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Clathrin-coated vesicles: Isolation, dissociation and factor-dependent reassociation of clathrin baskets
Cell, Vol. 16,303-312, February 1979, Copyright 0 1979 by MIT
Clathrin-Coated Vesicles: Isolation, and Factor-Dependent Reassociation Baskets James H. Keen, Mark C. Willingham Ira H. Pastan Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20014
and
Summary The nature of the protein coat on clathrin-coated vesicles and the interactions responsible for the structure and reformation of clathrin baskets have been investigated. Coated vesicles were isolated from bovine brain using a rapid, one-day modification of the method of Pearse (1975). The vesicles are composed predominantly of clathrin (175,000) with smaller amounts of 110,000 and 55,000 molecular weight polypeptides. Clathrin was released in a solubilized form from these vesicles by treatment with 0.5 M Tris(hydroxymethyl)methyl ammonium chloride (Tris-Cl) or other protonated amines at neutral pH. It was not released on treatment with thiols, thiol reagents, Triton X-100 or sodium chloride, leading us to suggest that specific amino-carboxylate salt linkages are necessary for maintenance of the basket structure. When viewed by electron microscopy, the solubilized proteins in Tris-Cl are present in the form of filamentous aggregates and no basket structures are observed. Gel filtration of the extract in Tris-Cl resolves a clathrin-containing fraction (I) from one consisting predominantly of the 110,000 molecular weight polypeptide (II). We were able to reconstitute basket structures from the unfractionated Tris-Cl extract by dialyzing it against the vesicle isolation buffer, a solution of moderate ionic strength (r/2 = 0.11). Neither of the resolved fractions (I or II) alone yielded baskets on dialysis, but reconstitution was successful when both I and II were combined and dialyzed. The activity in II, which appears to be a basket-assembly factor, was heat-labile and could not be replaced by bovine serum albumin or brain calcium-dependent modulator protein. If a solution of low ionic strength (r/2 = 0.01) containing calcium was used as the dialysate, the clathrin fraction (I) alone was capable of reforming baskets. Thus the clathrin coat is a labile structure that can be solubilized by nondenaturing treatments, and baskets can be reformed from the extracted material.
Dissociation of Clathrin
oocytes. Coated pits and intracellular vesicles bearing similar coats on their cytoplasmic surfaces have since been reported in many other cell types (Pearse, 1976) and suggested to function in a variety of processes, including receptor-mediated protein uptake (Anderson, Goldstein and Brown, 1976; Anderson, Brown and Goldstein, 1977; M. C. Willingham, F. R. Maxfield and I. H. Pastan, manuscript submitted), nonspecific protein uptake (Friend and Farquhar, 1967) plasma membrane recycling (Heuser and Reese, 1973) and intracellular membrane transfer (Friend and Farquhar, 1967; Holtzman, Novikoff and Villaverde, 1967). Morphological studies on adsorptive endocytosis have indicated that endocytosed materials are initially found predominantly in coated vesicles and subsequently appear in smooth-surfaced vesicles, suggesting that either the coated vesicle rapidly loses its coat or it transfers its contents to another vesicle (Roth and Porter, 1964; Friend and Farquhar, 1967; Lagunoff and Curran, 1972; Anderson et al., 1977). Although many functions for coated membranes and vesicles have been suggested based on morphological observations, little biochemical information was available until coated vesicles were isolated from brain (Pearse, 1975) and other sources (Pearse, 1976). Examination of purified vesicle fractions by SDS gel electrophoresis revealed the predominance of a single protein species of about 175,000 subunit molecular weight. This protein was named clathrin because of its presumed role in the formation of the lattice-like protein coat structure. Urea (Blitz, Fine and Tosselli, 1977) and treatment at pH 10 (Roth, Woodward and Woods, 1977) have recently been reported to release clathrin from coated vesicles. We wished to investigate the interactions responsible for the coat structure with the aim of elucidafing the role played by coated regions in receptormediated endocytosis. Conditions were sought which would dissociate the coat structure and release clathrin in the absence of denaturants or extremes of pH. We report here that high concentrations (0.5 M) of protonated amines at pH 7 solubilize clathrin and other proteins. Clathrin solubilized by this procedure has been reconstituted into basket structures as well as purified from a separable basket-assembly factor. This factor is required for basket reconstitution under ionic conditions that approximate those found in vivo.
Introduction Results Unique regions of plasma membrane with a dense protein coat on their cytoplasmic surface were first described by Roth and Porter (1964) and suggested to play a role in uptake of yolk protein by mosquito
Clathrin-Coated Vesicle Isolation Dissociation A rapid, one-day procedure for
and the
isolation
of
Cell 304
clathrin-coated vesrcles from cow brain, requiring only two gradient centrifugation steps, is described in Experimental Procedures. It is a modification of the method of Pearse (1975). Thin-section electron micrographs (Figure 1) of each of the final fractions (E and F) reveal the presence of coated vesicles similar in size to those reported previously (Pearse, 1975), as well as some uncoated membranous contaminants. Aside from slightly larger amounts of uncoated membrane in the lower vesicle fraction (F), we did not see any other morphological differences between the fractions. SDS-polyacrylamide gels of the major fractions obtained on purification are shown in Figure 2. The uppermost fractions from the second gradient (E and F) were most highly enriched in clathrin, a protein of 175,000 subunit molecularweight. Clathrin comprised about 70% of the total protein in fractions E and F, although polypeptides of 110,000 and 55,000 daltons were also purified in these
fractions, as had been observed earlier (Blitz et al., 1977; Woods, Woodward and Roth, 1978). The mobility of the clathrin band was not altered when N-ethyl maleimide rather than a reducing agent was added before electrophoresis, indicating that clathrin is not disulfide-linked in the coated vesicle (not shown). Only a small amount of clathrin was released from coated vesicles when they were resuspended in isolation buffer (buffer A) (Figure 3). However, clathrin has been reported to be solubilized by treatment with 2 M urea (Blitz et al., 1977) or alkalinization to pH 10 (Roth et al., 1977). We sought conditions allowing solubilization at neutral pH in the absence of denaturants. To this end, various agents were tested for their ability to dissociate clathrin from coated vesicles as determined by release of the protein in a nonsedimentable form (Figure 3). Clathrin was progressively released by increasing concentrations of Tris(hydroxymethyl)methyl ammonium chloride (Tris-Cl) added to buffer A: approximately 80% was solubi-
ABCDEFG
Figure Figure
1. Coated
Vesicles
(A) Fraction E (magnification F (magnification 29,000X; vesicles.
from
Bovine
Brain
34,000X; bar = 0.2 pm); (B) fraction bar = 0.2 pm). Arrows denote coated
Molecular (37 pg); (39 pg); fraction second
2. Analysis
of Coated
Vesicle
Purification
by SDS-PAGE
weight scale (in daltons x~O-~) at left. (A) Homogenate (B) 20,000 x g supernatant (45 pg); (C) 85,000 x g pellet (D) first gradient pool (23 pg); (E) second gradient upper (7 rg): (F) second gradient middle fraction (5 pg); (G) gradient lower fraction (7 pg).
