Structural characterization of labeled clathrin and coated vesicles

ARCHIVES

OF BIOCHEMISTRY

Vol. 235, No. 2, December,

Structural

AND BIOPHYSICS

pp. 403-410, 1984

Characterization

of Labeled Clathrin and Coated Vesicles

K. PRASAD, A. ALFSEN, R. E. LIPPOLDT, P. K. NANDI, Clinical

Endocrimlogy

Branch, NIADDK,

National

Institutes

AND

H. EDELHOCH’

of Health, Bethesda, Ma&and

20205

Received August 2, 1934

Clathrin (8 S) and coated vesicles have been covalently labeled by using the sulfhydryl-labeling fluorescent probe N-(1-anilinonaphthalene)maleimide. A large increase in energy transfer from Trp to anilinonaphthalene (AN) residues was observed in clathrin in the pH range -6.5-6.0, where the rate of clathrin selfassociation increased rapidly. The change in energy transfer was indicative of a conformational rearrangement, which could be responsible for the initiation of the clathrin self-association reaction to form coat structure. The AN label was found in both the coat and membrane proteins after dissociation of coated vesicles at pH 8.5. The labeled coat and membrane proteins readily recombined to form coated vesicles after reducing the pH to 6.5, indicating that the labeling did not interfere with the ability of clathrin to self-associate and interact with uncoated vesicles to form coat structure. A comparison of the AN fluorescence with the Coomassie blue pattern after electrophoresis in sodium dodecyl sulfate-gels revealed that a 180,000-Da protein (clathrin) was mainly labeled in coated vesicles, while a llO,OOO-Da protein was also strongly labeled in uncoated vesicles. AN-labeled baskets and coated vesicles have been prepared. Trypsin digestion reduced the sedimentation rate of baskets from 150 S to 120 S and of coated vesicles from 200 S to 150 S. Gel electrophoresis of baskets and coated vesicles showed extensive conversion of clathrin (M, 180,000) to a product of M, g 110,000, suggesting equivalent structural organization of the coat in coated vesicles as in baskets. In both cases, the peptide released from the vesicles by digestion were essentially free of fluorescent label. In the case of the uncoated vesicles, tryptic digestion released most of the proteins remaining after coat removal. Q 1x34 Academic

Press. Inc.

Coated vesicles are distinct organelles occurring in all eucaryotic cells, whose function it is to transfer metabolites (including membrane components) either across or between cellular membranes (l5). Little is known about the mechanism by which clathrin forms a coat on the cytoplasmic side of the membrane, or about the signal which transforms coated pits to coated vesicles (6). Both types of coated structures are readily recognizable in electron micrographs by their characteristic polygonal coat structure (2, 7). Several proteins, clathrin, a llO,OOO-Da molecule, and light chains appear to be 1 To whom correspondence

should be addressed. 403

involved in the formation of coat structure (8-10). With the isolation of homogeneous preparations of CVs,’ it has been possible to compare the association of coat protein(s) with vesicles with its self-association in the absence of vesicles (11, 12). Though the pH and concentration dependence of the two processes of coat formation are similar, their dependence on 2 Abbreviations used: ANM, (N-(l-anilinonaphtha1ene)maleimide; AN, anilinonaphthalene; Mes, 4morpholineethanesulfonic acid, NaDodSO1, sodium dodecyl sulfate; CV, coated vesicle; UV, uncoated vesicle; DPH, 1,6-diphenyl-1,3,5-hexatriene; EGTA, ethylene glyeol his@-aminoethyl ether)-N,N’-tetraacetic acid. 0003-9861/&I Copyright All rights

$3.00

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

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404

ionic strength is very different (12). In order to continue our characterization of the properties of coat proteins and uncoated vesicles and their interactions, we have attached the covalent label, N-(lanilinonaphthalene)maleimide, to a few sulfhydryl groups in clathrin baskets, and coated and uncoated vesicles. This fluorochrome provides an extrinsic, highly fluorescent label which permits some new types of experiments and, in some cases, greater sensitivity in detecting protein components. By sedimenting CVs, UVs, and baskets and their dissociation and digestion products on sucrose gradients, we have been able to analyze for the various components present in solution. In this way we can account for all the molecular species present and not just the predominant species, as seen, for example, by electron micrography. Moreover, each species can be isolated from the gradient and analyzed independently for its chemical or protein composition. MATERIALS

