[30] Dissociation and reassociation of clathrin

350

INTERNALIZATION

OF PLASMA

[30] D i s s o c i a t i o n

MEMBRANE

COMPONENTS

and Reassociation

[30]

of Clathrin

By W. SCHOOK and S. PUSZKIN One o f the most basic processes for cell survival is that ofendocytosis. In order to interact with the external environment, cells must have a mechanism for sensing important changes, especially in the level o f appropriate signal molecules, for example, hormones. The cellular mechanism for processing this information has been under study for some time, but only recently has the requisite fusion o f biochemical and morphological techniques occurred, with the resultant explosion in information as evidenced by the variety o f review articles and texts available'-3 and by chapters o f this volume. Coated vesicles have been investigated as the probable mediator o f endocytotic events since their identification in the mid-1960s. 4 After the isolation o f coated vesicles by Pearse: a surge in studies on their formation and function began, leading to the isolation and partial characterization o f the coat protein, clathrin, by various investigators. 6- 8 Clathrin was shown to exist as a trimer of 180,000 Mr polypeptides that copurifies with a doublet o f polypeptides o f approximately 30,000 Mr. 9,1° The likely n u m b e r o f these copurifying polypeptides is one 36,000 and two 33,000 per clathrin trimer, based on dye-binding intensity o f the protein bands on dodecyl sulfatepolyacrylamide gels.t~ They are known as CAPS (clathrin-associated proteins) or light chains. The clathrin trimer is capable o f assembling into the geodesic d o m e structure observed on coated vesicles by simply decreasing the p H o f clathrin solutions from 7.5 to some value between 6 and 6.8, as long as chaotropic anions are absent and the ionic strength is not too

C. D. Ocklefordand A. Whyte, eds., "Coated Vesicles."CambridgeUniv. Press, London and New York, 1980. 2 j. L. Goldstein, R. G. W. Anderson, and M. S. Brown, Nature(London) 279, 679 (1979). 3 B. M. F. Pearse and M. S. Bretscher,Annu. Rev. Biochem. 50, 85 (1981). 4 T. F. Roth and K. R. Porter, J. CellBiol20, 313 (1964). 5 B. M. F. Pearse, J. Mol. Biol. 97, 93 (1975). 6 W. Schook, S. Puszkin, W. S. Bloom, C. Ores, and S. Kochwa,Proc. Natl. Acad. Sci. U.S.A. 76, 116 (1979). 7j. H. Keen, M. C. Willingham, and I. H. Pastan, Cell 16, 303 (1979). 8 M. P. Woodward and T. F. Roth, Proc. Natl. Acad. Sci. U.S.A. 75, 4394 (1978). 9 H. T. Pretorius, P. K. Nandi, R. E. Lippoldt, M. L. Johnson, J. H. Keen, I. Pastan, and H. Edelhoch, Biochemistry 20, 2777 ( 1981). ~oE. Ungewickelland D. Branton, Nature (London) 289, 420 (1981). tt j. KJrchhausenand S. C. Harrison, Cell23, 755 (1981). METHODS IN ENZYMOLOGY, VOL. 98

Copyright © 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181998-1

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DISSOCIATION AND REASSOCIAT1ON OF CLATHRIN

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high. 6-8,12 The 30,000-Mr doublet, which we call CAPs, is required for the proper formation of the basket structure13; since brief treatment with chymotrypsin, which partially degrades the CAPs, without apparent effect upon clathrin, destroys the ability of clathrin to assemble correctly. These observations have been supported by others ~1 using elastase, which apparently cleaves the CAPs into smaller fragments than chymotrypsin. The importance of understanding the mechanism of clathrin recruitment, in vivo assembly, and interaction with the cellular cytoskeleton is self-evident, and our approach to this problem basically has been a biochemical one starting with the isolation of clathrin. The available procedures for clathrin purification depend upon the relative enrichment of this protein by virtue of its tendency to form or remain assembled as macromolecular aggregates under conditions of slightly acidic pH. In addition, cosedimentation of both coated vesicles and clathrin baskets in sucrose gradients allows a further enrichment from the starting homogenate. A second property utilized is clathrin's sensitivity to conditions of basic pH. When assemblies ofclathrin are incubated in buffers of low ionic strengths and basic pH (7.5 or somewhat higher), they disassemble into trimers (triskelions), which can be separated from the bulk of the vesicular membrane and other proteins by sedimentation and column chromatography. There are two principal approaches to clathrin purification described in the literature: one utilizes 2 M urea following low-ionicstrength extraction of a crude coated vesicle fraction; the other utilizes a high concentration of Tris buffer and a more highly purified coated vesicle preparation. Our procedure will be described in detail, and the method of Keen and co-workers will be described briefly, since it is basically quite similar. Purification

