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[24] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f F u n c t i o n a l Clathrin-Coated Endocytic Vesicles
By PHILIP G. WOODMAN and GRAHAM WARREN Introduction The transferrin cycle is well characterized, and serves as a model of receptor-mediated endocytosis. ~ Halotransferrin binds to cell surface receptors located in clathrin-coated pits. These pits invaginate and pinch off to form endocytic, clathrin-coated vesicles, which carry the transferrin into the cell. Removal of at least part of the clathrin coat enables transferrin to be delivered to the endosome by a membrane fusion event. Once exposed to the low pH of the endosome, iron dissociates from transferrin and the apoprotein is recycled back to the plasma membrane by vesicular transport. For the transferrin cycle to work efficiently, vesicles must recognize and fuse with their target membrane. The biochemical specificity that underlies this selection has allowed us to reconstitute fusion of endocytic vesicles in a cell-free system. The assay for vesicle fusion requires the preparation of "donor" endocytic vesicles containing ~25I-labeled transferrin. These are mixed with "acceptor" endocytic vesicles, containing internalized antitransferrin antibody, in the presence of a cytosol fraction and an ATP-regenerating cocktail. Vesicle fusion permits the formation of a radiolabeled immunocomplex, which is isolated on Staphylococcus aureus cells after solubilization of the vesicle membrane. Measurement of the fusion of endocytic vesicles within crude preparations has been described in detail elsewhere.2 However, further analysis of the interaction between cytosolic and membrane components during the fusion reaction requires use of purified membranes as substrates. We have chosen to isolate a donor preparation of clathrin-coated vesicles, 3 because this is the best characterized endocytic compartment. In addition, the unique composition of coated vesicles simplifies both purification and identification. Here, we describe the isolation of functional clathrin-coated vesicles. Isolation is monitored by the ability to fuse with acceptor endocytic vesicles.
z R. D. Klausner, G. Ashwell, J. v a n Renswoud¢, J. B. Harford, a n d K. R. Bridges, Proc Natl. Acad. Sci. USA 80, 2263 (1980). 2 p. G. W o o d m a n a n d G. Warren, Methods Cell Biol. 31, 197 0 9 8 9 ) . 3 p. G. W o o d m a n a n d G. Warren, J. Cell Biol. 112, 1133 (1991).
METHODS IN ENZYMOLOGY, VOL. 219
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Isolation of Donor Clathrin-Coated Vesicles
Cells All membrane and cytosol fractions used in this study are prepared from A431 cells, a human cell line rich in transferrin receptors? A431 cells are grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal calf serum and 100 U/ml of penicillin and streptomycin, in an atmosphere of 95% air/5% CO2. After trypsinization cultures are split 1:5 every 48 hr. All tissue culture media and supplements were bought from Northumbria Biologicals, Ltd, Northumberland, UK.
Radiolabeling Human transferrin is radiolabeled with iodine-125 according to the method of Fraker and Speck.5 Dissolve lodogen (1,3,4,6-tetrachloro3ot,6c~-diphenylglycoluril; Pierce Chemical Co., Rockford, IL) to 0.5 mg/ ml in chloroform. Evaporate 20/zl in a glass tube under a stream of nitrogen. Add 30/tl sodium phosphate buffer, pH 7.2, containing 100/zg human transferrin. To this add 2.5 mCi (25/~1) NanSI (16 mCi/#g; Amersham, UK). Incubate for 15 min on ice, then stop the reaction by addition of 166 mg/ml unlabeled KI (50 ~1) and 2.5 /xg/ml sodium metabisulfite (50/zl). Remove the free iodine by gel filtration over a BioGel P-6 column (Bio-Rad Laboratories, Richmond, CA), prewashed with sodium phosphate buffer containing 0.1 mg/ml bovine serum albumin (BSA) and equilibrated with BSA-free phosphate buffer, followed by dialysis against 400 vol of the same buffer. This method should achieve a specific activity of 2-3 × 107 counts per minute (cpm)//zg transferrin.
Isolation of Coated Vesicles Donor coated vesicles are prepared using a modification of the method of Pearse, 6 and isolation is monitored by the cell-free assay for vesicle fusion (see below). Care must be taken to avoid pelleting the membranes, because this leads to aggregation and loss of activity. The preparation is carried out at pH 6.6 throughout, to stabilize the clathrin coat. For a standard preparation, grow A431 cells to near confluence on four 24 × 24 cm tissue culture dishes, supplied by GIBCO (Paisley, Scotland). For each dish, wash cells with ice-cold Dulbecco's phosphate-buffered saline (PBS), then incubate on a slowly rocking platform at 4 ° with 15 ml bind4 C. R. Hopkins and I. S. Trowbridge, J. CellBiol. 97, 508 (1983). s p. j. Fraker and J. C. Speck, Biochem. Biophys. Res. Commun. 80, 849 (1978). 6 B. M. F. Pearse, Proc. Natl. Acad. Sci. USA 79, 451 (1982).
