes of Health Grant Airfuge at 20 p.s.i. for 10 min and resuspended in Buffer A containing GM-25662. The costs of publication of this article were defrayed in 10 pg/ml of trypsin inhibitor. A smaller family of tryptic peptides (40 part by the payment of page charges. This article must therefore be The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperahereby marked "advertisement" in accordance with 18 U.S.C. Section zineethanesulfonic acid EGTA, ethylene glycol bis(@-aminoethyl 1734 solely to indicate this fact. $Present address: Department of Cell Biology, Yale University ether)-N,N,N',N'-tetraaceticacid SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. School of Medicine, P. 0.Box 3333,New Haven, CT 06510.
10050
Binding of Uncoating Protein to Clathrin
10051
and 50 kDa) were released into the supernatant. Thereleased proteolysis products were concentrated by precipitation in 50% saturated (NH,),S04, resuspended in Buffer B containing 10 pg/ml of trypsin inhibitor and dialyzed against 50 volumes of Buffer B. Clathrin light chains, which are degraded during proteolysis, could be added back to trypsin-digested cages (9). Light chains (at a ratio of 0.6 mg/mg of clathrin) were incubated for 1 h on ice with trypsintreated cages. Reconstituted, light chain-containing trypsin-treated cages werecollected by sedimentation inthe airfuge and resuspended in Buffer A containing 10 pg/ml of trypsin inhibitor. Other Methods-Protein was determined as described by Bradford (lo), using bovine serum albumin as the standard. SDS-PAGE was by the procedure of Laemmli (11).
1
I
1
2
RESULTS
Recycling of Uncoating Protein-Two approaches were taken to characterize the stability of cage-derived complexes of clathrin with uncoating protein and to determine if the uncoatingproteintheycontainis available for additional rounds of uncoating. First, the complexes were isolated and then tested for the ability of the bound uncoating protein to catalyze further rounds of uncoating. Second, the stability of these complexes to furtherincubation was examined. We found that uncoating protein present in complexes had exactly the same specific activity as free, unused uncoating protein, measured either by clathrin release (Fig. lA)or cage-dependent ATP hydrolysis (Fig. 1B). Thus, eitheruncoating protein retains full activity while bound to triskelions or it can dissociate from the triskelions, recycling to thecages. The latter possibility was suggested by the second approach, in which the isolated cage-derived clathrin-uncoatingprotein complexes were reincubated under assay conditions identical to those in which they had initially been formed (30 min at 37 "C). Following this incubation, only about 30% of the uncoating protein still remained bound to clathrin (Fig. 2b) as judged by gel filtration. In contrast, all but a trace of the uncoating protein remained bound to triskelions when the complexes were kept at 4 "C (Fig. 2a). Together these data demonstrate that the uncoating protein acts asan enzyme in that it is capable of recycling from its product (triskelions) to its substrate (cages). To explain this recycling, it seems necessary to postulate that, although uncoating protein binds to triskelions, it prefers cages. When allof the cage substrate hasbeen dissociated (in an ATP-dependent process), then the uncoating protein will be associated with the triskelions, since there isno longer an alternative. However, as soon as fresh cages are supplied, uncoating protein once again rapidly transfers to this preferred substrate. These inferences are supported by several lines of evidence. First,there is no detectable lag in the initiation of a second round of uncoating when uncoating protein is added as a complex. Second, even a 5-fold weight excess of triskelions fails to detectably inhibit the interaction of uncoating protein with cages, as measured by the elicited ATP hydrolysis (data not shown). Third, theK, for cages (as measured in both the clathrin release and ATPase assays (2, 5)) is 0.19 pM clathrin (moles of triskelions). Fig. 3 shows the fraction of uncoating protein bound to triskelions as a function of the concentration of clathrin. The binding constant is 0.9 ~ L clathrin M (as moles of triskelions). Thus, the apparent affinity of uncoating proteinfor clathrin assembled into cages is almost five times higher than that for unassembled triskelions. Nature of the Binding Equilibrium-We were struck by the finding that even when a vast excess of clathrin is added, no more than about half of the uncoating protein isrecovered in complex with clathrin following gelfiltration chromatography at 4 "C (Figs. 3 and 4). To explain this, it is possible that the
0
UNCOATING ATPase (pg) I
20
-
15
-
1
-
(B)
0
10
20
TIME (min)
FIG. 1. Clathrin release and ATPase activities of bound and free uncoating enzyme. Uncoating protein-clathrin complexes were prepared as described under "Experimental Procedures" using trace labeled 3H-labeled uncoating protein to facilitate quantitation of bound enzyme. Panel A , clathrin release activity of bound (0)and 3H-labeled free (0)uncoating enzyme.Assays contained 10pgof empty cages, 2.5 nmol of ATP, and theindicated amount of uncoating enzyme in 50 pl of Buffer B. Incubations were for 15 min at 37 "Cas described elsewhere (2). Panel B, cage-dependent ATPase activities of bound (0)and free (0)uncoating enzyme. Assayscontained 0.4 pg of uncoating enzyme, 5 pg of empty cages, and 80 pmol of [3H]ATP in 20 pl of Buffer B and were performed exactly as described (5).