Procedure

The purification scheme presented below 6,14 results in a preparation which is 90-95% pure; the major copurifying proteins ae the CAPs that are bound to clathrin. Step 1. Fresh bovine brains, packed in ice, are obtained from a local slaughterhouse. The usual preparation requires three to five brains, and the exact volume of solutions for four brains is given. Meninges are removed, ~2 p. p. Van Jaarsveld, P. K. Nandi, R. E. Lippoldt, H. Saroff, and H. Edelhoch, Biochemistry 20, 4129 (1981). ~3 M. P. Lisanti, W. Schook, N. Moskowitz, K. Beckenstein, W. S. Bloom, C. Ores, and S. Puszkin, Eur. J. Biochem. 121,617 (1982). ~4 W. S. Bloom, W. Schook, E. Feageson, C. Ores, and S. Puszkin, Biochim. Biophys. Acta 598, 447 (1980).

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INTERNALIZATION OF PLASMA MEMBRANE COMPONENTS

[30]

along with the cerebellum and brainstem. The gray matter is separated from the white by suction through a suction flask kept on ice. The tissue is suspended into 0.1 2-(N-morpholino)ethanesulfonic acid M (MES) buffer, pH 6.5, containing 1 m M EGTA, 0.5 m M MgCI2, 7 m M 2-mercaptoethanol, and 0.02% sodium azide. Approximately half the tissue suspension is transferred to a Waring blender and homogenized for 10 sec at top speed followed by a 1-min rest period. This procedure is repeated twice, and the homogenate is transferred to a 2-liter beaker. The second half is treated similarly, and all the homogenates are combined. With this number of brains, 1 liter of buffer is used and the resulting homogenate volume is approximately 2.0 liters. The homogenate is centrifuged at 30,000 g for 15 rain in a Sorvall SS-34 rotor, and the supernatant is saved for the next step. Step 2. The low-speed supernatants obtained above (approximately 900 ml) are combined and loaded into a Beckman 30 rotor and centrifuged at 80,000 g for 1 hr. The pellets obtained are suspended in 500 ml of 20 m M Tris, pH 7.5, containing 1 m M EDTA and 2 m M 2-mercaptoethanol and extracted overnight with stirring at 4 °. The extract is centrifuged for 1 hr at 100,000 g to remove membranous debris and fractionated with solid ammonium sulfate. The salt is added to 30% saturation with stirring, and the solution is left on ice for 30 min and finally centrifuged for 10 min at 30,000 g to pellet the protein. Protein pellets are suspended in a minimal volume, approximately 20 ml of 20 mMTris, pH 7.5 buffer, containing 2 Murea and 7 m M 2-mercaptoethanol (buffer A) and dialyzed against 20 volumes of the same buffer overnight. Step 3. The dialyzate is centrifuged at 30,000 g for 10 min to remove any denatured protein and loaded onto a 5 X 100-cm column of Sepharose 4B equilibrated with buffer A. A flow rate of 15 -20 ml/hr is maintained with a peristaltic pump, and fractions of 8 ml are collected. Those containing proteins are detected by UV absorbance at 280 nm. A peak eluting in the void volume containing residual membranes and other aggregates is discarded; the second peak observed contains clathrin, and these fractions are pooled, adjusted to 50% saturation by the addition of solid ammonium sulfate, and sedimented at 30,000 g for 10 min after a 30-min incubation on ice. The pellets obtained are resuspended in a minimal volume of buffer A (approximately 10 ml) and dialyzed against 20 volumes of 20 m M Tris, pH 7.6, containing 0.5 m M MgC12, 7 m M 2-mercaptoethanol, and 0.02% sodium azide (buffer B), followed by a buffer change and overnight dialysis. Step 4. Dialyzed protein from two large column runs (120- 140 mg) is centrifuged at 100,000 g for 1 hr to remove aggregates and loaded on another Sepharose 4B column (2.5 X 100 cm) equilibrated with buffer B; 5-ml fractions are collected. Material eluting in the void volume is composed mainly of aggregated clathrin and much of the copurifying proteins. The second peak observed by UV absorbance at 280 nm is composed of 90% pure clathrin, and major copurifying polypeptides consisting of the CAPs