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ing medium [BM; DMEM containing 20 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, and 0.2% (w/v) BSA] containing 1.5 #g/ml 125I-labeled transferrin. After 2 hr, wash the cells three times in PBS, and incubate for 2 min at 31 ° with 50 ml prewarmed BM. The short period for internalization should ensure that the greatest proportion of internalized transferrin is in coated vesicles. Wash the dish four times in 50 ml ice-cold vesicle buffer [140 m M sucrose, 0.5 m M MgC12, l m M EGTA, 20 raM 2-(N-morpholino)ethanesulfonic acid (MES), 70 m M potassium acetate, pH 6.6]. Drain for 2 min at 4 ° to remove excess buffer, then scrape the cells from the dish with a rubber policeman. Suspensions from four plates should be combined (approximately 3 - 4 ml), supplemented with dithiothreitol (DTT; 1 raM) and protease inhibitors [ 1 #g/ml antipain, 1 #g/ml chymostatin, 1 #g/ml pepstatin, 2 #g/ml E64, 40 #g/ml phenylmethylsufonyl fluoride (PMSF), all stored in a 1000× concentrate in dimethyl sulfoxide (DMSO)], and broken in a stainless-steel homogenizer. Cells are passed 10 times through a 0.2540-in. bore containing a 0.2530-in. ball. 7 These conditions should break 80-90% of cells with little damage to nuclei, as assessed by trypan blue staining and microscopy. Prepare a postnuclear supernatant by centrifuging at 500 g,v for 5 min at 4 °. Polyribosomes, potential contaminants, are disassembled by incubating the extract with ribonuclease A (50 #g/ml; Worthington Enzymes, Ltd., Freehold, NJ)for 30 min at 4 °. Centrifuge at 7000 g,v for 30 min at 4 °, and then apply the supernatant (approximately 3 ml) to a 10-ml continuous deuterium oxide (D20) rate sedimentation gradient of 10-90% (w/v) D20 in vesicle buffer, containing 1 m M DTT throughout. Centrifuge for 30 min at 45,000 g,v in an SW40 rotor (Beckman Instruments, Inc., Palo Alto, CA) and collect l-ml fractions from the bottom by tube puncture. The coated vesicles sediment slowly, and should remain in the top 5 ml of the gradient. This should be monitored by assaying 50-#1 samples for vesicle fusion activity (see below). Pool the peak fractions, dilute to 18 ml in vesicle buffer containing 1 m M DTT, and apply to the top of an equilibrium density gradient (20 ml) of 2% (w/v) FicoU/9% (w/v) D20-20% (w/v) Ficoll/90% (w/v) 1)20 in vesicle buffer containing I m M DTT. (Before use, the Ficoll should be dissolved in water, dialyzed extensively to remove low molecular weight contaminants, and lyophilized.) Centrifuge for 16 hr at 80,000 g,, in a Beckman SW28 rotor and remove twenty l-ml fractions from the bottom by using a fine capillary tube attached to a peristaltic pump. Samples (50 #1) from each fraction should be analyzed for vesicle fusion activity. As indicated below, to dilute
7 W. E. Balch, W. G. Dunphy, W. A. Braell, and J. E. Rothman, Cell 39, 405 (1984).
254
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out the viscous Ficoll and vesicle isolation buffer, samples should be diluted at least 1 : 4 into the fusion assay mix. Analysis of Donor Coated Vesicle Preparations
Endocytic VesicleFusion Activity Because fusion occurs only between endocytic compartments,2 only those fractions supporting fusion activity should contain clathrin-coated vesicles. ~25I-labeled transferrin migrates as two peaks on the equilibrium gradient (Fig. 1). Only the lower peak possesses fusion activity and, therefore, contains endocytic vesicles. Observed etficiency of fusion is dependent on the concentration of acceptor membranes; with an excess of acceptor membranes, up to about 40% of total L2SI-labeled transferrin should be precipitated in an ATP-dependent manner. The peak migrates to the same position as placental clathrin-coated vesicles isolated on similar gradients. The unique properties of clatbrin-coated vesicles permit several independent methods of confirming that vesicles in this peak are coated, and of assessing the purity of the preparation. Analysis can include detection of coated vesicle proteins by Western blotting with anti-(coat protein)
150 -
3000
loo-
l
20o0 c
:.,
E:
i
~'==
0
o j
E
e,s •
, 10
•
0 20
Fraction number
FIG. l. Isolation of endocytic coated vesicles. Fractions (1 ml) from the D20/Ficoll density gradient are sampled (100/A) for '2~I-labeled tmnsferrin and endocytic vesicle fusion activity. Fusion activity is expressed as the ATP-dependent immunoprecipitation o f l:~Ilabeled transferrin.
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255
antisera, electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and susceptibility of the preparation to the action of the clathrin-uncoating ATPase3
Western Blotting To obtain sufficient material to detect coat proteins by Western blotting, supplement the normal donor vesicle preparation with twelve 24 × 24 cm dishes of untreated cells. After collecting the fractions from the equilibrium gradient, sediment vesicles by diluting 10-fold in vesicle buffer and centrifuging for 2 hr at 100,000 g,v in a Beckman SW40 rotor. Wash the pellets carefully with vesicle buffer, and resuspend in sample buffer [1 M sucrose, 200 mM Tris-HC1, pH 6.5, 5 mM ethylene diamine tetraacetic acid (EDTA), 0.04% (w/v) bromphenol blue, 10 mM DTT, 4% (w/v) SDS], boil for 5 min, cool, add 5 m M iodoacetamide, and electrophorese overnight (70 V, constant voltage) on a 10% (w/v) polyacrylamide gel. Transfer to nitrocellulose, according to the method of Towbin, for 2 hr at 1.5 A. Incubate the nitrocellulose on a rocking platform in Tris/salt buffer (200 mM Tris-HC1, pH 7.4, 150 mM NaC1) containing 0.2% (w/v) polyoxyethylene sorbitan monolaurate (Tween 20) and 0.1% (w/v) fish skin gelatin. Incubate with the appropriate dilution of an anti-(coat protein) antiserum (e.g., anti-clathrin light chain) in the same buffer for 90 min at room temperature, then wash three times in 1 hour with Tris/salt containing 0.05% (w/v) Tween 20, followed by Tris/salt without Tween 20. Incubate for 60 min at room temperature with t2Sl-labeled protein A [0.1 ltCi/ ml in Tris/salt containing 5% (w/v) BSA]. Wash three times in Tris/salt containing 0.05% (w/v) Tween 20, dry, and expose to photographic film.