binding process is a complex equilibrium involving more than a single step ormore than a single binding site. Alternatively, the uncoating protein could be heterogeneous with respect to the binding process. The latter possibility was eliminated by examining the binding activity of the unbound uncoating protein and testingwhether the equilibrium achieved in Figs. 3 and 4 could be approached equally from both directions. The population of uncoating protein, which did not bind to excess clathrininafirst incubation, was able to bind to clathrin in a second incubation to the same extent as the starting uncoating protein (Table I). The population of uncoating protein released from complexes during reincubation at 37 "C (Fig. 2b) also bound to clathrin to the same extent as the startinguncoating protein (Table I). Complexes which survived this first reincubation could be reincubated for a second time at 37 "C, and the uncoating protein once again equilibrated between free and bound forms to about the same extentas before (Table I). In each of these incubations, uncoating protein and clathrinwere kept at thesame concentration to avoid any possible concentration effects. Uncoating protein exists as a mixture of monomers and dimers, both ofwhich are active (2). The dimers can be
Binding of Uncoating Protein to Clathrin
10052
”-I
n
a) no 37O incubation
a 6 0
5
4
z
2
B w
u
x
0
1
2
3
I
I
I
I
I
4
5
6
7
0
ICLATHRIN] @MI
0
4
8
12
16
20
24
FRACTION NUMBER FIG. 2. Stability of uncoating protein-clathrin complexes. Uncoating protein-clathrin complexes were prepared as described under “Experimental Procedures” and concentrated by precipitation with 50% saturated (NH&SOI. Pam1 a, uncoating protein-clathrin complexes rechromatographed on Bio-Gel A-1.5m after storage for 24 h a t 4 “C. Panel b, uncoating protein-clathrin complexes rechromatographed after incubation for 30 min at 37 “C in Buffer B containing 50 pM ATP.
quantitatively cross-linked with glutaraldehyde under conditions in which monomers are not cross-linked at all (2). To see whether uncoating protein was bound to clathrin as monomers or dimers, the [3H]uncoatingprotein-clathrin complexes were cross-linked with glutaraldehyde at 37 “C as described (2). No uncoating protein was cross-linked into dimers, implying that the bound form is entirely monomeric, even at theearliest time points (data not shown). Also, none of the bound uncoating protein was cross-linked to clathrin, facilitating the interpretations of the experiment. Two Kinds of Binding Sites on Clathrin-The simplest explanation for the partial binding of uncoating protein would be an artifact of the gel filtration analysis. That is, although all of the uncoating protein would actually be bound during the incubation with excess clathrin, only approximately half would be bound in such a way as to survive the gel filtration procedure. If so, there would then be at least two modes of binding to clathrin, most simply thought of as two kinds of binding sites. Binding of uncoating protein to one kind of site would form a complex that dissociates slowly at 4 “C and thus would be stable during gel filtration. Binding to the second kind of site would be unstable at 4 ”C, so that dissociation would occur during analysis by gel filtration. Even though all of the uncoating protein would be bound in the presence of excess clathrin, only that population of uncoating protein bound to the“stable” siteswould survive gel filtration and be
FIG. 3. Effect of clathrin concentration on uncoating protein-clathrin binding. Binding assays containing 2.5 pg (36 prnol) of [3H]uncoating protein, 1.25 nmol of ATP, and the indicated concentrations of clathrin triskelions in 25 p1 of Buffer B, pH 7.6, were performed as described under “Experimental Procedures.” The double reciprocal plot of the data, expressed as theconcentration of clathrin bound &e. the concentration of uncoating protein-clathrin complexes) uersus the concentration of free clathrin, is shown in the inset.