AND

METHODS

Chemicals. N-(1-Anilinonaphthalene)maleimide (ANM) was obtained from Polysciences. 1,6-Diphenyl1,3,5-hexatriene (DPH) was from Aldrich Chemicals. Sucrose and Tris were purchased from Bethesda Research Laboratories. Mes and soybean trypsin inhibitor were Sigma products. NaDoSOl was purchased from BDH. Trypsin was obtained from Worthington. Preparatimz of coated vesicles. The method of preparing coated vesicles is described in detail elsewhere (13). This procedure was based on the one developed by Pearse (14). Preparation of uncoated vesicles and clathrin. Coated vesicles (CV), prepared in 0.10~ Mes, pH 6.5, were dialyzed for -15 h against 0.01 M Tris, pH 8.5, 2 M urea solutions. The solution was centrifuged at 36,000 rpm for 1 h in a Type 70.1 Ti rotor in a Beckman L2-65B centrifuge. The pellet was dispersed in 0.01 M Tris, pH 8.0, in a Dounce homogenizer, and then centrifuged again at 10,000 rpm for 10 min to remove the undissolved pellet. The supernatant was dialyzed for 12 h against the same buffer, and was used directly for uncoated vesicle (UV) experiments. The supernatant of the centrifugation at 36,000 rpm was dialyzed against 0.01 M Tris, pH 8.0, for 12 h to give a preparation of clathrin which was routinely used.

ET AL. Preparation of baskets. Clathrin baskets were made by titrating clathrin (1 mg/ml) with 1 M Mes (pH 5.9) to pH 6.0. This pH ensures almost complete conversion of clathrin to baskets (11). After several hours, the pH was changed to 6.5 by dialyzing against a solution of 0.10 M Mes, pH 6.5, for 18 h. Labeling of clathrin baskets was at pH 6.5. Raising the pH from 6.0 to 6.5 did not dissociate the baskets. Tryptic digestion Trypsin digestion occurred at a weight ratio of l/100 for about 8 h at 23°C. The digestion was stopped by addition of a weight ratio of 1.80 soybean trypsin inhibitor. CVs and baskets were digested in 0.10~ Mes, pH 6.5. UVs were digested in 0.01 M Tris, pH 8.0. The digestion products were monitored by NaDodSO,-gel electrophoresis. Reassociation of CV emponents. Recombination of uncoated vesicles (undigested and digested) and clathrin was accomplished by mixing solutions of each component in 0.10 M Tris, pH 8.0, and dialyzing for at least 12 h against a large volume of 0.1 M Mes, pH 6.5, 0.5 mM MgClz, 1 mM EGTA, 3 mM azide. We have used an absorbance ratio (at 280 nm) of 6:l of clathrin to uncoated vesicles as the stoichiometric ratio of these components in CVs. Fluorescence measurements. Fluorescence intensities were measured on a Perkin-Elmer MPF3 Buorometer. Protein concentration was measured from the emission intensity at 340 nm after exciting at 280 nm. Phospholipid was evaluated from the fluorescent intensity of the noncovalent probe DPH in unlabeled preparations. DPH was excited at 366 nm and measured at 430 nm (13). It has been shown that clathrin does not react with DPH. The fluorescence of the product of the sulfhydryl reaction with ANM was excited at 360 and recorded at 430 nm. Labeling of sulfhydryl groups with anilinonaphthalene maleimide. ANM was reacted with CVs and baskets at pH 6.5, and with UVs and clathrin at pH 7.9, for 2 h at 23°C using sixfold molar excess calculated for clathrin subunits (Af, lSO,OOO),following the procedure of Kanaoka et al. (15). The labeled proteins and vesicles were isolated from unbound reagent by chromatography on a G-25 Sephadex column. The number of bound ANM groups was determined from the absorption at 355 nm (e = 1.4 was obX lo4 Me1 cm-i). The protein concentration tained from the absorption at 280 nm in 6 M guanidinium chloride for CVs and UVs, and in aqueous buffer, pH 8.0, for clathrin and baskets. The value %, = 10.9 (16) obtained for clathrin was also of E’% used for CVs, UVs, and baskets. (We have obtained values of 12.6, 12.3, and 10.8 for CVs, UVs, and clathrin, respectively, using the Lowry method to measure protein concentration.) The number of thiol groups which have reacted with ANM is given for 100,000 g protein (see Figure Legends). Sucrose gradient centrifugaticm. The suspensions of CVs or UVs were sedimented on a 10-30s linear

CHARACTERIZATION

OF LABELED

CLATHRIN

AND

COATED

VESICLES

405

-

sucrose gradient in a SW 40 rotor at 27,000 rpm for 110 min in a Beckman Model L2-65B centrifuge (12). The baskets, however, were sedimented on a lo-30% linear sucrose gradient in a SW 2’7 rotor at 24,000 rpm for 110 min. The fractions from the gradient were collected from the bottom of the tube using a peristaltic pump. The elution was monitored by the fluorescence intensities of the various probes. Gel electmphoresis. This procedure has been described previously by us (16).