SDS-PAGE The unique protein composition of coated vesicles (clathrin heavy chain, M r 180,000; clathrin light chain, M r 34,000-36,000; adaptins, Mr approximately 100,000, 50,000, and 16,000) makes SDS-PAGE an ideal method of assessing the purity of a preparation. Because the quantity of material isolated is very small, the best method of detection is to add carrier untreated, metabolically labeled cells to the preparation. Wash one 24 × 24 cm dish of semiconfluent A431 cells three times with sterile PBS, and add 80 mI of labeling medium [DMEM containing 0.75 mg/liter methionine (make up from an MEM-Selectamine kit, supplied by GIBCO
s D. M. Schlossman, S. L. Schmid, W. A. Braell, and J. E. Rothman, J. Cell Biol. 99, 723 (1984).
256
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Laboratories, Grand Island, NY) and supplemented with 10% (v/v) dialyzed fetal calf serum, and 5 mCi [35"S]methionine]. Label overnight at 37 ° in an atmosphere of 95% air/5% CO2. Wash the cells four times with vesicle buffer, scrape from the dish, and combine with cells containing labeled transferrin before homogenization. Follow the isolation procedure, and process the fractions of peak fusion activity for SDS-PAGE as described for Western blotting. Run the 10% gel overnight at 70 V, constant voltage, then fix the gel in 10% acetic acid, 20% methanol for 1 hr at room temperature. Discard the fixative, replace with Amplify (Amersham), and incubate for a further 1 hr. Dry the gel and expose to film. The autoradiograph (for example, see Fig. 2, lane c) should clearly show clathrin heavy chain and the adaptin proteins. A Coomassie blue-stained gel of a placental coated vesicle preparation is shown for comparison (Fig. 2, lane d). Clathrin light chains are less easily distinguished, because they label poorly with [3SS]methionine but can be visualized after longer exposure (Fig. 2,
a 200
b
c
d athrin /y chain ~
..... /--1
OOkD 'aptins "-'--"-4 ~
97--
8O ~100
nsferdn 69 n ibulin 48
~0kD - - - - - - - Japtin
46--
--36 athrin ~ I chains
~
--34
!: ~:~ii~ 30--
Fio. 2. SDS-PAGEof coated vesicles.Autoradiograph of an SDS-PAGEof a postnuclear supcrnatant (lane a) and coated vesiclepreparation (lane c) from metabolicallylabeledcells. Clathrin light chains are distinguished on longer exposure of the film (lane b). A Coomassie blue-stained gel of a placental coated vesiclepreparation is shown for comparison (lane d). [Reproduced from the Journal of Cell Biology 112, 1133-1141 (1991).]
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lane b). A major contaminant is tubulin (Mr 55,000). This can be reduced by addition of 10 #g/ml colchicine before the final density gradient.
Electron Microscopy Clathrin-coated vesicles are readily identified by electron microscopy, owing to their polygonal protein coat. Again, it is advisable to include carrier untreated ceils in the preparation. Use 15 dishes of untreated cells and 1 dish of cells labeled with ~25I-labeled transferrin. Dilute the peak fractions from the D 20/FicoU gradient 1 : 9 in vesicle buffer, then centrifuge for 2 hr at 100,000 gay in a Beckman SW40 rotor. Wash the pellet twice in vesicle buffer, then fix at room temperature in 3% (w/v) glutaraldehyde, in the same buffer. Rinse the pellet three times in 0.1 M sodium cacodylate, pH 7.4, then postfix in I% (w/v) osmium tetroxide and 1.5% (w/v) potassium ferrocyanide for 30 rain at 4 ° . Wash the pellet three more times in sodium cacodylate, then dehydrate, using standard procedures, in graded ethanol. Embed in Epon and cut 70-nm sections. Stain the sections with alcoholic uranyl acetate and Reynold's lead citrate. A typical preparation is shown in Fig. 3.
........
¸
FIG. 3. An electron micrograph of a typical coated vesicle preparation. Bar: 0.2/tm.
258
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Uncoating A TPase
Clathrin is removed from coated vesicles by an uncoating ATPase3 Therefore the most direct method of showing that all of the ~25I-labeled transferrin is within coated vesicles is to incubate the preparation with uncoating ATPase. The clathrin coat confers a high density to the vesicle, permitting purification on D20/Ficoll density gradients. ATP-dependent removal of the coat will lower the density of the vesicle, which will migrate to a higher position on a similar D20/Ficoll equilibrium gradient. No change in density will result from incubations lacking uncoating ATPase, or performed without ATP, or at pH 6.6. For each sample, use 25/tl donor coated vesicles. Add ATP-regenerating or -depleting cocktails (25 kd), as for the fusion assay. Add 10/tg uncoating ATPase, prepared from brain or placenta as described by Schlossman et aL s Dilute to 250/tl in HEPES buffer/1 m M DTT, or vesicle buffer/1 m M DTT, containing protease inhibitors, and incubate for 15 rain at 37 °. Stop the reaction by diluting to 2 ml in ice-cold vesicle buffer/l m M D T T . Each sample is analyzed on a 10-ml gradient of 2% (w/v) Ficoll/9% (w/v) I)20-20% (w/v) Ficoll/90% (w/v) D20, in vesicle buffer containing 1 m M D T T . Centrifuge for 16 hr at 80,000 gay in a Beckman SW40 rotor, and collect fractions from the bottom using a capillary tube and peristaltic pump. Figure 4 demonstrates the uncoating ATPase-dependent change in vesicle density.