V 0 2 3
* 00
10
O
20
30
X
TRISKELIONS : UNCOATING PROTEIN (moles per mole)
FIG. 4. Uncoating protein binding in the presence of excess clathrin. [3H]Uncoatingprotein, 1 pg, was incubated for 40 min at 37 “Cin 25 pl of Buffer B containing 50 PM ATPrS and increasing concentrations of clathrin triskelions. Uncoating protein binding was analyzed as described under “Experimental Procedures.” The molar ratio of clathrin to uncoatingprotein is expressed as 180,000M,heavy chains to 70,000 M,uncoating protein monomer.
measured. This model would further require that theuncoating protein have similar affinity for the two kinds of binding sites at 37 “C. Only then would the equilibrium established during incubation at 37 “C give rise to anapproximately even distribution of uncoating protein between the stableand “unstable” sites, consistentwith the data in Fig. 4. If the putative stable and unstable sites were to reside on different domains of the clathrin molecule, it might be possible to separate them by proteolytic dissection. As shown in Fig. 5, limited proteolysis of cages using trypsin dissects the clathrin heavy chaininto two distinct domains; the light chains are rapidly degraded. One domain consists of a family
Binding Clathrin of Uncoating toProtein
10053
TABLE I The uncoating protein-clathrin binding reaction is a readily reversible equilibrium The various populations of uncoating protein and uncoating protein-clathrin complexes were prepared by gel filtration chromatography on Bio-Gel A-1.5m columns following 30-min incubations at 37 “C asdescribed in Fig. 2 and under “Experimental Procedures.” Uncoating protein-clathrin association reactions (Reactions 1, 2, and 4) and dissociation reactions (Reactions 3 and 5) contained approximately 15 pg of clathrin, 1 pgof [3H]uncoating Drotein. and 1.0 nmol of ATP in 20 rrl of Buffer B. Species incubated
32.5
2. Unbound [3H]uncoating protein from 1 + triskelions 3. [3H]uncoating protein-clathrin complexes from 1
30.6 32.6
4. Released [3H]uncoating proteinfrom 3 + triskelions 5. Remaining [3H]uncoating protein-clathrin complexes from 3
31.8 43.8
205 1189468-
46 -
28-
a
b
c
d
Species isolated
3’6 of total
+ triskelions
1. [3H]uncoating protein
Uncoating protein bound
Unbound [3H]uncoatingprotein and [3H]uncoating protein-clathrin complexes None Released [3H]uncoating protein and remaining [3H]uncoating proteinclathrin complexes None None
e
I
-Clathrin -110 kDa
-50 kDa 40 kDa Light Chains
4-
1
I
I
I
1
I
I
a) Intact Triskelions
1
z
W
FIG.5. SDS-polyacrylamide gel of proteolytic domains of clathrin. Empty cages (1 mg/ml) were incubated with trypsin (0.01 mg/ml) for 30 min a t 37 “C. The proteolytic domains of clathrin were isolated as described under “Experimental Procedures.” Lane a, intact clathrin (12 pg); lane b, terminal domain fragments (12 pg); lane c, truncated triskelions (12 pg); lane d, truncated triskelions reconstituted with intact light chains (12 pg); lane e, clathrin light chains (4 pg), prepared by boiling purified clathrin triskelions as described (3, 4). Molecular weight markers used and theirmolecular weights, were myosin (205,000), Escherichia coli @-galactosidase(118,000), phosphorylase b (94,000), bovine serum albumin (68,000), ovalbumin (46,000). and carbonic anhydrase (28,000). The gel was stained with Coomassie Blue.