-

55K

RESULTS

Fluorescent Spectra of AN-Labeled Proteins The labeling of proteins with ANM is due to the formation of a covalent bond between SH groups and the maleimide ring. The emission peak of anilinonaphthalene (AN) label was at 433 nm when bound to clathrin but, was at 430 nm in CVs, UVs, and baskets when excited at 360 nm (Fig.lA). According to the polarity scale given by Kanaoka et al. (15) for ANlabeled N-acetylcysteine in different solvents, the emission at 430 nm corresponds to the solvent of lowest polarity that was I 4

I AN

I

I----

l

II A

III

IV

v

VI

B

FIG. 2. (A) NaDodSO,-gel electrophoresis of ANCV (I), fluorescence; AN-CV (II), Coomassie; and AN-UV, (III) Coomassie. Comparison of CVs (II) and UVs (III) was at the same lipid concentration. (B) AN-UV (IV), fluorescence; AN-UV (V), Coomassie; and AN-UV digest (VI), Coomassie. In (B), the gel has been overloaded with respect to protein.

employed, i.e., tertiary butanol. The environment of the labeled SH groups should be highly hydrophobic in all the above cases.

Gel Electrophoretic Characterization of AN-labeled Proteins

I

1

I

400

440

483

WAVELENGTH

(nm)

FIG. 1. (A) Emission spectrum of AN-clathrin (- - -), AN-baskets (O), AN-CV (0), and AN-UV (-). In all cases the excitation wavelength was 360 nm. There were 2.0, 1.5, 1.5, and 2.5 labels per 100,000 g protein, respectively.

The protein composition of AN-labeled CVs and UVs was analyzed by gel electrophoresis in NaDodS04. Coomassie blue stains of CVs and UVs, using the same amount of lipid, are seen in Fig. 2A, II and III. The gels indicated that about half of the -llO-kDa proteins and most or all of the 55-kDa proteins remained with the UVs after dissociating the coat proteins from the CVs. It has been reported by others (8-10, 20, 21), and we have also found that the standard preparations of clathrin (extracting by raising the pH to 7.5-8.5) contain, in addition to clathrin, mainly the light chains, 30-36,000 Da. The reaction of the proteins in CVs with ANM permitted us to determine those proteins which have the most exposed SH groups. We have, therefore, compared the Coomassie blue and AN

406

PRASAD

fluorescence pattern for CVs (Fig. 2A, I and II) and UVs (Fig. 2B, IV and V) after gel electrophoresis. In accord with that observed by Coomassie blue stain, most of the AN fluorescence of CVs was found in clathrin, i.e., 180-kDa position on the gel, with a much smaller amount in the llO-kDa region. After tryptic digestion of CVs, all of the fluorescence at 180 kDa disappeared, and was largely replaced by a family of fluorescent bands in the region of 110 kDa. The conversion of 180-kDa clathrin in baskets to -llO-kDa proteins by tryptic digestion has been reported (10, 20). In order to observe the proteins remaining in UVs, the gels shown in Fig. 2B were overloaded by comparison with those shown in Fig. 2A. In UVs, a small protein band was observed in the 180-kDa region, in addition to the usual group of bands observed in -llOand 55-kDa regions. After tryptic digestion, almost all of the Coomassie blue-stained protein disappeared, except for small amounts in the 180-kDa region (Fig. 2B, VI). The loss of most of the protein in UVs with digestion seemed surprising, since receptors should not be digested. However, if the amounts of each receptor present were too small, they would not have been observed. The pattern of AN labeling of UVs corresponded approximately to that observed by Coomassie blue staining, except that there was relatively little AN label in the 55,000-Da region (Fig. 2B, IV). Tryptic digestion of UVs resulted in the disappearance of essentially all of the AN fluorescence.

ET AL.