Clathrin-Coated 305
Vesicles
lized by 0.5 M Tris-Cl (Tris-buffer A). At 0.5 M and pH 6.5, triethanolamine chloride, ammonium chloride and imidazole chloride were also effective, whereas uncharged hydroxylamine was not. Release was not simply due to elevated ionic strength or the hydroxyl groups of Tris since neither 0.5 M NaCl nor 0.5 M glycerol were effective. Other ineffective compounds include Triton X-100 (l%), dithiothreitol (5 mM), glutathione (5 mM), p-chloromercuribenzoate (5 mM), protamine sulfate (1 mg/ml), D-glucosamine (0.1 M), benzocaine (1 mM), dibucaine (1 mM), colchicine (50 pg/ml) and histone 2a (7.5 mg/ml). Clathrin was not the only protein released by these treatments; it was accompanied by other proteins whose relative amounts depend upon the agent used. Treatment at pH 10 released all polypeptides uniformly, whereas Tris extracts were enriched in clathrin and to a lesser extent in a polypeptide of 110,000 daltons. Lower molecular weight material was retained in the pellet. Since coated vesicles also contain lipid, we measured the amount of phospholipid released by Tris-Cl treatment. A coated vesicle preparation containing 110 pg of protein and 47 nmole of lipid phosphorous was treated with Tris-Cl and centrifuged. After centrifugation, 42 nmole of lipid phosphorous and 54 pg of protein remained in the pellet. Thus TrisCl treatment solubilized no more than 10% of the phospholipid. Morphological evidence for disruption of the clathrin basket structure was obtained by negativestaining electron microscopy. The Tris extract displayed a pattern of linear aggregates (Figure 4A). Intact clathrin coats or basket-like structures were not observed. These structures were also not found in the Tris-treated vesicle pellet when examined by thin-section electron microscopy (not shown). Reconstitution of Clathrin Baskets Tris-extracted vesicles were centrifuged to remove uncoated vesicle remnants, and the resulting supernatant was dialyzed overnight at room temperature against buffer A to remove Tris-Cl. When the retentate was examined by electron microscopy (Figure 4B), it was apparent that ordered “basketlike” structures were reformed (compare Figures IA and 4A). The reconstituted structures were of similar size to that of the intact coated vesicle. Unlike intact vesicles, however, they did not possess lipid bilayer membranes when examined by electron microscopy. In fact, when the entire Tristreated solution was dialyzed, rather than the soluble extract alone, protein basket structures reformed entirely separately from the membranous material present (not shown). Reconstituted baskets prepared as described above had an unusual property: they were not
readily sedimentable. Unlike the parent coated vesicles, these reconstituted baskets did not sediment at 130,000 X g for 60 min. On 5-20% sucrose gradients in buffer A, intact vesicles had sedimentation constants in excess of 150s. Under the same conditions, clathrin in reconstituted baskets sedimented only slowly as a discrete 12s species. This may reflect a pressure-dependent dissociation under these conditions of ionic strength since negative-staining electron microscopy revealed linear protein aggregates but no basket structures in the clathrin peak after centrifugation. S. Puszkin (personal communication) has reported, and we have confirmed, that basket reconstitution may also be accomplished by dialysis against very dilute buffers. The reformed baskets obtained under these conditions of low ionic strength were morphologically identical to those described above, but were more stable to centrifugation and were entirely sedimentable at 100,000 X g for 30 min. Fractionation of the Tris Extract When the Tris-treated vesicle extract was chromatographed on a Sepharose CL-4B column equilibrated with Tris-buffer A, two protein fractions were obtained (Figure 5, lower panel). SDS gel electrophoresis of these fractions on 6% (Figure 5, upper panel) and 13% (not shown) acrylamide gels revealed the first peak (I) to be composed predominantly of clathrin, with only trace amounts of polypeptide of 90,000 molecular weight. The following peak (II) was almost entirely devoid of clathrin but greatly enriched in a peptide of about 110,000 daltons and, to a lesser and variable extent, in one of 55,000 daltons. By comparison with a standard curve prepared using marker proteins (Tanford et al., 1974), the elution volume of fraction II indicates a Stokes radius of 68 A and, assuming a globular conformation, a molecular weight of approximately 420,000 daltons. It was not possible to perform the same calculations confidently for the clathrin peak (fraction I) because its elution volume was beyond the range of the standard curve (Nozaki et al., 1976). As an approximation for the purpose of comparison, however, a Stokes radius of about 160 A was estimated by extrapolation, corresponding to a molecular weight of 1,200,OOO daltons. Dialysis of fraction I against buffer A yielded only filamentous aggregates of protein with no basket structures reformed (Figure 6A). Similarly, dialysis of fraction II did not result in basket formation (Figure 6B). However, when equal volumes of each of these fractions were combined and then dialyzed against buffer A, ordered basket structures were clearly obtained (Figure 6C). Fraction II could not be replaced by varied amounts of either bovine serum albumin or pig brain calcium-dependent
Cell 306
1 s
2 s
P
P
s
P
s
6
5
4
3
P
s
P
s
P
68
A Figure
3. Analysis
of Clathrin
Release
from
Coated
Vesicles
by SDS-PAGE
Coated vesicles from three preparations (gels 1-4, 5-9 and 10-12) were used. Vesicles (25-50 pg protein) were resuspended in 100 /.LI of the indicated solutions prepared in buffer A and centrifuged after 15 min. Each pair of gel lanes represents equal aliquots of the resultant supernatants (S) and pellets (P) from one treatment of vesicles. (1) Control [buffer A: 0.1 M Na MES (pH 6.5), 1 mM EGTA, 0.5 mM MgCI,, 0.02% sodium azide]; (2) 0.05 M Tris-Cl (pH 7.0); (3) 0.03 M Tris-Cl (pH 7.0); (4) 0.5 M Tris-Cl (pH 7.0); (5) 0.5 M triethanolamine chloride (pH 6.5); (6) 0.5 M ammonium chloride (pH 6.5); (7) 0.5 M imidazole chloride (pH 6.5); (8) 0.5 M hydroxylamine (pH 6.5); (9) 25 mM sodium carbonate (pH 10) without buffer A; (10) 0.5 M sodium chloride; (11) 0.5 M glycerol: (12) 1% Triton X-100.
modulator protein (Klee, 1977), and its ability to promote basket reassembly was abolished by heating at 100°C for 4 min. This suggests that one or more basket assembly factors are present in II and are required for reassembly under these conditions. Based on the relative amounts of protein in the two fractions, we estimate that there are at least 3 clathrin monomers per 110,000 dalton polypeptide in the coated vesicle preparation. We further investigated the dependence of basket reassembly on fraction II as a function of ionic strength and the presence or absence of divalent cations. Aliquots of fraction I were mixed with equal volumes of fraction II or Tris-buffer A and then dialyzed against either buffer A or a more dilute buffer [5 mM Na MES (pH 6.5)]. CaCI,, MgCI,, NaCI, KCI, EDTA or ethyleneglycol-bis-(p-aminoethyl ether)N,N’-tetraacetic acid (EGTA) were added to the dialysates as indicated in Table 1.
After dialysis, the retentates were assayed for reconstituted basket structures by negative-staining electron microscopy. Baskets were reformed when fraction I alone was dialyzed against the dilute MES buffer (r/Z -0.01) containing Ca++ (Table 1, line 1). No reassembly for Ca++ was observed when Mg ++ was substituted (line 5) or when both were omitted (line 3). The combination of fraction I with II was found to promote basket reassembly under these conditions of ionic strength irrespective of the presence of divalent cations (lines 2, 4 and 6). That the action of fraction II was not due to calcium contained in the fraction is indicated by its activity despite the presence of EGTA (lines 4 and 6). When dialysis was performed against buffer A, a solution of moderate ionic strength (r/2 -O.ll), baskets were not reformed from fraction I alone in the presence or absence of Ca++ or Mg++ (Table 1,
Clathrin-Coated 307
Vesicles
8
7 s
P
s
9 P
s
10 P
s
11 P
s
12 P
s
P
250
165
94
68
lines 7, 9 and 11); the addition of fraction II was required for reassembly under these conditions (lines 8, IO and 12). The requirement for fraction II is a consequence of the ionic strength of the medium rather than of the MES concentration. When the ionic strength of the dilute MES buffer was made equal to that of buffer A by the addition of either KCI or NaCl (line 13), fraction I alone was no longer capable of basket reassembly and fraction II was required (lines 14), as observed with buffer A. Discussion To understand the structure and function of coated membrane regions, we have attempted to explore some of the physical interactions within isolated coated vesicles. As found by Pearse (1975) and other investigators (Blitz et al., 1977), bovine brain is a rich source of coated vesicles, and the modified isolation procedure reported here is rapid and should simplify further experimentation. The protein coat of clathrin-coated vesicles is a labile structure held together by noncovalent bonds. Clathrin monomers are not disulfide-linked
to other proteins in the coated vesicle, since their electrophoretic mobility on denaturing gels was not effected by prior treatment with either alkylating or reducing agents. Furthermore, clathrin was not solubilized from coated vesicles by treatment with either free thiols or thiol reagents. There were, however, a wide variety of agents that effectively dissociated it from the lipid vesicle. That Tris and other amines were effective, rather than Triton X100, indicates that clathrin is not an instrinsic membrane protein (Singer and Nicolson, 1972). Solubilized clathrin in Tris-Cl was not fully dissociated but was a discrete aggregate of approximately 6 monomers that may represent a protomerit functional unit. It was easily separable by gel filtration from another major protein fraction eluting in the molecular weight range of 420,000 daltons. The latter fraction (II) played a role in promoting reconstitution and had a more varied composition, with a 110,000 dalton polypeptide predominating. The Tris effect is specific for the amine function, rather than being an effect of either the hydroxyl moiety or elevated ionic strength, since neither glycerol nor sodium chloride were effective in re-
Cell
308
Figure 5. Gel Filtration ibrated with Tris Buffer
of Tris Extract A
on Sepharose
CL-4B
Equil-
6.7 mg of protein in 0.6 ml of Tris-buffer A were applied to a 0.9 x 57 cm column, and fractions of 0.9 ml were collected. Lower panel: protein elution profile. Column standards used were blue dextran 2000 (V,), myosin, thyroglobulin, y-globulin, ovalbumin and “C-leucine (V,). Upper panel: SDS-PAGE analysis of eluted fractions. Fractions 21-26 were pooled and designated I, while II consisted of fractions 29-31.