300 -
e"E
200-
~.,.,
-?7" 100
|
i
i
i
i
i
i
3
5
7
9
11
13
15
Fraction number
FIG. 4. Action of uncoating ATPase. Vesicleslabeled with z25I-labeledtransferrinare incubatedwith MgATP,with (!1)or without(I~ uncoatingATPase.Samplesare loadedonto D20/Ficoll gradients(10 ml) and fractionated(0.5 ml) from the bottom.
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FUNCTIONAL CLATHRIN-COATED ENDOCYTIC VESICLES
259
Vesicle Fusion Assay
Materials Creatine phosphate (CP) and creatine phosphokinase (CPK) are obtained from Boehringer Mannheim (Indianapolis, IN). Sheep anti-transferrin antiserum is supplied by the Scottish Antibody Production Unit (Carluke, Scotland) and heat inactivated by incubating for 30 rain at 56 °. All other reagents are supplied by Sigma Chemical Co. (St. Louis, MO) unless specified.
Acceptor Membrane Preparations For a standard acceptor membrane preparation grow cells to near confluence on four 24 × 24 cm tissue culture dishes. For each plate, wash the cells four times in ice-cold PBS and incubate on a rocking platform at 4 ° for 1 hr with 10 #g/ml unlabeled human transferrin in 25 ml BM. Wash the cells a further four times with ice-cold PBS, then incubate for 1 hr with 1 ml sheep anti-transferrin antiserum diluted to 25 ml with BM. Wash the cells four times with PBS, and incubate in a water bath for 5 rain at 37 ° with 50 ml of prewarmed BM. Wash the cells four times with approximately 50 mi ice-cold HEPES buffer (140 m M sucrose, 20 mM HEPESKOH, 70 mM potassium acetate, pH 7.2) to cool rapidly. Drain the dishes, then scrape the cells, add DTT and protease inhibitors, and homogenize as for donor coated vesicles. Centrifuge the homogenate for 5 rain at 500 gay at 4 °, and apply the supernatant to the tops of two discontinuous sucrose gradients (it is important not to overload the gradients; extracts from not more than two dishes of cells should be applied to each gradient, to prevent membrane aggregation) of 2 ml 40% (w/v) sucrose in HEPES buffer containing 1 mM DTT overlaid with 8 ml 20% (w/v) sucrose in the same buffer. Centrifuge for 2 hr at 155,000 gay in a Beckman SW40 rotor and recover the crude membrane preparation at the 20%/40% interface by tube puncture. Membranes can be snap-frozen and stored in 100-/A aiiquots in liquid nitrogen. Preparations contain approximately 2 - 3 mg]ml protein.
Cytosol Fractions Wash four 24 × 24 cm dishes of near-confluent A431 cells four times with HEPES buffer, drain the dishes, and scrape off the cells. Add DTT to 1 mM, together with protease inhibitors, and homogenize as described above. Centrifuge the extract for 30 rain at 400,000 gay in a Beckman TL100 benchtop ultracentrifuge and carefully remove the supernatant. Apply 3.5 ml to a 10-ml BioGel P-6 desalting column, equilibrated with
260
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ice-cold HEPES buffer containing 1 m M DTT, and collect the fractions of peak protein concentration. Combine, then freeze and store in liquid nitrogen. Protein concentrations should be 5- 10 mg/ml. Fusion Assay Conditions The fusion assay conditions are similar to those described previously,2 but modified to account for the need to dilute the donor coated vesicle preparations (see below). A typical incubation contains 50/~l donor coated vesicles (normally 2000-5000 cpm) and 2 mg/ml cytosol (final concentration). Add 25/~1 unlabeled transferrin (1 mg/ml in HEPES buffer) to prevent immunoprecipitation of any 125I-labeled transferrin present outside the sealed coated vesicles, and 50/~l of acceptor membranes. The incubation should include an ATP-regenerating cocktail (added in 25 gl as a 10 X concentrate of l0 m M MgATP, 50 m M C P , 80 IU/ml CPK), and the total incubation is diluted to 250 gl with HEPES buffer/l m M DTT. Include a control sample with an ATP-depleting cocktail (25 gl) of 500 1U/ml hexokinase in 50 m M glucose, to give a background ATP-independent signal. This value is typically 2% or less of that obtained in the standard incubation. Incubate for 2 hr at 37 °, then dilute to l ml with immunoprecipitation buffer [0.1 M Tris-HCl, pH 8.0, 0.1 MNaC1, 5 mMMgCI2, I% (w/v) Triton X-100, 0.5% (w/v) SDS, 1% (w/v) sodium deoxycholate, 0.1% (v/v) BSA]. Add 20 ill S. aureus cells (Calbiochem, San Diego, CA), washed three times in immunoprecipitation buffer, and incubate on ice for a further 1 hr. Pellet the cells at low speed in a "microcentaur" microfuge (Measuring & Scientific Equipment, London, England) for 4 rain at room temperature and carefully remove the supcrnatant with a syringe needle. Repeat the washing procedure, then count the pellet for radioactivity. Samples are normally counted for 10-30 rain. Future Prospects This chapter describes the isolation of a functional transport intermediate. It should be possible to use this in combination with inhibitors of vesicle fusion to isolate intermediates in the fusion pathway. For example, incubation with a specific inhibitor of fusion may lead to the association of cytosolic proteins with the vesicle membrane, to form a fusion intermediate that cannot be consumed. Reisolation of these vesicles should result in enrichment of these proteins.