Moreover, the data in 0.01 M Tris were very similar to that in 0.01 M Tris, 0.20 M NaCl, where the polymerization was strongly inhibited. The fluorescence of Trp was slightly quenched in unlabeled clathrin between pH 7.6 and 6.0 (Fig 3). However, AN fluorescence in labeled clathrin increased about 20% in the same pH range, with most of the change occurring between pH 6.5 and 6.0. We have also evaluated the effect of AN labeling on Trp emission. In this case, the degree of Trp quenching in the same pH interval was about fourfold greater than in the absence of label. It appears that the greater quenching resulted from energy transfer to AN groups. This was evident from a corresponding threefold increase in AN fluorescence intensity when Trp was excited. The large increase in energy transfer in clathrin can be explained by a pHdependent conformation change in which either the distances between donors and acceptors or the angles between their transition moments decreased (or a combination of the two possibilities). In the absence of the AN label, the very small

,fiL

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I

I -I

I.”

s G d

1.4

f

1.2

-

TRANSFER

1.0 I

pH Dependence of Fluorescence of AN-Labeled Clathrin The rate of self-association of clathrin (8 S) to form baskets increased rapidly with acidity between pH ‘7 and 6 (16). It is not known whether the rate is controlled by the reduction in net charge of clathrin or by other factors. We have measured the changes in fluorescence intensity of the AN label and the Trp residues of clathrin with acidification in 0.01 M Tris at concentrations of clathrin where selfassociation was too slow to measure.

I

I

I

I

I

6.0

7.0

6.0

PH

FIG. 3. Energy transfer in AN-labeled clathrin. (0) Trp fluorescence(280 - 340 nm) in unlabeled clathrin; (0) Trp fluorescence (280 - 340 nm) in AN-labeled clathrin; (Cl) AN fluorescence (360 - 430 nm); (A) AN fluorescence (280 - 430 nm) in labeled clathrin.

CHARACTERIZATION

OF LABELED

quenching of Trp residues cannot be readily interpreted, since protonation of neighboring histidyl (or carboxylate) groups would also produce quenching. The increase in AN fluorescence also suggests a conformational origin. Its marked enhancement by transfer from Trp residues strongly supports a conformational transition.

CLATHRIN

AND

COATED

VESICLES

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symmetrical curve was obtained with a peak at fraction 19-20. When the pH was raised from 6.5 to 8.5 by dialyzing for 36 h against 0.01 M Tris, pH 8.5, at 4”C, two sedimenting bands appeared with AN fluorescent peaks at fractions 4 and 10. It has been shown previously, using tryptophan and DPH fluorescence as probes for protein and phospholipid, respectively, that fractions 4-5 and lo-11 correspond to protein and UVs (12). There was almost Reassociation of Labeled no lipid in the protein peak, and about Components of CVs 10% of the protein fluorescence appeared in the lipid peak. It is of interest that A. AN-Labeled CVs. We have shown much more of the AN fluorescence was that the dissociation of CVs and the re- seen in the UVs than in the clathrin band, association of UVs and clathrin can be suggesting that either there are more followed conveniently on lo-30% sucrose labeling sites in the former than in the gradients (12). The distribution of AN- latter or that the quantum yield is higher labeled proteins in CVs on a 10-30s gra- for the UV proteins. In the case of Trp dient is shown in Fig. 4. A single, nearly emission of dissociated CVs, much more of the fluorescence is in the clathrin band than in the UVs (see Fig. 5 in Ref. (12)). I I I I When the pH was brought back to 6.5, CVs were reformed with almost the same sedimentation pattern as the native species (Fig. 4). Only about 10-15s of the 1 AN fluorescence remained at the position of the dissociated components. The labeling with AN therefore does not appear to affect the reversibility of the dissociation. IB. AN-Labeled UVs. The sedimentation pattern of AN-labeled UVs was also analyzed by sucrose gradient sedimentation (Fig. 5). The principal protein boundary, as determined by either AN or Trp fluoI rescence, had a peak at fraction 11. However, there were present, in addition, small, slower sedimenting protein boundaries observed by AN and Trp fluorescence. 1 The latter probably represented small amounts of protein which adhered to the suspension of UVs and have slowly dissociated from them. AN-labeled UVs proI I vided a second label and therefore an 4 20 B unambiguous method of following the inFRACTION teraction of UVs with unlabeled clathrin FIG. 4. Reversibility of AN-coated vesicle dissociaor other proteins. In Fig. 5, we see the tion. Sucrose gradient analysis (lo-30%) of native sedimentation pattern of AN-labeled UVs AN-coated vesicles (0, pH 6.5, 0.10 M Mes), of when recombined with unlabeled, native dissociated AN-coated vesicles (- - -, pH 8.5, 0.01 M 8 S clathrin. The peak at fraction Tris), and of reassociated AN-coated vesicles (--, pH 6.5, 0.10 M Mes). Centrifugation was at 27,000 20-21 corresponded to the position of native CVs. rpm for 110 min at 23°C.