Figure 4. Structure and after Dialysis
of Extracted
Coated
Vesicle
Protein
before
(A) Electron micrograph of Tris extract of coated vesicles, negatively stained (magnification 79,000X; bar = 0.1 pm). (B) Electron micrograph (thin section) of reconstituted clathrin baskets formed by dialysis of Tris extract (magnification 65,000X; bar = 0.1 pm).
leasing clathrin. Although amines could act through a variety of mechanisms, we suggest that specific amino-carboxylate salt bridges may be necessary for the integrity of the clathrin basket structure. According to this hypothesis, disruption of the basket structure by high concentrations of protonated amines results from competition with protein-bound amino groups, while disruption by treatment at pH 10 reflects deprotonation of the protein amino groups. Hydroxylamine is probably ineffective in releasing clathrin because it is largely unprotonated at pH 6.5 (pKa = 6.03). Although an ionic strength effect might also be anticipated, we saw no release of clathrin from coated vesicles by sodium chloride at concentrations up to 0.5 M. Ionic strength did, however, have a critical effect on basket structure in determining both the extent of reconstitution and the stability of reassembled
baskets. The effect of urea is more difficult to interpret. It may reflect disruption of critical salt linkages or a more general denaturing action upon coat protein structures (Jencks, 1969). It is probable that the important interactions which Tris-Cl disrupts in solubilizing basket proteins are clathrin-clathrin rather than clathrin-vesicle interactions, since reconstitution of the basket structure occurred freely although the stripped vesicles had been removed. In contrast to the absence of an effect of the stripped vesicles, reconstitution was strongly dependent upon the ionic strength of the medium, fraction II and Ca++. In solutions of moderate ionic strength (r/2 ~0.11) similar in magnitude to those found in vivo, purified clathrin alone has not been observed to reform baskets upon removal of Tris-Cl under any of the conditions tested. However, the addition of a partially purified heat-labile factor promoted reconstitution. The data indicated that the requirement was specific. In the presence of fraction II, clathrin reassociated regardless of the presence of either Ca++, Mg++ or either of their chelators. Further characterization of fraction II is in progress. Following the report of S. Puszkin (personal
Clathrin-Coated
Vesicles
309
Figure
6. Reconstitution
of Clathrin
Baskets
from
Extract
Fractionated
Negatively stained electron micrographs of Sepharose fractions (A) Fraction I alone (magnification 66,000X); (6) fraction II alone
by Gel Filtration
I and II dialyzed against (66,000X); (C)fractions
communication), we have also been able to reconstitute baskets by dialysis against very dilute buffer solutions. Using purified clathrin and calcium-containing medium of low ionic strength (r/2 =O.Ol), we found that baskets reformed without the addition of other components, indicating that clathrin, rather than one of the other polypeptides in the coated vesicle fraction (Blitz et al., 1977; Roth et al., 1977), is the structural component of the protein coat. Morphological studies have demonstrated that the coat protein structure is associated with only limited regions of the plasma membrane of most cells (Friend and Farquhar, 1967; Anderson et al., 1976). Low-density lipoprotein (Anderson et al., 1977), aZ-macroglobulin, insulin and epidermal growth factor (Maxfield et al., 1978; Schlessinger et al., 1978; M. C. Willingham et al., manuscript
buffer A to remove Tris-Cl (bar = 0.1 pm in I and II combined (79,000X).
submitted) have been shown to cluster specifically over coated regions prior to internalization, suggesting that the protein coat may be involved in the process of patching. Furthermore, it has been speculated that the coat serves as an anchorage point (Kanaseki and Kadota, 1969) in the process of invagination that results in the formation of a coated pit and, ultimately, a coated vesicle. At early times, ferritinor peroxidase-labeled ligands are seen predominantly in these coated vesicles, but there is a rapid and progressive appearance of ligand in smooth-surfaced vesicles (Roth and Porter, 1964; Friend and Farquhar, 1967; Lagunoff and Curran, 1972; Anderson et al., 1977). The molecular properties of isolated clathrincoated vesicles and reconstituted baskets reported here are consistent with these observations. That the clathrin-coat structure survives the several iso-
Cell 310
Table 1. Clathrin Basket Reconstitution as a Function of Buffer Concentration and the Presence or Absence of Divalent Cations Fraction II
Dialysate (1) (2)
(3) (4)
(5) (6)
(7)
(8)
5 mM Na CaCI, + 5 mM Na CaCI, +
MES (pH 6.5) + 2 mM 1 mM EDTA MES (pH 6.5) + 2 mM 1 mM EDTA
5 mM Na MES (pH 6.5) EGTA + 1 mM EDTA 5 mM Na MES (pH 6.5) EGTA + 1 mM EDTA
Buffer A [IO0 mM Na MES 6.5) + 0.5 mM MgClz + 1 EGTA + 0.02% Na azide] Buffer A [lo0 mM Na MES 6.5) + 0.5 mM MgCI, + 1 EGTA + 0.02% Na azide] Buffer Buffer
(11)
BufferA+2mMEDTA+2mM CaCI, Buffer A + 2 mM EDTA CaCI,
(13)
(14)
-
+
+
+
-
-
+
+
+ 1 mM
5 mM Na MES (pH 6.5) + 2 mM MgCI, + 1 mM EGTA 5 mM Na MES (pH 6.5) + 2 mM MgCI, + 1 mM EGTA
(9) (10)
(12)
+ 1 mM
+
(pH mM (pH mM
A + 2 mM EDTA A + 2 mM EDTA
-
+ -
-
+
+
-
-
+
+
Equal aliquots of fraction I and Tris-buffer dialyzed against the solutions indicated. assayed for reconstituted basket structures electron microscopy.
Experimental
function, depend which are likely
Procedures
Materials Electrophoresis grade reagents for SDS gel electrophoresis were from Bio-Rad. The molecular weight standards used are described elsewhere (Wallach, Davies and Pastan, 1978). Sepharose CL-48 was a product of Pharmacia, and ultra-pure Tris base and sucrose were from Schwartz/Mann or Bethesda Research Laboratories. Goat anti-rabbit immunoglobulin was from Miles, and ovalbumin and bovine serum albumin were obtained from Sigma. Pig brain calcium-dependent modulator protein was a gift from C. Klee (NIH); bovine thyroglobulin was a gift from A. Burkhardt (NIH); and rabbit skeletal muscle myosin was a gift from L. Greene and E. Eisenberg (NIH).
+
+ +
+
+ 2 mM
5 mM Na MES (pH 6.5) + 2 mM CaCI, + 1 mM EDTA + 90 mM NaCl or KCI 5 mM Na MES (pH 6.5) + 2 mM CaCI, + 1 mM EDTA + 90 mM NaCl or KCI
Reconstituted Baskets
coat, and probably its specific upon these additional factors, candidates for in vivo modulation.