FUNCTIONAL CLATHRIN-COATED ENDOCYTIC VESICLES
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[24] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f F u n c t i o n a l Clathrin-Coated Endocytic Vesicles
By PHILIP G. WOODMAN and GRAHAM WARREN Introduction The transferrin cycle is well characterized, and serves as a model of receptor-mediated endocytosis. ~ Halotransferrin binds to cell surface receptors located in clathrin-coated pits. These pits invaginate and pinch off to form endocytic, clathrin-coated vesicles, which carry the transferrin into the cell. Removal of at least part of the clathrin coat enables transferrin to be delivered to the endosome by a membrane fusion event. Once exposed to the low pH of the endosome, iron dissociates from transferrin and the apoprotein is recycled back to the plasma membrane by vesicular transport. For the transferrin cycle to work efficiently, vesicles must recognize and fuse with their target membrane. The biochemical specificity that underlies this selection has allowed us to reconstitute fusion of endocytic vesicles in a cell-free system. The assay for vesicle fusion requires the preparation of "donor" endocytic vesicles containing ~25I-labeled transferrin. These are mixed with "acceptor" endocytic vesicles, containing internalized antitransferrin antibody, in the presence of a cytosol fraction and an ATP-regenerating cocktail. Vesicle fusion permits the formation of a radiolabeled immunocomplex, which is isolated on Staphylococcus aureus cells after solubilization of the vesicle membrane. Measurement of the fusion of endocytic vesicles within crude preparations has been described in detail elsewhere.2 However, further analysis of the interaction between cytosolic and membrane components during the fusion reaction requires use of purified membranes as substrates. We have chosen to isolate a donor preparation of clathrin-coated vesicles, 3 because this is the best characterized endocytic compartment. In addition, the unique composition of coated vesicles simplifies both purification and identification. Here, we describe the isolation of functional clathrin-coated vesicles. Isolation is monitored by the ability to fuse with acceptor endocytic vesicles.
z R. D. Klausner, G. Ashwell, J. v a n Renswoud¢, J. B. Harford, a n d K. R. Bridges, Proc Natl. Acad. Sci. USA 80, 2263 (1980). 2 p. G. W o o d m a n a n d G. Warren, Methods Cell Biol. 31, 197 0 9 8 9 ) . 3 p. G. W o o d m a n a n d G. Warren, J. Cell Biol. 112, 1133 (1991).
METHODS IN ENZYMOLOGY, VOL. 219
Copyright© 1992by AcademicPrms, Inc. All rightsof reproductionin any form reserved.
252
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[24]
Isolation of Donor Clathrin-Coated Vesicles
Cells All membrane and cytosol fractions used in this study are prepared from A431 cells, a human cell line rich in transferrin receptors? A431 cells are grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal calf serum and 100 U/ml of penicillin and streptomycin, in an atmosphere of 95% air/5% CO2. After trypsinization cultures are split 1:5 every 48 hr. All tissue culture media and supplements were bought from Northumbria Biologicals, Ltd, Northumberland, UK.
Radiolabeling Human transferrin is radiolabeled with iodine-125 according to the method of Fraker and Speck.5 Dissolve lodogen (1,3,4,6-tetrachloro3ot,6c~-diphenylglycoluril; Pierce Chemical Co., Rockford, IL) to 0.5 mg/ ml in chloroform. Evaporate 20/zl in a glass tube under a stream of nitrogen. Add 30/tl sodium phosphate buffer, pH 7.2, containing 100/zg human transferrin. To this add 2.5 mCi (25/~1) NanSI (16 mCi/#g; Amersham, UK). Incubate for 15 min on ice, then stop the reaction by addition of 166 mg/ml unlabeled KI (50 ~1) and 2.5 /xg/ml sodium metabisulfite (50/zl). Remove the free iodine by gel filtration over a BioGel P-6 column (Bio-Rad Laboratories, Richmond, CA), prewashed with sodium phosphate buffer containing 0.1 mg/ml bovine serum albumin (BSA) and equilibrated with BSA-free phosphate buffer, followed by dialysis against 400 vol of the same buffer. This method should achieve a specific activity of 2-3 × 107 counts per minute (cpm)//zg transferrin.
Isolation of Coated Vesicles Donor coated vesicles are prepared using a modification of the method of Pearse, 6 and isolation is monitored by the cell-free assay for vesicle fusion (see below). Care must be taken to avoid pelleting the membranes, because this leads to aggregation and loss of activity. The preparation is carried out at pH 6.6 throughout, to stabilize the clathrin coat. For a standard preparation, grow A431 cells to near confluence on four 24 × 24 cm tissue culture dishes, supplied by GIBCO (Paisley, Scotland). For each dish, wash cells with ice-cold Dulbecco's phosphate-buffered saline (PBS), then incubate on a slowly rocking platform at 4 ° with 15 ml bind4 C. R. Hopkins and I. S. Trowbridge, J. CellBiol. 97, 508 (1983). s p. j. Fraker and J. C. Speck, Biochem. Biophys. Res. Commun. 80, 849 (1978). 6 B. M. F. Pearse, Proc. Natl. Acad. Sci. USA 79, 451 (1982).