408

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ET AL.

similar patterns. When the baskets were dissociated to 8 S clathrin in 0.01 M Tris, pH 8.5, they could be reformed by dialyzing the solution back in 0.10 M Mes, pH 6.5. Tryptic digestion produced a symmetrical sedimenting boundary with a peak at fraction 13 without evidence of a shoulder (Fig. 6). The AN fluorescence pattern closely followed the Trp pattern. A significant fraction of the Trp fluorescence was also present in a slower moving boundary, which did not separate from the meniscus and showed almost no AN fluorescence. This component represented the clathrin fragment(s) split out by trypsin. NaDodSO,-gel electrophoresis of the digested basket preparation showed the El

T FRACTION

FIG. 5. Characterization of AN-uncoated vesicles by AN fluorescence (0) (exe, 360 nm; em, 430 nm), and tryptophan fluorescence (A) (exe, 280 nm; em, 340 nm), by sucrose gradient analysis (lo-30%) in 0.01 M Tris, pH 8.5. Line without points with peak at fraction 21 was drawn from by AN fluorescence analysis, and represents the reassociation of ANuncoated vesicles with native clathrin in 0.1 M Mes pH 6.5. Centrifugation was at 27,000 rpm in a SW40 rotor for 110 min at 20°C. The degree of AN labeling was 2/100,000 g protein.

Tryptic Digestion of AN-Labeled CVs, UV.., and Baskets A. Baskets. It has been shown by Ungewickell et al. (10) and Schmid et al. (20) that, when baskets were digested by trypsin, clathrin was fragmented into species of -110 k and 40-50 kDa. The baskets nevertheless retained their characteristic polygonal coat structure, but the length of the arms of the triskelions were appreciably reduced. We have repeated this experiment using AN-labeled baskets, and have analyzed the sedimentation behavior of digested baskets. The undigested basket preparation gave a symmetrical boundary by sucrose gradient sedimentation, with a peak at fraction 1’7 (Fig. 6). After AN labeling, a small shoulder was observed on the leading edge of the gradient pattern. The principal peak was found at the same fraction as unlabeled baskets (Fig. 6). AN and Trp fluorescence gave very

FRACTION

FIG. 6. (A) Sucrose gradient sedimentation of clathrin baskets in lo-30% gradient in 0.1 M Mes pH 6.5. 0, Trp fluorescence. It should be noted that a different rotor was used for sedimenting baskets, i.e., SW 27. The time of sedimentation was 110 min at 24,000 rpm and 23’C. (B) Sedimentation of ANlabeled baskets. 0, Trp fluorescence; A, AN fluorescence. (C) Sedimentation of trypsin digested ANlabeled baskets. 0, Trp fluorescence; A, AN fluorescence. The degree of AN labeling of baskets was 2 m01/100,000 g protein.

CHARACTERIZATION

OF LABELED

disappearance of most of the 180-kDa clathrin band and all of the light chains (M,. 30-36,000), and the formation of -llOkDa bands and several bands migrating slower than and faster than the light chains, in accord with the results of Ungewickell et aZ. (10) and Schmid et al. (20). In the velocity untracentrifuge, the average sedimentation coefficient of the baskets was reduced from 150 S to 120 S by tryptic digestion. This reduction in sedimentation rate was readily accounted for by the loss of -70 kDa mass from clathrin if the structure remained the same. B. AN-Labeled CVs. It was of interest to see if clathrin in coated vesicles could be similarly transformed by trypsin digestion. CVs were labeled with AN, and were analyzed by sucrose gradient centrifugation. As seen in Fig. ‘7, the AN emission profile paralleled that of Trp, with a peak at fractions 19-20. Digestion with trypsin produced results similar to those observed with baskets. The fluorescent peak of the digested vesicles was at fractions 15-16, a shift of four fractions from that of undigested CVs. A second boundary was found with a peak at fraction 3, which showed Trp but no AN fluorescence. NaDodSO,-gel electrophoresis of digested CVs showed changes similar to those observed with baskets, i.e., the loss of clathrin and light chains and the appearance of --IlO-kDa bands and faster migrating components. The shift in sedimentation rate observed on sucrose gradients after digestion was also observed by velocity ultracentrifugation. The sedimentation rate of CVs decreased from -200 S to 150 S after tryptic digestion. CT.AN-Labeled UV... Digested UVs (unlabeled) gave a symmetrical boundary by DPH analysis (not shown) after sucrose gradient sedimentation, with its peak shifted one to two fractions from that of undigested UVs, i.e., fractions 9-10. Very little, if any, of the llO- or 55-kDa proteins remained after digestion (Fig. 2B, VI). Unanue et aZ. (9) have shown that 3Hlabeled clathrin did not bind to their trypsin-digested, stripped vesicles. When AN-labeled clathrin, prepared from ANlabeled CVs, was added to a tryptic digest of unlabeled UVs (containing soybean