+ 0 Ey
z:
I
a €
y”
4-
321
1
0
p, \:
-
I
fJl
of closely similar cleavage products of the heavy chain (-110 kDa) which are derived from the proximal portion of the triskelion leg and which remain assembled into cages (8, 9). This domain retains a trimericstructure and contains binding sites for light chains (9). Thus, light chains can be quantitatively rebound to this domain to generate “truncated triskelions.” Two smaller cleavage products (-40 and -50 kDa) are derived from the distal portionof the triskelion leg, designated the “terminal domain” (12) and are released from cages following proteolysis (8,9). The truncated triskelions and the terminal domains of clathrin were prepared and each tested for its ability to bind uncoating protein. Binding of uncoating protein to the truncated triskelions could not be detected by gel filtration (Fig. 6), even though the truncated triskelions were well resolved from free uncoating protein. This would mean either that there was no binding, or that thecomplexes had formed but had all disassociated during the gel filtration. To help resolve this ambiguity, we tested the ability of truncated triskelions to inhibit thebinding of uncoating protein to thestable sites on intact triskelions,using gel filtration analysis. Fig. 7 (dashed line) shows the curve predicted for inhibition if the truncated triskelions have the same affinity as intact triskelions for the uncoatingprotein. The truncated triskelions
I
Chains
3
&
m -X
I
b) Truncated Triskelions + Light
0 0
4
8
12
16
20
24
FRACTION NUMBER
FIG. 6. Truncated triskelions as substrates for uncoating protein binding. Light chain-containing truncated triskelions were prepared and binding assays were performed as described under “Experimental Procedures.” Shown are the elution profiles from BioGel A-1.5m columns. Panel a, 3H-uncoating-protein binding to intact triskelions. Panel b, 3H-uncoating-protein binding to light chaincontaining truncatedtriskelions.
(closed triangles) indeed inhibit binding in the expected fashion. This inhibition requires the light chains, since truncated triskelions prepared without light chains do not inhibit (open triangles). It is important to note that binding occurs very slowly, if at all, at 4 “C and so the uncoating protein can not re-equilibrate between binding sites during the gel filtration analysis (cf. Fig. 2a). These results strongly suggest that truncated triskelions containlightchain-dependent binding sites for uncoating protein that arepurely of the unstable type, i.e. that dissociate during gel filtration. This is intriguing since clathrin cages have also been shown to contain a light chain-dependent site for interaction with uncoating protein, asite thatelicits ATP hydrolysis (7). Binding to this siteis also transient (1). We next examined whether the terminal domain has bind-
Binding of Uncoating Protein to Clathrin
10054 n
r
I
1
I
I
I 1
PROTEOLYTIC FRAGMENT : INTACT TRlSKELlON (moles per mole)
FIG. 7. Inhibition of uncoating protein binding to intact triskelions by proteolytic domains of clathrin. The various proteolytic domains of clathrin wereprepared as describedunder "Experimental Procedures." Incubations were for 20 min at 37 "C and contained intact triskelions (15 pg), [3H]uncoatingprotein (2 pg), and 1.25 nmol of ATP in 25 pl of Buffer B, pH 7.6. Assays were performed as described under "Experimental Procedures." The dashed line is the predicted curve for inhibition of uncoating protein binding to intact triskelions by a competitor which has the same affinity for uncoating protein. Data points shown are for truncated triskelions (A), truncated triskelions containing light chains (A), and terminal domain fragments (0).