A or fraction II were The retentates were by negative-staining
lation procedures suggests that it is rigid enough to fulfill a structural role intracellularly in the process of pinocytosis, but the clathrin-coat structure is also a product of noncovalent interactions that are readily dissociable under nondenaturing conditions. This property of reversible disaggregation of clathrin over an entire vesicle could account for rapid transformation of a coated to a smooth-surfaced vesicle. Alternatively, a localized dissociation of the coat could allow selective fusion with an uncoated vesicle and subsequent transfer of endocytosed contents. More direct information on the fate of coated vesicles is required to distinguish between these possibilities. The reassociation process in vitro does not require lipid vesicles but is sensitive to Ca++, ionic strength and the presence of a separable factor. Thus while information for the protein coat structure resides in clathrin, the site and stability of the
Purification of Coated Vesicles Bovine brains were obtained from Frederick County Products (Frederick, Maryland) immediately after slaughter and placed on ice. All operations were performed at 0-4°C unless otherwise noted. Within 2 hr, purification was begun. The meninges were removed from the gray matter, the medulla was discarded, and the remaining whole brain (generally about 500 g) was minced coarsely and placed in a Waring blender with an equal volume of 0.1 M sodium MES (pH 6.5) 1 mM EGTA, 0.5 mM MgCI,, 0.02% sodium azide (buffer A). Homogenization was performed with three IO set bursts at full speed. The homogenate (fraction A) was centrifuged at 20,000 x g (all values are average centrifugal forces) for 30 min in a Sorvall GSA rotor, and the resulting pellet was discarded. The supernatant (fraction B) was centrifuged at 85,000 x g for 60 min in a Beckman 45Ti rotor; the soluble fraction was removed, and the vesicle pellet (fraction C) was resuspended in about 40 ml of buffer A with the aid of several strokes of a loose-fitting Dounce homogenizer. This suspension was divided into six equal aliquots, and each was carefully layered upon a sucrose step gradient in a 1 x 3.5 in cellulose nitrate tube. All sucrose solutions were prepared in buffer A and concentrations were (w/v) %. Each gradient consisted of 4.5 ml of 5% sucrose, 9.0 ml each of IO and 40% sucrose, and 4.5 ml each of 50 and 60% sucrose. Centrifugation was performed in a Beckman SW27 rotor at 50,000 x g for 2 hr with the brake on. Coated vesicles were obtained by pooling the middle section of the gradient found below the reddish soluble fraction and above the dense material packed upon the 60% sucrose solution. Generally the band at the 10/40% sucrose interface and the IO and 40% sucrose solutions were saved, diluted 3 fold with buffer A and centrifuged at 85,000 x g for 60 min. The concentrated vesicles were resuspended in 6 ml of buffer A with the aid of a loose-fitting Dounce homogenizer and 1 ml (fraction D) layered carefully upon each of 6 5/8 x 4 in tubes containing 11 ml of 5% sucrose, 4 ml of 30% sucrose and 1 ml of 60% sucrose. The samples were centrifuged at 55,000 x g for 45 min in a Beckman SW27.1 rotor (brake on) generating three fractions. Coated vesicles were most highly enriched in the upper 5% sucrose solution (fraction E) and somewhat less so in the band obtained from the 5/30% sucrose interface (fraction F). Typical examples are shown in Figure 2. A variable amount of material containing clathrin but contaminated by other proteins was always recovered upon the 60% sucrose cushion (fraction 6) (Figure 2). When this material was resuspended and recentrifuged on new gradients, it was found on the 60% sucrose cushion. Purified coated vesicles from fractions E and F were stored at 4°C directly or after resuspension in buffer A. Dissociation of Clathrin Various compounds were tested for their ability to release clathrin in a nonsedimentable form. Coated vesicles (25-50 pg of protein)
Clathrin-Coated 311
Vesicles
were sedimented for 30 min in a Beckman Airfuge at room temperature at 130,000 x g. Vesicles were resuspended and incubated for 15 min at room temperature in 100 ~1 of the indicated solution prepared in buffer A adjusted to pH 6.5; resuspension in buffer A was used as a control. The tubes were then recentrifuged as above, and equal aliquots of the supernatants and pellets were analyzed by SDS-polyact-ylamide electrophoresis.
Note
Added
M. Woodward and T. Roth (1978, Proc. Nat. Acad. Sci. USA 75, 4394-4398) have recently reported that basket structures can be reformed from a urea extract of brain-coated vesicles by dilution with urea-free buffer. These results are consistent with our reassembly experiments using unfractionated Tris-Cl extracts. References
Gel Filtration of Solubilized Clathrin Fractions E and F (3.9 and 2.6 mg of protein, respectively, in a representative experiment) were combined, diluted with buffer A and centrifuged at 100,000 x g for 60 min in a Beckman 45Ti rotor. The resulting pellet was resuspended in 3.5 ml of Trisbuffer A [consisting of a mixture of equal volumes of buffer A and 1.0 M Tris-Cl (pH 7.0)] with the aid of a loose-fitting Dounce, incubated for 15 min at room temperature and recentrifuged at 100,000 x g for 45 min as above. The extract was concentrated by the addition of ammonium sulfate to 70% saturation, centrifuged and resuspended in 0.6 ml of Tris-buffer A. This sample was applied to a column (0.9 x 57 cm) containing Sepharose CL-48 equilibrated and eluted by descending flow at 25°C with Trisbuffer A. Fractions of 0.9 ml were collected at a flow rate of 2.5 ml/hr. Column markers used were blue dextran 2000 (v,), rabbit skeletal muscle myosin, bovine thyroglobulin (85 A), rabbit yglobulin (52 A), ovalbumin (30 A) and “C-leucine (v~). Values for Stokes radii were obtained from sources described elsewhere (Yamadaet al., 1977). Electron
Microscopy
Samples of the coated vesicle fractions were fixed by resuspension in 2% glutaraldehyde (Tousimis) in phosphate-buffered saline (PBS) for 10 min at 23°C. These were then centrifuged at 10,000 x g into a pellet. After washing, the samples were postfixed in 1.5% OsOr in PBS for 30 min at 23°C. The pellets were dehydrated in graded ethanol followed by propylene oxide and embedded in Epon 812. Thin sections were cut using a diamond knife and Sorvall MTP-B ultramicrotome, and viewed after staining with uranyl acetate and lead citrate with a Hitachi Hu-12A electron microscope at 50 KV. For negative staining, grids were prepared by coating 250 mesh copper grids with a 0.25% Formvar film followed by glow discharging just prior to use. 10 ~1 samples were applied directly to the grids for 3 min at 23°C and the excess was removed by suction with filter paper. Before allowing the grid to dry, either 1% glutaraldehyde in H,O followed by 3% phosphotungstic acid, or 1% uranyl acetate in H,O directly, was added and immediately removed. The grids were then allowed to air-dry and were viewed immediately at 50 kV. Miscellaneous Procedures Dialysis of small volumes was performed overnight at room temperature using collodion bags, grade UH 100125, obtained from Schleicher and Schuell. Protein was determined using a Coomassie dye binding assay (Bradford, 1978) and Bio-Rad reagent with immunoglobulin standard. SDS gel electrophoresis was performed using a system described earlier (Wallach et al., 1978) with 3% stacking and 6% resolving acrylamide gels. Phosphate was determined by the method of Ames (1966) after ashing the samples with magnesium nitrate. Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received
October
2,1978;
revised
November
22,1978
Ames, B. N. (1966). Assay of inorganic phate and phosphatases. In Methods York: Academic Press), pp. 115-l 18.
phosphate, total phosin Enzymology, 8 (New
Anderson, R. G. W., Goldstein, J. L. and Brown, M. S. (1976). Localization of low density lipoprotein receptors on plasma membrane of normal human fibroblasts and their absence in cells from a familial hypercholesterolemia homozygote. Proc. Nat. Acad. Sci. USA 73, 2434-2438. Anderson, R. G. W., Brown, Role of the coated endocytic bound low density lipoprotein 364.
M. S. and Goldstein, vesicle in the uptake in human fibroblasts.
J. L. (1977). of receptorCell IO, 351-
Blitz, A. L., Fine, R. E. and Tosselli, P. A. (1977). Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum. J. Cell Biol. 75, 135-147. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Friend, D. S. and Farquhar, M. G. (1967). Functions of coated vesicles during protein absorption in the rat vas deferens. J. Cell Biol. 35, 357-376. Heuser, J. E. and Reese, T. S. (1973). Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315-344. Holtzman, E., Novikoff, A. B. and Villaverde, H. (1967). Lysosomes and GERL in normal and chromatolytic neurons of the rat ganglion nodosum. J. Cell Biol. 33, 419-435. Jencks, W. P. (1969). Catalysis (New York: McGraw Hill), p. 360. Kanaseki, T. and Kadota, Cell Biol. 42, 202-220.
I. (1969).
in Chemistry The
“vesicle
and
Enzymology
in a basket”.
J.
Klee, C. (1977). Conformational transitions accompany the binding of calcium to the protein activator of 3’,5’-cyclic adenosine monophosphate diesterase. Biochemistry 76, 1017-1024. Langunoff, D. and Curran, D. E. (1972). Role of bristle-coated membrane in the uptake of ferritin by rat macrophages. Exp. Cell Res. 75, 337-346. Maxfield, F. R., Schlessinger, J., Shechter, Y., Pastan, I. and Willingham, M. C. (1978). Collection of insulin, EGF and ol,macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization. Cell 74, 805-810. Nozaki, Y., Schechter, N. M., Reynolds, J. A. and Tanford, C. (1976). Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemistry 75, 3884-3890. Pearse, B. M. F. (1975). Coated vesicles from pig brain: purification and biochemical characterization. J. Mol. Biol. 97, 93-98. Pearse, B. M. F. (1976). Clathrin: a unique protein associated intracellular transfer of membrane by coated vesicles. Proc. Acad. Sci. USA73, 1255-1259. Roth, T. F. and Porter, K. R. (1964). oocyte of the mosquitoAedes aegypti
with Nat.