[:30]

DISSOCIATION AND REASSOCIATION OF CLATHRIN

353

that are associated with clathrin in the triskelion. The clathrin peak is concentrated again by ammonium-sulfate fractionation (50% using the solid salt), and the protein pellets are resuspended in 10-15 ml of buffer A, dialyzed overnight against at least 20 volumes of buffer A, followed by two changes of buffer B. The dialyzed protein can be stored frozen in 10% sucrose for 6 months with no loss in its ability to reassemble into baskets. Figure 1 demonstrates the purity obtainable from a typical clathrin preparation. Keen and co-workers7 have used 0.5 MTris buffer to effect solubilization of clathrin from a more purified starting material. This buffer results in the solubilization of more protein from the vesicle membrane, and this results in a third clearly discernible peak following chromatography. Starting with a more purified coated vesicle fraction is more important in this procedure, as higher levels of contamination are likely to be observed in clathrin preparations because the buffer extracts a greater amount of protein from the membrane. Usually quite pure clathrin can be obtained with only one pass through a 1 × 90 cm column, using the coated vesicles obtained from one bovine brain; however, the yield is quite low (approximately 3 - 5 mg). The assembly properties of clathrin prepared by this procedure are also somewhat different from preparations using 2 M urea, and this will be discussed fully later. Biophysical Properties of Clathrin Irrespective of the procedure used to purify clathrin, it is always associated with the CAPs of molecular weights, as reported by various investigators, ranging from 30,000 to 36,0003 °-~3 Preparations of clathrin contain approximately 2 tool of faster-moving polypeptide to one of the slower, as judged by densitometry of Coomassie Blue-stained bands in polyacrylamide gels. When stored in solutions that favor depolymerization, clathrin has been shown by several groups to have a sedimentation constant of 8.6 S20,w~ o which, when combined with other biophysical data, suggests that clathrin exists as a trimer or triskelion in solution. This proposal is supported by electron microscopic evidence using rotary shadowing ~° and traditional ~5 negative staining techniques that clearly show three bent legs radiating from a central point. If such triskelions are assembled into baskets and briefly treated with chymotrypsin, ~3 the CAPs are partially proteolyzed, whereas the clathrin is apparently unaffected. When such preparations are examined 0 value of 8.2 is obtained, which in the analytical ultracentrifuge, an s20,w clearly demonstrates that removal of the susceptible segment of the CAPs polypeptide chain does not result in disassembly of the triskelion. The partial removal of the CAPs alters the ability of the modified triskelions to 15 R. A. Crowther and B. M. F. Pearse, J. Cell Biol. 91,790 (1981).

354

INTERNALIZATION OF PLASMA MEMBRANE COMPONENTS

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[30]