CLATHRIN

AND

COATED

VESICLES

409

Pb

An-Labeled

40 7 a 6‘20 L. t ti 5 0 li km 2 2 40

20 0

10

20 FRACTION

30

FIG. 7. (A) Sucrose gradient sedimentation of CVs in 10-30s gradient in 0.1 M Mes, pH 6.5. The rotor was SW 40. The time of sedimentation was 110 min at 27,000 rpm at 20°C. 0, Trp fluorescence; A, AN fluorescence. (B) Sedimentation of trypsin digested AN-labeled CVs. 0, Trp fluorescence; A, AN fluorescence. The degree of labeling was 2.5 AN/lOO,OOO g protein.

trypsin inhibitor) in 0.01 M Tris, pH 8.5, and the solution brought to pH 6.5 in 0.01 M Mes by dialysis for 20 h, sucrose gradient analysis did not show any change in the sedimentation behavior of the ANlabeled clathrin, i.e., peak at fractions 45. Since only the clathrin is labeled by the AN probe, this method of analysis is more sensitive than using Trp fluorescence, where both components are fluorescent. This result is in agreement with that reported by Unanue et al. (9), using a completely different experimental approach. DISCUSSION

We have prepared AN-labeled CVs, UVs, and baskets. It has been shown that the sedimentation behavior of the labeled CVs and UVs remains unchanged after label-

410

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ing, while a small amount of association occurred with labeled baskets. AN-Labeled CVs dissociated normally at pH 8.5. The AN-fluorescence intensity of the UVs was greater than that of the coat proteins (Fig. 4). The reverse was true with respect to Trp fluorescence in dissociated CVs (12). Thus, the AN labeling provides a more highly fluorescent marker of UVs. The AN labeling of both components has little effect on the ability of the two components to recombine, since almost complete reassociation was observed by reducing the pH from 8.5 to 6.5 (Fig. 4). The 180- and llO-kDa proteins are the most strongly AN-labeled in CVs, although, in overloaded gels, the 55-kDa protein and light chains (30-36 kDa) were also labeled (not shown). Apparently, all of the major protein components of CVs contain free -SH groups which are accessible to ANM in the native state of the vesicle. In UVs, the 180-kDa band(s) and the -llO-kDa proteins are strongly labeled, although weak AN fluorescence was also observed in the 55-kDa region and in minor satellite bands near the 180- and llO-kDa proteins (Fig. 2B). We have used AN-labeled clathrin to reveal a conformational change in the pH region where it self-associates. This was possible by energy transfer experiments which were made feasible by the AN label. It has been shown by Ungewickell et al. (10) and Schmid et al. (18) that clathrin baskets can be digested with trypsin to liberate a 70-kDa fragment without destroying the polyhedral organization of the basket structure. We have found that the fragment(s) that was digested and released was not labeled with AN. It is evident that this fragment either does not contain free SH groups or its conformation in the basket structure prevents reaction. It seems likely, consequently, that this fragment of clathrin will not represent a homologous fraction of the molecule with respect to either composition or function. We have shown that CVs are also digested by trypsin in much the same way as baskets, since a llO-kDa protein was produced and the liberated 70-kDa unit(s) lacked AN fluorescence. This correspon-

ET AL.

dence in susceptibility to tryptic digestion between CVs and baskets indicates that the conformation of clathrin in both structures is similar. This similarity in behavior is even more striking since free clathrin is hardly affected when digested by trypsin (at pH 8.0), as judged by NaDodSO,-gel electrophoresis experiments (unpublished experiments by the authors). REFERENCES 1. GOLDSTEIN, J. L., ANDERSON, R. G. W., AND BROWN, M. S. (1979) Nature (London) 279,6’79685. 2. HEUSER, J. (1980) J. Cell BioL 84, 560-583. 3. ROTHMAN, J. E., AND FINE, R. E. (1980) P~oc.

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