Yolk protein uptake in the L. J. Cell Biol. 20, 313-332.
Roth, T. F., Woodward, M. P. and Woods, J. (1977). Comparison and selective dissociation of coated vesicles isolated from brain and oocytes. Abstracts Am. Sot. Cell Biol. 75, 3722.
Cell 312
Schlessinger, J., Shechter, Y., Willingham, M. C. and Pastan, I. (1978). Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc. Nat. Acad. Sci. USA 75, 2659-2663. Singer, S. J. and Nicolson, G. L. (1972). The fluid mosaic of the structure of cell membranes. Science 775, 720-731. Tanford, C., Nozaki, Y., Reynolds, J. A. and Makino, Molecular characterization of proteins in detergent Biochemistry 13, 2369-2376. Wallach, D., Davies, mammalian filamin.
P. J. A. and Pastan, I. (1978). J. Biol. Chem.253, 3328-3335.
Woods, J. W., Woodward, M. P. and Roth, features of coated vesicles from dissimilar and structure. J. Cell Sci. 30, 87-97. Yamada, K. M., Schlesinger, D. H., Kennedy, (1977). Characterization of a major fibroblast protein. Biochemistry 16, 5552-5559.
model
S. (1974). solutions.
Purification
of
T. F. (1978). Common tissues: composition D. W. and Pastan, I. cell surface glyco-
Clathrin-Coated Vesicles: Isolation, and Factor-Dependent Reassociation Baskets James H. Keen, Mark C. Willingham Ira H. Pastan Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20014
and
Summary The nature of the protein coat on clathrin-coated vesicles and the interactions responsible for the structure and reformation of clathrin baskets have been investigated. Coated vesicles were isolated from bovine brain using a rapid, one-day modification of the method of Pearse (1975). The vesicles are composed predominantly of clathrin (175,000) with smaller amounts of 110,000 and 55,000 molecular weight polypeptides. Clathrin was released in a solubilized form from these vesicles by treatment with 0.5 M Tris(hydroxymethyl)methyl ammonium chloride (Tris-Cl) or other protonated amines at neutral pH. It was not released on treatment with thiols, thiol reagents, Triton X-100 or sodium chloride, leading us to suggest that specific amino-carboxylate salt linkages are necessary for maintenance of the basket structure. When viewed by electron microscopy, the solubilized proteins in Tris-Cl are present in the form of filamentous aggregates and no basket structures are observed. Gel filtration of the extract in Tris-Cl resolves a clathrin-containing fraction (I) from one consisting predominantly of the 110,000 molecular weight polypeptide (II). We were able to reconstitute basket structures from the unfractionated Tris-Cl extract by dialyzing it against the vesicle isolation buffer, a solution of moderate ionic strength (r/2 = 0.11). Neither of the resolved fractions (I or II) alone yielded baskets on dialysis, but reconstitution was successful when both I and II were combined and dialyzed. The activity in II, which appears to be a basket-assembly factor, was heat-labile and could not be replaced by bovine serum albumin or brain calcium-dependent modulator protein. If a solution of low ionic strength (r/2 = 0.01) containing calcium was used as the dialysate, the clathrin fraction (I) alone was capable of reforming baskets. Thus the clathrin coat is a labile structure that can be solubilized by nondenaturing treatments, and baskets can be reformed from the extracted material.
Dissociation of Clathrin
oocytes. Coated pits and intracellular vesicles bearing similar coats on their cytoplasmic surfaces have since been reported in many other cell types (Pearse, 1976) and suggested to function in a variety of processes, including receptor-mediated protein uptake (Anderson, Goldstein and Brown, 1976; Anderson, Brown and Goldstein, 1977; M. C. Willingham, F. R. Maxfield and I. H. Pastan, manuscript submitted), nonspecific protein uptake (Friend and Farquhar, 1967) plasma membrane recycling (Heuser and Reese, 1973) and intracellular membrane transfer (Friend and Farquhar, 1967; Holtzman, Novikoff and Villaverde, 1967). Morphological studies on adsorptive endocytosis have indicated that endocytosed materials are initially found predominantly in coated vesicles and subsequently appear in smooth-surfaced vesicles, suggesting that either the coated vesicle rapidly loses its coat or it transfers its contents to another vesicle (Roth and Porter, 1964; Friend and Farquhar, 1967; Lagunoff and Curran, 1972; Anderson et al., 1977). Although many functions for coated membranes and vesicles have been suggested based on morphological observations, little biochemical information was available until coated vesicles were isolated from brain (Pearse, 1975) and other sources (Pearse, 1976). Examination of purified vesicle fractions by SDS gel electrophoresis revealed the predominance of a single protein species of about 175,000 subunit molecular weight. This protein was named clathrin because of its presumed role in the formation of the lattice-like protein coat structure. Urea (Blitz, Fine and Tosselli, 1977) and treatment at pH 10 (Roth, Woodward and Woods, 1977) have recently been reported to release clathrin from coated vesicles. We wished to investigate the interactions responsible for the coat structure with the aim of elucidafing the role played by coated regions in receptormediated endocytosis. Conditions were sought which would dissociate the coat structure and release clathrin in the absence of denaturants or extremes of pH. We report here that high concentrations (0.5 M) of protonated amines at pH 7 solubilize clathrin and other proteins. Clathrin solubilized by this procedure has been reconstituted into basket structures as well as purified from a separable basket-assembly factor. This factor is required for basket reconstitution under ionic conditions that approximate those found in vivo.
Introduction Results Unique regions of plasma membrane with a dense protein coat on their cytoplasmic surface were first described by Roth and Porter (1964) and suggested to play a role in uptake of yolk protein by mosquito
Clathrin-Coated Vesicle Isolation Dissociation A rapid, one-day procedure for
and the
isolation
of
Cell 304
clathrin-coated vesrcles from cow brain, requiring only two gradient centrifugation steps, is described in Experimental Procedures. It is a modification of the method of Pearse (1975). Thin-section electron micrographs (Figure 1) of each of the final fractions (E and F) reveal the presence of coated vesicles similar in size to those reported previously (Pearse, 1975), as well as some uncoated membranous contaminants. Aside from slightly larger amounts of uncoated membrane in the lower vesicle fraction (F), we did not see any other morphological differences between the fractions. SDS-polyacrylamide gels of the major fractions obtained on purification are shown in Figure 2. The uppermost fractions from the second gradient (E and F) were most highly enriched in clathrin, a protein of 175,000 subunit molecularweight. Clathrin comprised about 70% of the total protein in fractions E and F, although polypeptides of 110,000 and 55,000 daltons were also purified in these
fractions, as had been observed earlier (Blitz et al., 1977; Woods, Woodward and Roth, 1978). The mobility of the clathrin band was not altered when N-ethyl maleimide rather than a reducing agent was added before electrophoresis, indicating that clathrin is not disulfide-linked in the coated vesicle (not shown). Only a small amount of clathrin was released from coated vesicles when they were resuspended in isolation buffer (buffer A) (Figure 3). However, clathrin has been reported to be solubilized by treatment with 2 M urea (Blitz et al., 1977) or alkalinization to pH 10 (Roth et al., 1977). We sought conditions allowing solubilization at neutral pH in the absence of denaturants. To this end, various agents were tested for their ability to dissociate clathrin from coated vesicles as determined by release of the protein in a nonsedimentable form (Figure 3). Clathrin was progressively released by increasing concentrations of Tris(hydroxymethyl)methyl ammonium chloride (Tris-Cl) added to buffer A: approximately 80% was solubi-
ABCDEFG
Figure Figure
1. Coated
Vesicles
(A) Fraction E (magnification F (magnification 29,000X; vesicles.
from
Bovine
Brain
34,000X; bar = 0.2 pm); (B) fraction bar = 0.2 pm). Arrows denote coated
Molecular (37 pg); (39 pg); fraction second
2. Analysis
of Coated
Vesicle
Purification
by SDS-PAGE
weight scale (in daltons x~O-~) at left. (A) Homogenate (B) 20,000 x g supernatant (45 pg); (C) 85,000 x g pellet (D) first gradient pool (23 pg); (E) second gradient upper (7 rg): (F) second gradient middle fraction (5 pg); (G) gradient lower fraction (7 pg).