ing sites for uncoating protein by testing for its ability to inhibit binding to triskelions. Terminal domains also inhibited binding in the predicted fashion (Fig. 7, open circles). The binding of the 70-kDa uncoating proteinto either the 40or 50-kDa terminal domain fragments could not be directly analyzed by gel filtration because of the similarity in molecular weights. Because of the stoichiometric inhibition, it seems likely that theterminal domains also possess binding sites for uncoating protein. Thesesites would not be light chaindependent, since there are no light chains in the terminal domain. The close fit of these data to the predicted curve (Fig. 7) indicates that the uncoating proteinhas approximately the same affinity for the sites in bothtruncated triskelions and terminaldomains. It is likely that the binding sites on truncated triskelions are indeed distinct from those in terminal domains and do not represent cross-contaminationof proteolytic domains for two reasons: first, the two sites are differentially dependent on the presence of light chains; second, since the two preparations of fragments are equally effective inhibitors, there would have to be 50% cross-contamination between the two domains. Analysis by SDS-PAGE (Fig. 5) indicates that the two domains are almost completely resolved by sedimentation. These two domains of clathrin also differ in their ability to inhibit cage-dependent ATP hydrolysis by the uncoating enzyme. The uncoating ATPase activity was inhibited by the terminal domain of clathrin (Fig. 8, open circles), but not by light chaincontaining-truncatedtriskelions (Fig. 8, closed triangles). The basis for this interesting difference is not yet clear. Nonetheless, it seems likely that the terminal domain inhibits by sequestering the uncoating protein from its cage substrate, thereby reducing the rate of ATP hydrolysis. Indeed, as wouldbe predicted from the 5-fold difference in affinity (Fig. 3), a 5-fold molar excess of terminal fragments to clathrin in cages wasnecessary to achieve a 50% inhibition of cage-dependent ATPase activity. The initial stages of the uncoating process require the transient interaction of uncoating protein with a light chaincontaining site on the cage substrate to elicit ATP hydrolysis (1). Therefore, cages comprised of light chain-containing truncated triskelions were tested to determine if they were sufficient to act as substrate for the uncoating enzyme. In
0
2
4
6
8
10
1 2 1 4
PROTEOLYTIC FRAGMENTS : CLATHRIN (moles per mole)
FIG.8. Inhibition of cage-dependent ATP hydrolysis by the uncoating protein in the presence of proteolytic fragments of Buffer B, were performedexactly clathrin. ATPase assays, in 20 pl of as described (5) and contained empty cages, 4 pg, uncoating enzyme, 0.4 pg, 80 pmol of [3H]ATP, and the indicated amount of light chaincontaining truncated triskelions (A) or terminal domain fragments (0).Incubations were for 20 min at 37 "C.
'C 0
-k 20 O
w
N
>
i
2n
10
>
I
ea
0 0
10
20
TIME (rnin) FIG. 9. Trypsin-digested cages as substrates of uncoating enzyme for cage-dependent ATP hydrolysis. ATPase assays contained the indicated cage substrate (4 pg), uncoating enzyme (0.5 pg) and 80 pmol of [3H]ATP in 20 pl of Buffer B. 0, control empty cages; A, trypsin-digested cages; A, trypsin-digested cages reconstituted with light chains.
fact, however, these trypsin-digested, light chain-containing cages were unable to elicit ATP hydrolysis by the uncoating enzyme (Fig. 9); nor were they dissociated in the presence of uncoating protein and ATP (data not shown). DISCUSSION
The uncoating protein is a very abundant protein,comprising -1% of total cytosolic protein in bovine brain. Clathrin is also abundant in brain, comprising 0.1-0.5% of total protein (14,15). Since -20% of clathrin is membrane-bound in brain (15), cytosolic clathrin would represent 0.2-1% of total cytosolic protein. Thus, the molar ratio of uncoating protein to cytosolic clathrin would be at least 3:l in brain. This amount of uncoating protein would be sufficient to saturate allof the binding sites on cytosolic clathrin. The existence of a readily reversible equilibrium that would allowthe rapid dissociation of uncoating protein at these siteswould bea great advantage. When no other protein is available to bind or utilize clathrin, uncoating protein would keep the clathrin sequestered, preventing it from reassembling unproductively or binding to membranes in an uncontrolled fashion. Yet, as soon as proteins with a higher affinity for, or slower dissociation from,