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ing medium [BM; DMEM containing 20 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, and 0.2% (w/v) BSA] containing 1.5 #g/ml 125I-labeled transferrin. After 2 hr, wash the cells three times in PBS, and incubate for 2 min at 31 ° with 50 ml prewarmed BM. The short period for internalization should ensure that the greatest proportion of internalized transferrin is in coated vesicles. Wash the dish four times in 50 ml ice-cold vesicle buffer [140 m M sucrose, 0.5 m M MgC12, l m M EGTA, 20 raM 2-(N-morpholino)ethanesulfonic acid (MES), 70 m M potassium acetate, pH 6.6]. Drain for 2 min at 4 ° to remove excess buffer, then scrape the cells from the dish with a rubber policeman. Suspensions from four plates should be combined (approximately 3 - 4 ml), supplemented with dithiothreitol (DTT; 1 raM) and protease inhibitors [ 1 #g/ml antipain, 1 #g/ml chymostatin, 1 #g/ml pepstatin, 2 #g/ml E64, 40 #g/ml phenylmethylsufonyl fluoride (PMSF), all stored in a 1000× concentrate in dimethyl sulfoxide (DMSO)], and broken in a stainless-steel homogenizer. Cells are passed 10 times through a 0.2540-in. bore containing a 0.2530-in. ball. 7 These conditions should break 80-90% of cells with little damage to nuclei, as assessed by trypan blue staining and microscopy. Prepare a postnuclear supernatant by centrifuging at 500 g,v for 5 min at 4 °. Polyribosomes, potential contaminants, are disassembled by incubating the extract with ribonuclease A (50 #g/ml; Worthington Enzymes, Ltd., Freehold, NJ)for 30 min at 4 °. Centrifuge at 7000 g,v for 30 min at 4 °, and then apply the supernatant (approximately 3 ml) to a 10-ml continuous deuterium oxide (D20) rate sedimentation gradient of 10-90% (w/v) D20 in vesicle buffer, containing 1 m M DTT throughout. Centrifuge for 30 min at 45,000 g,v in an SW40 rotor (Beckman Instruments, Inc., Palo Alto, CA) and collect l-ml fractions from the bottom by tube puncture. The coated vesicles sediment slowly, and should remain in the top 5 ml of the gradient. This should be monitored by assaying 50-#1 samples for vesicle fusion activity (see below). Pool the peak fractions, dilute to 18 ml in vesicle buffer containing 1 m M DTT, and apply to the top of an equilibrium density gradient (20 ml) of 2% (w/v) FicoU/9% (w/v) D20-20% (w/v) Ficoll/90% (w/v) 1)20 in vesicle buffer containing I m M DTT. (Before use, the Ficoll should be dissolved in water, dialyzed extensively to remove low molecular weight contaminants, and lyophilized.) Centrifuge for 16 hr at 80,000 g,, in a Beckman SW28 rotor and remove twenty l-ml fractions from the bottom by using a fine capillary tube attached to a peristaltic pump. Samples (50 #1) from each fraction should be analyzed for vesicle fusion activity. As indicated below, to dilute
7 W. E. Balch, W. G. Dunphy, W. A. Braell, and J. E. Rothman, Cell 39, 405 (1984).
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IDENTIFICATION OF TRANSPORT INTERMEDIATES
out the viscous Ficoll and vesicle isolation buffer, samples should be diluted at least 1 : 4 into the fusion assay mix. Analysis of Donor Coated Vesicle Preparations
Endocytic VesicleFusion Activity Because fusion occurs only between endocytic compartments,2 only those fractions supporting fusion activity should contain clathrin-coated vesicles. ~25I-labeled transferrin migrates as two peaks on the equilibrium gradient (Fig. 1). Only the lower peak possesses fusion activity and, therefore, contains endocytic vesicles. Observed etficiency of fusion is dependent on the concentration of acceptor membranes; with an excess of acceptor membranes, up to about 40% of total L2SI-labeled transferrin should be precipitated in an ATP-dependent manner. The peak migrates to the same position as placental clathrin-coated vesicles isolated on similar gradients. The unique properties of clatbrin-coated vesicles permit several independent methods of confirming that vesicles in this peak are coated, and of assessing the purity of the preparation. Analysis can include detection of coated vesicle proteins by Western blotting with anti-(coat protein)
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FIG. l. Isolation of endocytic coated vesicles. Fractions (1 ml) from the D20/Ficoll density gradient are sampled (100/A) for '2~I-labeled tmnsferrin and endocytic vesicle fusion activity. Fusion activity is expressed as the ATP-dependent immunoprecipitation o f l:~Ilabeled transferrin.
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FUNCTIONAL CLATHRIN-COATED ENDOCYTIC VESICLES
255
antisera, electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and susceptibility of the preparation to the action of the clathrin-uncoating ATPase3
Western Blotting To obtain sufficient material to detect coat proteins by Western blotting, supplement the normal donor vesicle preparation with twelve 24 × 24 cm dishes of untreated cells. After collecting the fractions from the equilibrium gradient, sediment vesicles by diluting 10-fold in vesicle buffer and centrifuging for 2 hr at 100,000 g,v in a Beckman SW40 rotor. Wash the pellets carefully with vesicle buffer, and resuspend in sample buffer [1 M sucrose, 200 mM Tris-HC1, pH 6.5, 5 mM ethylene diamine tetraacetic acid (EDTA), 0.04% (w/v) bromphenol blue, 10 mM DTT, 4% (w/v) SDS], boil for 5 min, cool, add 5 m M iodoacetamide, and electrophorese overnight (70 V, constant voltage) on a 10% (w/v) polyacrylamide gel. Transfer to nitrocellulose, according to the method of Towbin, for 2 hr at 1.5 A. Incubate the nitrocellulose on a rocking platform in Tris/salt buffer (200 mM Tris-HC1, pH 7.4, 150 mM NaC1) containing 0.2% (w/v) polyoxyethylene sorbitan monolaurate (Tween 20) and 0.1% (w/v) fish skin gelatin. Incubate with the appropriate dilution of an anti-(coat protein) antiserum (e.g., anti-clathrin light chain) in the same buffer for 90 min at room temperature, then wash three times in 1 hour with Tris/salt containing 0.05% (w/v) Tween 20, followed by Tris/salt without Tween 20. Incubate for 60 min at room temperature with t2Sl-labeled protein A [0.1 ltCi/ ml in Tris/salt containing 5% (w/v) BSA]. Wash three times in Tris/salt containing 0.05% (w/v) Tween 20, dry, and expose to photographic film.