DISSOCIATION AND REASSOCIATION OF CLATHRIN

355

assemble into baskets.11,13 There is a marked shift in the equilibrium from assembled structures to that of trimers under conditions usually conducive to cage assembly. This change in assembly properties points to a pivotal role for the CAPs in guiding the way triskelions interact with each other during the formation of cages. On the basis of the proposed model for clathrin assembly by Crowther and Pearse 15 and on data obtained with trypsintreated clathrin by Schmid et al., 16 it seems likely that the CAPs are found near the vertex of the triskelion and interact with the triskelion's legs to determine their orientation and therefore guide the formation of points of association during clathrin assembly. Utilizing chymotrypsin bound to the surfaces of polystyrene beads, we have shown that the CAPs are oriented to the outside of the basket structure in coated vesicles and clathrin baskets ~7and that the fragmentation of CAPs by this protease results in the retention of a portion of the CAP molecule by clathrin. We have devised a procedure for the purification of CAPs from triskelions that reveals two interesting properties of these polypeptides - - their heat stability and binding affinity for calmodulin.18 Step I. Clathrin preparations or even crude coated vesicles (2 - 5 mg/ml) are placed in buffer B and heated for 5 min in a boiling water bath and centrifuged at 100,000 g for 60 min to remove denatured material. The supernatant is concentrated to approximately 1 mg/ml with dry Sephadex G-200 or Ficoll. An estimate of the amount of CAPs expected from such treatment can be obtained by assuming that CAPs constitute 5 - 10% of the starting clathrin concentration. Step 2. A calmodulin affinity column is prepared as previously described 19using CNBr-activated Sepharose and calmodulin. The CAPs preparation is dialyzed overnight against 20 mMTris, pH 6.5, buffer containing 1 m M C a 2÷, 2 mM2-mercaptoethanol, and 0.02O/osodium azide and allowed to bind to this column for 3 0 - 6 0 min at room temperature. The column then is washed with more buffer to remove unbound protein, washed with a similar buffer containing 0.5 M NaCI to remove nonspecifically bound protein, and finally with a similar buffer containing 2 m M E G T A in place of Ca 2+. The CAPs are eluted in the EGTA buffer and are dialyzed against 50 volumes of buffer B overnight. They are concentrated again and stored in 10% sucrose. The protein composition at each step in this procedure is illustrated in Fig. 2. ~6S. L. Schmid, A. K. Matsumoto, and J. E. Rothman, Proc. Natl. Acad. Sci. U.S.A. 79, 91 (1982). ~7 M. P. Lisanti, W, Schook, N. Moskowitz, C. Ores, and S. Puszkin, Biochem. J. 201, 297 (1982). ~8 M. P. Lisanti, L. Shapiro, N. Moskowitz, E. Hua, S. Puszkin, and W. Schook, Eur. J. Biochem. 125, 463 (1982). ~9 D. M. Watterson and T. C. Vanaman, Biochem. Biophys. Res. Commun. 73, 40 (1976).

356

INTERNALIZATION OF PLASMA MEMBRANE COMPONENTS

[30]

Step 2 is necessary when crude preparations of starting material are used rather than purified clathrin. This purification procedure will allow preparation of CAPs adequate for investigation of the chemical and biophysical properties of these regulatory proteins that attach to clathrin. The properties already observed, i.e., heat stability, requirement for efficient cage assembly, ability to bind calmodulin, and their presence in all coated vesicle or clathrin preparations, suggest that these polypeptides are important for clathrin function and have not been formed as a result ofproteolytic degradation, as suggested by immunological cross-reactivity with antibodies purified using a

BOVINE BRAIN CORTEX

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PURIFIED COATED VESICLE PELLET EXTRACTED, 20 MM TRIS, 2 MM EDTA, PH 7,5

~

CRUDE CLATHRIN_a_

~ HEATINACTIVATION,5 MINUTES a

b

O

PURIFIED CAPs SOLUTION _b_

FIG. 2. Schematic summary of the purification steps leading to a homogeneous solution of clathrin-associated proteins (CAPs): The protein composition of selected steps is illustrated by the Coomassie Blue-stained bands on SDS-gradient slab gels. Lane a: Extract of purified coated vesicles containing clathrin prior to heat treatment (100/tg); lane b: concentrated supernatant after heat treatment of clathrin (30/~g); lane c: snpernatant of an extract of crude coated vesicles after heat treatment. Note that the preparation shown in lane c is considerably less pure than that shown in lane b. The purity of this CAP preparation can be improved to that illustrated in lane b by use of affinity chromatography using immobilized calmodulin as described in the text (30/~g).

[30]

DISSOCIATION AND REASSOCIATION OF CLATHRIN

357

denatured clathrin affinity column. 2° Furthermore, recent results from our laboratory have demonstrated that the CAPs colocalize with clathrin at both the light and electron microscopic levels, 18,21,22and, although these studies failed to detect a soluble pool of CAPs, the possibility that these proteins may exist independently from clathrin has been raised by the conditions of their interaction with calmodulin. This possibility has received support from a recent report that CAP-like polypeptides present in a cytosol fraction derived from adrenal chromaffin cells are bound to the chromaffin granule membrane in a Ca 2+- and calmodulin-dependent manner. 23 C o n d i t i o n s C o n d u c i v e to B a s k e t A s s e m b l y