Binding Cluthrin of Uncoating toProtein clathrinare made available, uncoatingprotein wouldbe quickly displaced and thecytoplasmic pool of clathrin rapidly mobilized, perhaps for coated-pit assembly. Our studies have revealed that a rapidly reversible, albeit complex, equilibrium indeed defines the interaction of uncoating protein with clathrin. Uncoating protein can rapidly and spontaneously dissociate from triskelions, and preferentially transfer to cages when they are present. This spontaneous recycling establishes that the uncoating protein is an enzyme, capable of repeated rounds of action. Its marked preference for cages is entirely appropriate for an uncoating enzyme whose substrate is a cage and whose product is a triskelion. Binding involves at least two different kinds of interactions between uncoating protein and clathrin triskelions. The uncoating protein interacts transiently with clathrin at a site requiring light chains located on the proximal leg of triskelions. The rapid dissociation of uncoating protein from these unstable binding sites precludes detection bygel filtration assays. The second kind of interaction of uncoating protein with clathrin is stable to gel filtration and seems to occur at a location on the terminal domain of the triskelion legs. Light chains are not a component of this stable binding site. To explain how the two kinds of sites can have similar affinities but very different dissociation rates, we envision that the rates of association and dissociation are both much faster for the unstable sites than for the stable sites. The ratio of these rates to each other (i.e. the equilibrium constant) would be similar for the two sites, so that these two sites would have similar affinity for the uncoating protein.Therefore, following an incubation at 37 "C, the uncoating protein would be approximately equally distributed between the two sites. Most likely, the "dissociation" observed during reincubation of complexes (Fig. 2b and Table I) is only apparent. In actuality,the isolated complexes would contain uncoating protein bound only to thestable sites. When thesecomplexes are reincubated at 37 "C, uncoating protein redistributes between the stable and unstable sites. Upon gel filtration (Fig. 2 b ) , those uncoating protein molecules that had redistributed to unstable sites would now dissociate. Thus, even the stable sites must dissociate rapidly at 37 "C. Therefore, the term stable refers only to the slow dissociation of these complexes at 4 "C. The unstable sites would dissociate much more rapidly at 4 "C, and (presumably) at 37 "C. In the previous paper we showed that theuncoating process can be separated into two stages. Recognition of cages in the first stage requires the presence of light chains and results in ATP-driven displacement of a portionof a clathrintriskelion. Uncoating protein captures the displaced triskelion, in the second stage, by binding to a light chain-independent site previously buried in the cage lattice (1). Because the first stage involves transient interactions that require light chains and the second stage produces complexes of clathrinand uncoating protein that are stable to gel filtration, it seems likely that the light chain-dependent binding site on the truncated triskelions is used in the first stage of uncoating, while the binding in the second stage would involve the site isolated in the terminal domain. The rapid dissociation from the light chain-dependent site would then facilitate the capture of the terminal domain by uncoating protein afterATPdependent displacement. Clearly, additional evidence is needed to test the correctness of this proposition. Note that even if this assignment is valid, the light chain-containing site in cages would have to be more complex than the light chain-containing site in triskelions, since the former elicits ATP hydrolysis, while the latter does not (5, 7).