Clathrin-Coated 305
Vesicles
lized by 0.5 M Tris-Cl (Tris-buffer A). At 0.5 M and pH 6.5, triethanolamine chloride, ammonium chloride and imidazole chloride were also effective, whereas uncharged hydroxylamine was not. Release was not simply due to elevated ionic strength or the hydroxyl groups of Tris since neither 0.5 M NaCl nor 0.5 M glycerol were effective. Other ineffective compounds include Triton X-100 (l%), dithiothreitol (5 mM), glutathione (5 mM), p-chloromercuribenzoate (5 mM), protamine sulfate (1 mg/ml), D-glucosamine (0.1 M), benzocaine (1 mM), dibucaine (1 mM), colchicine (50 pg/ml) and histone 2a (7.5 mg/ml). Clathrin was not the only protein released by these treatments; it was accompanied by other proteins whose relative amounts depend upon the agent used. Treatment at pH 10 released all polypeptides uniformly, whereas Tris extracts were enriched in clathrin and to a lesser extent in a polypeptide of 110,000 daltons. Lower molecular weight material was retained in the pellet. Since coated vesicles also contain lipid, we measured the amount of phospholipid released by Tris-Cl treatment. A coated vesicle preparation containing 110 pg of protein and 47 nmole of lipid phosphorous was treated with Tris-Cl and centrifuged. After centrifugation, 42 nmole of lipid phosphorous and 54 pg of protein remained in the pellet. Thus TrisCl treatment solubilized no more than 10% of the phospholipid. Morphological evidence for disruption of the clathrin basket structure was obtained by negativestaining electron microscopy. The Tris extract displayed a pattern of linear aggregates (Figure 4A). Intact clathrin coats or basket-like structures were not observed. These structures were also not found in the Tris-treated vesicle pellet when examined by thin-section electron microscopy (not shown). Reconstitution of Clathrin Baskets Tris-extracted vesicles were centrifuged to remove uncoated vesicle remnants, and the resulting supernatant was dialyzed overnight at room temperature against buffer A to remove Tris-Cl. When the retentate was examined by electron microscopy (Figure 4B), it was apparent that ordered “basketlike” structures were reformed (compare Figures IA and 4A). The reconstituted structures were of similar size to that of the intact coated vesicle. Unlike intact vesicles, however, they did not possess lipid bilayer membranes when examined by electron microscopy. In fact, when the entire Tristreated solution was dialyzed, rather than the soluble extract alone, protein basket structures reformed entirely separately from the membranous material present (not shown). Reconstituted baskets prepared as described above had an unusual property: they were not
readily sedimentable. Unlike the parent coated vesicles, these reconstituted baskets did not sediment at 130,000 X g for 60 min. On 5-20% sucrose gradients in buffer A, intact vesicles had sedimentation constants in excess of 150s. Under the same conditions, clathrin in reconstituted baskets sedimented only slowly as a discrete 12s species. This may reflect a pressure-dependent dissociation under these conditions of ionic strength since negative-staining electron microscopy revealed linear protein aggregates but no basket structures in the clathrin peak after centrifugation. S. Puszkin (personal communication) has reported, and we have confirmed, that basket reconstitution may also be accomplished by dialysis against very dilute buffers. The reformed baskets obtained under these conditions of low ionic strength were morphologically identical to those described above, but were more stable to centrifugation and were entirely sedimentable at 100,000 X g for 30 min. Fractionation of the Tris Extract When the Tris-treated vesicle extract was chromatographed on a Sepharose CL-4B column equilibrated with Tris-buffer A, two protein fractions were obtained (Figure 5, lower panel). SDS gel electrophoresis of these fractions on 6% (Figure 5, upper panel) and 13% (not shown) acrylamide gels revealed the first peak (I) to be composed predominantly of clathrin, with only trace amounts of polypeptide of 90,000 molecular weight. The following peak (II) was almost entirely devoid of clathrin but greatly enriched in a peptide of about 110,000 daltons and, to a lesser and variable extent, in one of 55,000 daltons. By comparison with a standard curve prepared using marker proteins (Tanford et al., 1974), the elution volume of fraction II indicates a Stokes radius of 68 A and, assuming a globular conformation, a molecular weight of approximately 420,000 daltons. It was not possible to perform the same calculations confidently for the clathrin peak (fraction I) because its elution volume was beyond the range of the standard curve (Nozaki et al., 1976). As an approximation for the purpose of comparison, however, a Stokes radius of about 160 A was estimated by extrapolation, corresponding to a molecular weight of 1,200,OOO daltons. Dialysis of fraction I against buffer A yielded only filamentous aggregates of protein with no basket structures reformed (Figure 6A). Similarly, dialysis of fraction II did not result in basket formation (Figure 6B). However, when equal volumes of each of these fractions were combined and then dialyzed against buffer A, ordered basket structures were clearly obtained (Figure 6C). Fraction II could not be replaced by varied amounts of either bovine serum albumin or pig brain calcium-dependent
Cell 306
1 s
2 s
P
P
s
P
s
6
5
4
3
P
s
P
s
P
68
A Figure
3. Analysis
of Clathrin
Release
from
Coated
Vesicles
by SDS-PAGE
Coated vesicles from three preparations (gels 1-4, 5-9 and 10-12) were used. Vesicles (25-50 pg protein) were resuspended in 100 /.LI of the indicated solutions prepared in buffer A and centrifuged after 15 min. Each pair of gel lanes represents equal aliquots of the resultant supernatants (S) and pellets (P) from one treatment of vesicles. (1) Control [buffer A: 0.1 M Na MES (pH 6.5), 1 mM EGTA, 0.5 mM MgCI,, 0.02% sodium azide]; (2) 0.05 M Tris-Cl (pH 7.0); (3) 0.03 M Tris-Cl (pH 7.0); (4) 0.5 M Tris-Cl (pH 7.0); (5) 0.5 M triethanolamine chloride (pH 6.5); (6) 0.5 M ammonium chloride (pH 6.5); (7) 0.5 M imidazole chloride (pH 6.5); (8) 0.5 M hydroxylamine (pH 6.5); (9) 25 mM sodium carbonate (pH 10) without buffer A; (10) 0.5 M sodium chloride; (11) 0.5 M glycerol: (12) 1% Triton X-100.
modulator protein (Klee, 1977), and its ability to promote basket reassembly was abolished by heating at 100°C for 4 min. This suggests that one or more basket assembly factors are present in II and are required for reassembly under these conditions. Based on the relative amounts of protein in the two fractions, we estimate that there are at least 3 clathrin monomers per 110,000 dalton polypeptide in the coated vesicle preparation. We further investigated the dependence of basket reassembly on fraction II as a function of ionic strength and the presence or absence of divalent cations. Aliquots of fraction I were mixed with equal volumes of fraction II or Tris-buffer A and then dialyzed against either buffer A or a more dilute buffer [5 mM Na MES (pH 6.5)]. CaCI,, MgCI,, NaCI, KCI, EDTA or ethyleneglycol-bis-(p-aminoethyl ether)N,N’-tetraacetic acid (EGTA) were added to the dialysates as indicated in Table 1.
After dialysis, the retentates were assayed for reconstituted basket structures by negative-staining electron microscopy. Baskets were reformed when fraction I alone was dialyzed against the dilute MES buffer (r/Z -0.01) containing Ca++ (Table 1, line 1). No reassembly for Ca++ was observed when Mg ++ was substituted (line 5) or when both were omitted (line 3). The combination of fraction I with II was found to promote basket reassembly under these conditions of ionic strength irrespective of the presence of divalent cations (lines 2, 4 and 6). That the action of fraction II was not due to calcium contained in the fraction is indicated by its activity despite the presence of EGTA (lines 4 and 6). When dialysis was performed against buffer A, a solution of moderate ionic strength (r/2 -O.ll), baskets were not reformed from fraction I alone in the presence or absence of Ca++ or Mg++ (Table 1,
Clathrin-Coated 307
Vesicles
8
7 s
P
s
9 P
s
10 P
s
11 P
s
12 P
s
P
250
165
94
68
lines 7, 9 and 11); the addition of fraction II was required for reassembly under these conditions (lines 8, IO and 12). The requirement for fraction II is a consequence of the ionic strength of the medium rather than of the MES concentration. When the ionic strength of the dilute MES buffer was made equal to that of buffer A by the addition of either KCI or NaCl (line 13), fraction I alone was no longer capable of basket reassembly and fraction II was required (lines 14), as observed with buffer A. Discussion To understand the structure and function of coated membrane regions, we have attempted to explore some of the physical interactions within isolated coated vesicles. As found by Pearse (1975) and other investigators (Blitz et al., 1977), bovine brain is a rich source of coated vesicles, and the modified isolation procedure reported here is rapid and should simplify further experimentation. The protein coat of clathrin-coated vesicles is a labile structure held together by noncovalent bonds. Clathrin monomers are not disulfide-linked
to other proteins in the coated vesicle, since their electrophoretic mobility on denaturing gels was not effected by prior treatment with either alkylating or reducing agents. Furthermore, clathrin was not solubilized from coated vesicles by treatment with either free thiols or thiol reagents. There were, however, a wide variety of agents that effectively dissociated it from the lipid vesicle. That Tris and other amines were effective, rather than Triton X100, indicates that clathrin is not an instrinsic membrane protein (Singer and Nicolson, 1972). Solubilized clathrin in Tris-Cl was not fully dissociated but was a discrete aggregate of approximately 6 monomers that may represent a protomerit functional unit. It was easily separable by gel filtration from another major protein fraction eluting in the molecular weight range of 420,000 daltons. The latter fraction (II) played a role in promoting reconstitution and had a more varied composition, with a 110,000 dalton polypeptide predominating. The Tris effect is specific for the amine function, rather than being an effect of either the hydroxyl moiety or elevated ionic strength, since neither glycerol nor sodium chloride were effective in re-
Cell
308
Figure 5. Gel Filtration ibrated with Tris Buffer
of Tris Extract A
on Sepharose
CL-4B
Equil-
6.7 mg of protein in 0.6 ml of Tris-buffer A were applied to a 0.9 x 57 cm column, and fractions of 0.9 ml were collected. Lower panel: protein elution profile. Column standards used were blue dextran 2000 (V,), myosin, thyroglobulin, y-globulin, ovalbumin and “C-leucine (V,). Upper panel: SDS-PAGE analysis of eluted fractions. Fractions 21-26 were pooled and designated I, while II consisted of fractions 29-31.