SDS-PAGE The unique protein composition of coated vesicles (clathrin heavy chain, M r 180,000; clathrin light chain, M r 34,000-36,000; adaptins, Mr approximately 100,000, 50,000, and 16,000) makes SDS-PAGE an ideal method of assessing the purity of a preparation. Because the quantity of material isolated is very small, the best method of detection is to add carrier untreated, metabolically labeled cells to the preparation. Wash one 24 × 24 cm dish of semiconfluent A431 cells three times with sterile PBS, and add 80 mI of labeling medium [DMEM containing 0.75 mg/liter methionine (make up from an MEM-Selectamine kit, supplied by GIBCO
s D. M. Schlossman, S. L. Schmid, W. A. Braell, and J. E. Rothman, J. Cell Biol. 99, 723 (1984).
256
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IDENTIFICATION OF TRANSPORT INTERMEDIATES
Laboratories, Grand Island, NY) and supplemented with 10% (v/v) dialyzed fetal calf serum, and 5 mCi [35"S]methionine]. Label overnight at 37 ° in an atmosphere of 95% air/5% CO2. Wash the cells four times with vesicle buffer, scrape from the dish, and combine with cells containing labeled transferrin before homogenization. Follow the isolation procedure, and process the fractions of peak fusion activity for SDS-PAGE as described for Western blotting. Run the 10% gel overnight at 70 V, constant voltage, then fix the gel in 10% acetic acid, 20% methanol for 1 hr at room temperature. Discard the fixative, replace with Amplify (Amersham), and incubate for a further 1 hr. Dry the gel and expose to film. The autoradiograph (for example, see Fig. 2, lane c) should clearly show clathrin heavy chain and the adaptin proteins. A Coomassie blue-stained gel of a placental coated vesicle preparation is shown for comparison (Fig. 2, lane d). Clathrin light chains are less easily distinguished, because they label poorly with [3SS]methionine but can be visualized after longer exposure (Fig. 2,
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Fio. 2. SDS-PAGEof coated vesicles.Autoradiograph of an SDS-PAGEof a postnuclear supcrnatant (lane a) and coated vesiclepreparation (lane c) from metabolicallylabeledcells. Clathrin light chains are distinguished on longer exposure of the film (lane b). A Coomassie blue-stained gel of a placental coated vesiclepreparation is shown for comparison (lane d). [Reproduced from the Journal of Cell Biology 112, 1133-1141 (1991).]
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FUNCTIONAL CLATHRIN-COATED ENDOCYTIC VESICLES
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lane b). A major contaminant is tubulin (Mr 55,000). This can be reduced by addition of 10 #g/ml colchicine before the final density gradient.
Electron Microscopy Clathrin-coated vesicles are readily identified by electron microscopy, owing to their polygonal protein coat. Again, it is advisable to include carrier untreated ceils in the preparation. Use 15 dishes of untreated cells and 1 dish of cells labeled with ~25I-labeled transferrin. Dilute the peak fractions from the D 20/FicoU gradient 1 : 9 in vesicle buffer, then centrifuge for 2 hr at 100,000 gay in a Beckman SW40 rotor. Wash the pellet twice in vesicle buffer, then fix at room temperature in 3% (w/v) glutaraldehyde, in the same buffer. Rinse the pellet three times in 0.1 M sodium cacodylate, pH 7.4, then postfix in I% (w/v) osmium tetroxide and 1.5% (w/v) potassium ferrocyanide for 30 rain at 4 ° . Wash the pellet three more times in sodium cacodylate, then dehydrate, using standard procedures, in graded ethanol. Embed in Epon and cut 70-nm sections. Stain the sections with alcoholic uranyl acetate and Reynold's lead citrate. A typical preparation is shown in Fig. 3.
........
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FIG. 3. An electron micrograph of a typical coated vesicle preparation. Bar: 0.2/tm.
258
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IDENTIFICATION OF TRANSPORT INTERMEDIATES
Uncoating A TPase
Clathrin is removed from coated vesicles by an uncoating ATPase3 Therefore the most direct method of showing that all of the ~25I-labeled transferrin is within coated vesicles is to incubate the preparation with uncoating ATPase. The clathrin coat confers a high density to the vesicle, permitting purification on D20/Ficoll density gradients. ATP-dependent removal of the coat will lower the density of the vesicle, which will migrate to a higher position on a similar D20/Ficoll equilibrium gradient. No change in density will result from incubations lacking uncoating ATPase, or performed without ATP, or at pH 6.6. For each sample, use 25/tl donor coated vesicles. Add ATP-regenerating or -depleting cocktails (25 kd), as for the fusion assay. Add 10/tg uncoating ATPase, prepared from brain or placenta as described by Schlossman et aL s Dilute to 250/tl in HEPES buffer/1 m M DTT, or vesicle buffer/1 m M DTT, containing protease inhibitors, and incubate for 15 rain at 37 °. Stop the reaction by diluting to 2 ml in ice-cold vesicle buffer/l m M D T T . Each sample is analyzed on a 10-ml gradient of 2% (w/v) Ficoll/9% (w/v) I)20-20% (w/v) Ficoll/90% (w/v) D20, in vesicle buffer containing 1 m M D T T . Centrifuge for 16 hr at 80,000 gay in a Beckman SW40 rotor, and collect fractions from the bottom using a capillary tube and peristaltic pump. Figure 4 demonstrates the uncoating ATPase-dependent change in vesicle density.