10055 Assuming that the light chain-containing site of truncated triskelions is part of the initial recognition site on cages for uncoating enzyme, it must be incomplete in that cages composed of light chain-containing truncated triskelions are not recognized by uncoating ATPase (Fig. 9). There are several possible explanations. It is possible that, although trypsindigested heavy chains still bindlight chains, they do not bind them in the correct manner. A second possibility is that proteolysis has destroyed a portion of the recognition site on the heavy chain. However, both of these possibilities seem unlikely, since light chains can restore uncoating protein binding when added back to truncated triskelions (Fig. 7). The third, more intriguing possibility is that the terminal domains are needed to take part in the ATP-dependent displacement reaction. Even though the binding site of the terminal domains is believedto be latent touncoating protein in cages, the domain might be necessary to allow displacement initiated at thelight chain binding site. Note that in the cage structure, three terminal domains from next-nearest neighbors are juxtaposed at each vertex (12, 13), quite close to where the light chains are thought to reside (16, 17). This arrangement only occurs in cages (which have next-nearest neighbors), and notfree triskelions. Thus, if terminal domains were needed as part of the ATP-dependent displacement mechanism, this mechanism could operate only in cages and not with unassembled triskelions. This would explain why cages but not triskelions elicit ATP hydrolysis by uncoating protein (5) and of course also explains why cages lacking terminal domains resist enzymatic uncoating and do not elicit ATP hydrolysis. The interaction of uncoating protein with distinct sites on clathrin has been inferred from the ability of each of the dissected regions to inhibit uncoatingproteinbinding to intact triskelions. It is unlikely that thisinhibition, or the inhibition of ATPase activity (Fig. 8), was due to proteolysis during the subsequent incubations, for several reasons: (a)trypsin inhibitor was present in excess of any residual trypsin throughout the incubations; ( b ) inhibition of binding by truncated triskelions was light chain-dependent, indicating a specificity not attributable to proteolysis; ( c ) SDS-PAGE analysis following standard incubations showed no detectable proteolysis of uncoating protein or of the highly sensitive light chains (data not shown). The nature of the binding process is likely to be more complex than can be accounted for by only two independent sites in clathrin for uncoating protein. Light chains are a necessary component of the unstable type of binding sites on truncated triskelions. If all unstable sites in triskelions were similarly light chain-dependent, then light chain-free triskelions would represent apure population of stable sites. In that case, all of the uncoating protein should remain bound to clathrin after gel filtration when uncoating protein is incubated with an excess of light chain-depleted triskelions. In contrast, the same amount of uncoating protein remains bound to a 15-fold excess of light chain-dependent triskelions after gel filtration as with intact triskelions (data notshown). Although this result does not negate the basic idea that there are stable and unstable sites in different parts of the triskelion, it does imply that additional interactions and/or equilibria are also involved. Acknowledgments-We wish to thank Dr. Roger Kornberg for his incisive interpretation of the initial binding experiments, and Drs. F. Randy Bryant, Donald Engelman, Peter Kim, I. Robert Lehman, and Michael O'Donnellforhelpful discussions. Dr. Suzanne Pfeffer is gratefully acknowledged for herpatient editing of this manuscript.
Binding Clathrin of to Uncoating Protein
10056 REFERENCES
1. Schmid, S. L., and Rothman, J. E. (1985) J. Biol. Chem. 260, 10044-10049 2. Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. J. Cell Biol. 99, 723-733 3. Lisanti, M. P., Shapiro, L. S. Morowitz, N., Hua, E. L., Puszkin, S., and Schook, W. (1982) Eur. J. Biochem. 125,463-470 4. Brodsky, F. M., Holmes, N. J., and Parham, P. (1983) J. Cell Biol. 96,911-914 5. Braell, W. A., Schlossman, D. M., Schmid, S. L., and Rothman, J. E. (1984) J. Cell Biol. 99, 734-741 6. Patzer, E. J., Schlossman, D. M., and Rothman, J. E. (1982) J. Cell Biol. 93, 230-236 7. Schmid, S. L., Braell, W. A., Schlossman, D. M., and Rothman, J. E. (1984) Nature 311, 228-231
8. Schmid, S. L., Matsumoto, A., and Rothman, J. E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 91-95 9. Ungewickell, E., Unanue,E. R., and Branton, D. (1982) Cold Spring Harbor Symp. Quant. Biol. 46, 723-731 10. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 11. Laemmli, U. K. (1970) Nature 227,680-685 12. Kirchhausen, T., and Harrison, S. C. (1984) J. Cell Biol. 99, 1725-1734 13. Crowther, R. A., and Pearse, B. M. F. (1981) J. Cell Biol. 91, 790-797 14. Pearse, B. M. F. (1975) J. Mol. Bwl. 97,93-98 15. Goud, B., Huet, C., and Louvard, D. (1985) J. Cell Biol. 100, 521-527 16. Kirchhausen, T.,Harrison, S. C., Parham, P., and Brodsky, F. M. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,2481-2485 17. Ungewickell, E. (1983) EMBO J. 2, 1401-1408