Figure 4. Structure and after Dialysis
of Extracted
Coated
Vesicle
Protein
before
(A) Electron micrograph of Tris extract of coated vesicles, negatively stained (magnification 79,000X; bar = 0.1 pm). (B) Electron micrograph (thin section) of reconstituted clathrin baskets formed by dialysis of Tris extract (magnification 65,000X; bar = 0.1 pm).
leasing clathrin. Although amines could act through a variety of mechanisms, we suggest that specific amino-carboxylate salt bridges may be necessary for the integrity of the clathrin basket structure. According to this hypothesis, disruption of the basket structure by high concentrations of protonated amines results from competition with protein-bound amino groups, while disruption by treatment at pH 10 reflects deprotonation of the protein amino groups. Hydroxylamine is probably ineffective in releasing clathrin because it is largely unprotonated at pH 6.5 (pKa = 6.03). Although an ionic strength effect might also be anticipated, we saw no release of clathrin from coated vesicles by sodium chloride at concentrations up to 0.5 M. Ionic strength did, however, have a critical effect on basket structure in determining both the extent of reconstitution and the stability of reassembled
baskets. The effect of urea is more difficult to interpret. It may reflect disruption of critical salt linkages or a more general denaturing action upon coat protein structures (Jencks, 1969). It is probable that the important interactions which Tris-Cl disrupts in solubilizing basket proteins are clathrin-clathrin rather than clathrin-vesicle interactions, since reconstitution of the basket structure occurred freely although the stripped vesicles had been removed. In contrast to the absence of an effect of the stripped vesicles, reconstitution was strongly dependent upon the ionic strength of the medium, fraction II and Ca++. In solutions of moderate ionic strength (r/2 ~0.11) similar in magnitude to those found in vivo, purified clathrin alone has not been observed to reform baskets upon removal of Tris-Cl under any of the conditions tested. However, the addition of a partially purified heat-labile factor promoted reconstitution. The data indicated that the requirement was specific. In the presence of fraction II, clathrin reassociated regardless of the presence of either Ca++, Mg++ or either of their chelators. Further characterization of fraction II is in progress. Following the report of S. Puszkin (personal
Clathrin-Coated
Vesicles
309
Figure
6. Reconstitution
of Clathrin
Baskets
from
Extract
Fractionated
Negatively stained electron micrographs of Sepharose fractions (A) Fraction I alone (magnification 66,000X); (6) fraction II alone
by Gel Filtration
I and II dialyzed against (66,000X); (C)fractions
communication), we have also been able to reconstitute baskets by dialysis against very dilute buffer solutions. Using purified clathrin and calcium-containing medium of low ionic strength (r/2 =O.Ol), we found that baskets reformed without the addition of other components, indicating that clathrin, rather than one of the other polypeptides in the coated vesicle fraction (Blitz et al., 1977; Roth et al., 1977), is the structural component of the protein coat. Morphological studies have demonstrated that the coat protein structure is associated with only limited regions of the plasma membrane of most cells (Friend and Farquhar, 1967; Anderson et al., 1976). Low-density lipoprotein (Anderson et al., 1977), aZ-macroglobulin, insulin and epidermal growth factor (Maxfield et al., 1978; Schlessinger et al., 1978; M. C. Willingham et al., manuscript
buffer A to remove Tris-Cl (bar = 0.1 pm in I and II combined (79,000X).
submitted) have been shown to cluster specifically over coated regions prior to internalization, suggesting that the protein coat may be involved in the process of patching. Furthermore, it has been speculated that the coat serves as an anchorage point (Kanaseki and Kadota, 1969) in the process of invagination that results in the formation of a coated pit and, ultimately, a coated vesicle. At early times, ferritinor peroxidase-labeled ligands are seen predominantly in these coated vesicles, but there is a rapid and progressive appearance of ligand in smooth-surfaced vesicles (Roth and Porter, 1964; Friend and Farquhar, 1967; Lagunoff and Curran, 1972; Anderson et al., 1977). The molecular properties of isolated clathrincoated vesicles and reconstituted baskets reported here are consistent with these observations. That the clathrin-coat structure survives the several iso-
Cell 310
Table 1. Clathrin Basket Reconstitution as a Function of Buffer Concentration and the Presence or Absence of Divalent Cations Fraction II
Dialysate (1) (2)
(3) (4)
(5) (6)
(7)
(8)
5 mM Na CaCI, + 5 mM Na CaCI, +
MES (pH 6.5) + 2 mM 1 mM EDTA MES (pH 6.5) + 2 mM 1 mM EDTA
5 mM Na MES (pH 6.5) EGTA + 1 mM EDTA 5 mM Na MES (pH 6.5) EGTA + 1 mM EDTA
Buffer A [IO0 mM Na MES 6.5) + 0.5 mM MgClz + 1 EGTA + 0.02% Na azide] Buffer A [lo0 mM Na MES 6.5) + 0.5 mM MgCI, + 1 EGTA + 0.02% Na azide] Buffer Buffer
(11)
BufferA+2mMEDTA+2mM CaCI, Buffer A + 2 mM EDTA CaCI,
(13)
(14)
-
+
+
+
-
-
+
+
+ 1 mM
5 mM Na MES (pH 6.5) + 2 mM MgCI, + 1 mM EGTA 5 mM Na MES (pH 6.5) + 2 mM MgCI, + 1 mM EGTA
(9) (10)
(12)
+ 1 mM
+
(pH mM (pH mM
A + 2 mM EDTA A + 2 mM EDTA
-
+ -
-
+
+
-
-
+
+
Equal aliquots of fraction I and Tris-buffer dialyzed against the solutions indicated. assayed for reconstituted basket structures electron microscopy.
Experimental
function, depend which are likely
Procedures
Materials Electrophoresis grade reagents for SDS gel electrophoresis were from Bio-Rad. The molecular weight standards used are described elsewhere (Wallach, Davies and Pastan, 1978). Sepharose CL-48 was a product of Pharmacia, and ultra-pure Tris base and sucrose were from Schwartz/Mann or Bethesda Research Laboratories. Goat anti-rabbit immunoglobulin was from Miles, and ovalbumin and bovine serum albumin were obtained from Sigma. Pig brain calcium-dependent modulator protein was a gift from C. Klee (NIH); bovine thyroglobulin was a gift from A. Burkhardt (NIH); and rabbit skeletal muscle myosin was a gift from L. Greene and E. Eisenberg (NIH).
+
+ +
+
+ 2 mM
5 mM Na MES (pH 6.5) + 2 mM CaCI, + 1 mM EDTA + 90 mM NaCl or KCI 5 mM Na MES (pH 6.5) + 2 mM CaCI, + 1 mM EDTA + 90 mM NaCl or KCI
Reconstituted Baskets
coat, and probably its specific upon these additional factors, candidates for in vivo modulation.