Since the discovery that clathrin was capable of in vitro assembly into cages, a number of groups have been investigating the environmental changes that can alter their rate and/or final state of assembly. The principal conclusions from these studies can be summarized as follows. Divalent cations can increase the rate of basket formation at a constant pH. Control o f p H is important, since the rate of assembly is dependent on this variable. 2 The order of effectiveness for divalent cations is as follows: Mn 2+ greater than Ca 2+ greater than Mg 2+ 2~; that for anions was found to follow basically the Hofmeister ranking, although sulfate seemed to exhibit more specific inhibitory properties.~2 Several drugs were shown to have stimulatory effects upon clathrin assembly ~3 and to share a common factor, hydrophobic structure and inherent basicity. They appear to stimulate the rate of assembly and possibly act by mimicking the effect of protons, making the environment appear more acidic than it actually is. Using a variety of polyamine bases, Nandi et al. 2~ have shown that a minimum of four basic groups appear to be required for significant binding unless a nonpolar region is present as in dansyl cadavarine. This group confirmed our observations of chlorpromazine effects upon basket assembly and found that lysozyme was capable of stimulating assembly, whereas other basic proteins tested had no effect. These studies all point to a region of the clathrin molecule capable of interacting with a variety of agents that can affect the conformation of the triskelion and stimulate the assembly process. These observations suggest that the assembly process depends upon both ionic and hydrophobic interactions. The type of buffer used also modulates the pH at which basket 2o j. H. Keen, M. C. Willingham, and 1. Pastan, J. Biol. Chem. 256, 2538 (1981). 2~ S. Puszkin, C. Ores, A. Andres, M. P. Lisanti, and W. J. Schook, Cell TissueRes. 231,495 (1983). 22 M. P. Lisanti, A. Andres, C. Ores, A. C. Puszkin, W. J. Schook, and S. Puszkin, Cell Tissue Res. 231, 507 (1983). 23 M. J. Geisow and R. D. Burgoyne, Nature (London) 301,432 (1983).

358

INTERNALIZATION OF PLASMA MEMBRANE COMPONENTS

[30]

assembly is normally observed, cacodylate appearing to enhance basket formation under more alkaline conditions. 6 Some differences in assembly properties have been reported by various laboratories for clathrin preparations obtained by different procedures. 22,24,25 It appears that clathrin prepared by 0.5 M Tris extraction is more sensitive to substances present in the assembly buffer and requires Ca 2+ as well as additional protein cofactors. Urea-purified clathrin assembles readily without such cofactors or similar requirements. An additional protein component of approximately 1 10,000 Mr z6 seems to be required for clathrin assembly onto membranes. This protein is probably responsible for the stimulatory effect of the peak III protein fraction observed by Keen et aL v We have compared the assembly capacity of clathrin obtained by both procedures and have found that Tris-treated clathrin yields quatitatively less assembly when tested under identical conditions, but that urea treatment will partially reverse the effects of the high Tris concentration used in the isolation procedure. These observations suggest that the effects of these agents upon clathrin's ability to assemble are complex and may involve changes in the properties of accessory factors as well as in clathrin itself. An especially promising recent development was the discovery of a cytosolic factor capable of effecting the disassembly of clathrin from isolated coated vesicles under conditions of neutral pH. This factor appears to be a protein that requires ATP and results in a modification of the clathrin triskelion which makes it refractory to in vitro reassembly under usually effective conditions. 2vThe identification of a membrane-bound protein as the probable site of attachment of clathrin triskelions to the membrane as well as the purification of the clathrin-associated proteins (the CAPs) and the discovery of their interaction with calmodulin suggest that studies on the interrelationship of these and the possibly other yet-to-be-described effectors will continue and that descriptions of the biochemical and biophysical properties of clathrin and its associated proteins will remain an important area for investigation.

24 p. K. Nandi, P. P. Van Jaarsveld, R. E. Lippoldt, and H. Edelhoch, Biochemistry 20, 6706 (1981). 25 M. P. Woodward and T. F. Roth, J. Suprarnol. Struct. 11,237 (1979). 26 E. R. Unanue, E. Ungewickell, and D. Branton, Cell26, 439 (1981). 27 E. J. Patzer, D. M. Schlossman, and J. E. Rothman, J. Cell Biol. 93, 230 (1982).