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9
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FIG. 4. Action of uncoating ATPase. Vesicleslabeled with z25I-labeledtransferrinare incubatedwith MgATP,with (!1)or without(I~ uncoatingATPase.Samplesare loadedonto D20/Ficoll gradients(10 ml) and fractionated(0.5 ml) from the bottom.
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FUNCTIONAL CLATHRIN-COATED ENDOCYTIC VESICLES
259
Vesicle Fusion Assay
Materials Creatine phosphate (CP) and creatine phosphokinase (CPK) are obtained from Boehringer Mannheim (Indianapolis, IN). Sheep anti-transferrin antiserum is supplied by the Scottish Antibody Production Unit (Carluke, Scotland) and heat inactivated by incubating for 30 rain at 56 °. All other reagents are supplied by Sigma Chemical Co. (St. Louis, MO) unless specified.
Acceptor Membrane Preparations For a standard acceptor membrane preparation grow cells to near confluence on four 24 × 24 cm tissue culture dishes. For each plate, wash the cells four times in ice-cold PBS and incubate on a rocking platform at 4 ° for 1 hr with 10 #g/ml unlabeled human transferrin in 25 ml BM. Wash the cells a further four times with ice-cold PBS, then incubate for 1 hr with 1 ml sheep anti-transferrin antiserum diluted to 25 ml with BM. Wash the cells four times with PBS, and incubate in a water bath for 5 rain at 37 ° with 50 ml of prewarmed BM. Wash the cells four times with approximately 50 mi ice-cold HEPES buffer (140 m M sucrose, 20 mM HEPESKOH, 70 mM potassium acetate, pH 7.2) to cool rapidly. Drain the dishes, then scrape the cells, add DTT and protease inhibitors, and homogenize as for donor coated vesicles. Centrifuge the homogenate for 5 rain at 500 gay at 4 °, and apply the supernatant to the tops of two discontinuous sucrose gradients (it is important not to overload the gradients; extracts from not more than two dishes of cells should be applied to each gradient, to prevent membrane aggregation) of 2 ml 40% (w/v) sucrose in HEPES buffer containing 1 mM DTT overlaid with 8 ml 20% (w/v) sucrose in the same buffer. Centrifuge for 2 hr at 155,000 gay in a Beckman SW40 rotor and recover the crude membrane preparation at the 20%/40% interface by tube puncture. Membranes can be snap-frozen and stored in 100-/A aiiquots in liquid nitrogen. Preparations contain approximately 2 - 3 mg]ml protein.
Cytosol Fractions Wash four 24 × 24 cm dishes of near-confluent A431 cells four times with HEPES buffer, drain the dishes, and scrape off the cells. Add DTT to 1 mM, together with protease inhibitors, and homogenize as described above. Centrifuge the extract for 30 rain at 400,000 gay in a Beckman TL100 benchtop ultracentrifuge and carefully remove the supernatant. Apply 3.5 ml to a 10-ml BioGel P-6 desalting column, equilibrated with
260
IDENTIFICATION OF TRANSPORT INTERMEDIATES
[24]
ice-cold HEPES buffer containing 1 m M DTT, and collect the fractions of peak protein concentration. Combine, then freeze and store in liquid nitrogen. Protein concentrations should be 5- 10 mg/ml. Fusion Assay Conditions The fusion assay conditions are similar to those described previously,2 but modified to account for the need to dilute the donor coated vesicle preparations (see below). A typical incubation contains 50/~l donor coated vesicles (normally 2000-5000 cpm) and 2 mg/ml cytosol (final concentration). Add 25/~1 unlabeled transferrin (1 mg/ml in HEPES buffer) to prevent immunoprecipitation of any 125I-labeled transferrin present outside the sealed coated vesicles, and 50/~l of acceptor membranes. The incubation should include an ATP-regenerating cocktail (added in 25 gl as a 10 X concentrate of l0 m M MgATP, 50 m M C P , 80 IU/ml CPK), and the total incubation is diluted to 250 gl with HEPES buffer/l m M DTT. Include a control sample with an ATP-depleting cocktail (25 gl) of 500 1U/ml hexokinase in 50 m M glucose, to give a background ATP-independent signal. This value is typically 2% or less of that obtained in the standard incubation. Incubate for 2 hr at 37 °, then dilute to l ml with immunoprecipitation buffer [0.1 M Tris-HCl, pH 8.0, 0.1 MNaC1, 5 mMMgCI2, I% (w/v) Triton X-100, 0.5% (w/v) SDS, 1% (w/v) sodium deoxycholate, 0.1% (v/v) BSA]. Add 20 ill S. aureus cells (Calbiochem, San Diego, CA), washed three times in immunoprecipitation buffer, and incubate on ice for a further 1 hr. Pellet the cells at low speed in a "microcentaur" microfuge (Measuring & Scientific Equipment, London, England) for 4 rain at room temperature and carefully remove the supcrnatant with a syringe needle. Repeat the washing procedure, then count the pellet for radioactivity. Samples are normally counted for 10-30 rain. Future Prospects This chapter describes the isolation of a functional transport intermediate. It should be possible to use this in combination with inhibitors of vesicle fusion to isolate intermediates in the fusion pathway. For example, incubation with a specific inhibitor of fusion may lead to the association of cytosolic proteins with the vesicle membrane, to form a fusion intermediate that cannot be consumed. Reisolation of these vesicles should result in enrichment of these proteins.