A or fraction II were The retentates were by negative-staining
lation procedures suggests that it is rigid enough to fulfill a structural role intracellularly in the process of pinocytosis, but the clathrin-coat structure is also a product of noncovalent interactions that are readily dissociable under nondenaturing conditions. This property of reversible disaggregation of clathrin over an entire vesicle could account for rapid transformation of a coated to a smooth-surfaced vesicle. Alternatively, a localized dissociation of the coat could allow selective fusion with an uncoated vesicle and subsequent transfer of endocytosed contents. More direct information on the fate of coated vesicles is required to distinguish between these possibilities. The reassociation process in vitro does not require lipid vesicles but is sensitive to Ca++, ionic strength and the presence of a separable factor. Thus while information for the protein coat structure resides in clathrin, the site and stability of the
Purification of Coated Vesicles Bovine brains were obtained from Frederick County Products (Frederick, Maryland) immediately after slaughter and placed on ice. All operations were performed at 0-4°C unless otherwise noted. Within 2 hr, purification was begun. The meninges were removed from the gray matter, the medulla was discarded, and the remaining whole brain (generally about 500 g) was minced coarsely and placed in a Waring blender with an equal volume of 0.1 M sodium MES (pH 6.5) 1 mM EGTA, 0.5 mM MgCI,, 0.02% sodium azide (buffer A). Homogenization was performed with three IO set bursts at full speed. The homogenate (fraction A) was centrifuged at 20,000 x g (all values are average centrifugal forces) for 30 min in a Sorvall GSA rotor, and the resulting pellet was discarded. The supernatant (fraction B) was centrifuged at 85,000 x g for 60 min in a Beckman 45Ti rotor; the soluble fraction was removed, and the vesicle pellet (fraction C) was resuspended in about 40 ml of buffer A with the aid of several strokes of a loose-fitting Dounce homogenizer. This suspension was divided into six equal aliquots, and each was carefully layered upon a sucrose step gradient in a 1 x 3.5 in cellulose nitrate tube. All sucrose solutions were prepared in buffer A and concentrations were (w/v) %. Each gradient consisted of 4.5 ml of 5% sucrose, 9.0 ml each of IO and 40% sucrose, and 4.5 ml each of 50 and 60% sucrose. Centrifugation was performed in a Beckman SW27 rotor at 50,000 x g for 2 hr with the brake on. Coated vesicles were obtained by pooling the middle section of the gradient found below the reddish soluble fraction and above the dense material packed upon the 60% sucrose solution. Generally the band at the 10/40% sucrose interface and the IO and 40% sucrose solutions were saved, diluted 3 fold with buffer A and centrifuged at 85,000 x g for 60 min. The concentrated vesicles were resuspended in 6 ml of buffer A with the aid of a loose-fitting Dounce homogenizer and 1 ml (fraction D) layered carefully upon each of 6 5/8 x 4 in tubes containing 11 ml of 5% sucrose, 4 ml of 30% sucrose and 1 ml of 60% sucrose. The samples were centrifuged at 55,000 x g for 45 min in a Beckman SW27.1 rotor (brake on) generating three fractions. Coated vesicles were most highly enriched in the upper 5% sucrose solution (fraction E) and somewhat less so in the band obtained from the 5/30% sucrose interface (fraction F). Typical examples are shown in Figure 2. A variable amount of material containing clathrin but contaminated by other proteins was always recovered upon the 60% sucrose cushion (fraction 6) (Figure 2). When this material was resuspended and recentrifuged on new gradients, it was found on the 60% sucrose cushion. Purified coated vesicles from fractions E and F were stored at 4°C directly or after resuspension in buffer A. Dissociation of Clathrin Various compounds were tested for their ability to release clathrin in a nonsedimentable form. Coated vesicles (25-50 pg of protein)
Clathrin-Coated 311
Vesicles
were sedimented for 30 min in a Beckman Airfuge at room temperature at 130,000 x g. Vesicles were resuspended and incubated for 15 min at room temperature in 100 ~1 of the indicated solution prepared in buffer A adjusted to pH 6.5; resuspension in buffer A was used as a control. The tubes were then recentrifuged as above, and equal aliquots of the supernatants and pellets were analyzed by SDS-polyact-ylamide electrophoresis.
Note
Added
M. Woodward and T. Roth (1978, Proc. Nat. Acad. Sci. USA 75, 4394-4398) have recently reported that basket structures can be reformed from a urea extract of brain-coated vesicles by dilution with urea-free buffer. These results are consistent with our reassembly experiments using unfractionated Tris-Cl extracts. References
Gel Filtration of Solubilized Clathrin Fractions E and F (3.9 and 2.6 mg of protein, respectively, in a representative experiment) were combined, diluted with buffer A and centrifuged at 100,000 x g for 60 min in a Beckman 45Ti rotor. The resulting pellet was resuspended in 3.5 ml of Trisbuffer A [consisting of a mixture of equal volumes of buffer A and 1.0 M Tris-Cl (pH 7.0)] with the aid of a loose-fitting Dounce, incubated for 15 min at room temperature and recentrifuged at 100,000 x g for 45 min as above. The extract was concentrated by the addition of ammonium sulfate to 70% saturation, centrifuged and resuspended in 0.6 ml of Tris-buffer A. This sample was applied to a column (0.9 x 57 cm) containing Sepharose CL-48 equilibrated and eluted by descending flow at 25°C with Trisbuffer A. Fractions of 0.9 ml were collected at a flow rate of 2.5 ml/hr. Column markers used were blue dextran 2000 (v,), rabbit skeletal muscle myosin, bovine thyroglobulin (85 A), rabbit yglobulin (52 A), ovalbumin (30 A) and “C-leucine (v~). Values for Stokes radii were obtained from sources described elsewhere (Yamadaet al., 1977). Electron
Microscopy
Samples of the coated vesicle fractions were fixed by resuspension in 2% glutaraldehyde (Tousimis) in phosphate-buffered saline (PBS) for 10 min at 23°C. These were then centrifuged at 10,000 x g into a pellet. After washing, the samples were postfixed in 1.5% OsOr in PBS for 30 min at 23°C. The pellets were dehydrated in graded ethanol followed by propylene oxide and embedded in Epon 812. Thin sections were cut using a diamond knife and Sorvall MTP-B ultramicrotome, and viewed after staining with uranyl acetate and lead citrate with a Hitachi Hu-12A electron microscope at 50 KV. For negative staining, grids were prepared by coating 250 mesh copper grids with a 0.25% Formvar film followed by glow discharging just prior to use. 10 ~1 samples were applied directly to the grids for 3 min at 23°C and the excess was removed by suction with filter paper. Before allowing the grid to dry, either 1% glutaraldehyde in H,O followed by 3% phosphotungstic acid, or 1% uranyl acetate in H,O directly, was added and immediately removed. The grids were then allowed to air-dry and were viewed immediately at 50 kV. Miscellaneous Procedures Dialysis of small volumes was performed overnight at room temperature using collodion bags, grade UH 100125, obtained from Schleicher and Schuell. Protein was determined using a Coomassie dye binding assay (Bradford, 1978) and Bio-Rad reagent with immunoglobulin standard. SDS gel electrophoresis was performed using a system described earlier (Wallach et al., 1978) with 3% stacking and 6% resolving acrylamide gels. Phosphate was determined by the method of Ames (1966) after ashing the samples with magnesium nitrate. Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received
October
2,1978;
revised
November
22,1978
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Klee, C. (1977). Conformational transitions accompany the binding of calcium to the protein activator of 3’,5’-cyclic adenosine monophosphate diesterase. Biochemistry 76, 1017-1024. Langunoff, D. and Curran, D. E. (1972). Role of bristle-coated membrane in the uptake of ferritin by rat macrophages. Exp. Cell Res. 75, 337-346. Maxfield, F. R., Schlessinger, J., Shechter, Y., Pastan, I. and Willingham, M. C. (1978). Collection of insulin, EGF and ol,macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization. Cell 74, 805-810. Nozaki, Y., Schechter, N. M., Reynolds, J. A. and Tanford, C. (1976). Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemistry 75, 3884-3890. Pearse, B. M. F. (1975). Coated vesicles from pig brain: purification and biochemical characterization. J. Mol. Biol. 97, 93-98. Pearse, B. M. F. (1976). Clathrin: a unique protein associated intracellular transfer of membrane by coated vesicles. Proc. Acad. Sci. USA73, 1255-1259. Roth, T. F. and Porter, K. R. (1964). oocyte of the mosquitoAedes aegypti
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model
S. (1974). solutions.
Purification
of
T. F. (1978). Common tissues: composition D. W. and Pastan, I. cell surface glyco-