Uncoating of Clathrin-Coated Vesicles by Uncoating ATPase ... · agarose was from Sigma, phenyl Sepharose CL-4B, Polybuf- fer Exchanger PBE 94, and Polybuffer 74 were from Phar- macia

Plant Physiol. (1993) 103: 205-212

Uncoating of Clathrin-Coated Vesicles by Uncoating ATPase from Developing Peas'

Thomas Kirsch and Leonard Beevers*

Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 7301 9

A cytosolic uncoating ATPase (an enzyme that dissociates clathrin from clathrin-coated vesicles in the presence of ATP) was isolated from developing pea (Pisum sativum 1.) cotyledons using chromatography on ATP-agarose. After chromatography on phenyl Sepharose, the fraction with uncoating activity was enriched in a doublet of 70-kD peptides. Using chromatofocusing, it was possible to produce fractions enriched in the upper component of the doublet of 70-kD peptides; these fractions st i l l retained ATP- dependent uncoating activity. In western blot analysis, antibodies against a member of the 70-kD family of heat-shock proteins interacted with the upper component of the doublet of the 70-kD peptides from the phenyl Sepharose-purified fractions. On the basis of these data, it appears that the uncoating ATPase may be a member of the 70-kD family of heat-shock proteins. l h e uncoating activity removed clathrin from both pea and bovine brain clathrin- coated vesicles. l h e uncoating ATPase from bovine brain also uncoated coated vesicles from peas. Pea clathrin-coated vesicles that were prepared by three different methods were uncoated to different extents by the plant uncoating ATPase. Different populations of clathrin-coated vesicles from the same preparation showed differential sensitivity to the uncoating ATPase. Limited proteolysis of the clathrin light chains in the protein coat abolished the susceptibility of the clathrin-coated vesicles to the uncoating ATPase. l h e properties of the uncoating ATPase isolated from developing pea cotyledons are similar to those of uncoating ATPases previously described from mammalian and yeast systems. It appears that despite dissimilarities in composition of the clathrin components of the vesicles from the respective sources, uncoating is achieved by a common mechanism.

In mammalian cells, the participation of clathrin-coated vesicles in receptor-mediated endocytosis and in the transport of lysosomal glycoproteins from the trans-Golgi nehvork to the lysosome is well established (Campbell et al., 1983; Goldstein et al., 1985). There is evidence that clathrin-coated vesicles are involved in endocytosis in plant cells also (Robin- son and Depta, 1988), and the demonstration of precursors of vacuolar storage proteins and lectins in clathrin-coated vesicles isolated from developing peas (Harley and Beevers, 1989b; Robinson et al., 1989) indicates that these vesicles are involved in intracellular transport from the trans-Gol@ network to the protein body/vacuole in plants. The participation of clathrin-coated vesicles in endocytosis and intracellular transport requires uncoating of the vesicle prior to fusion

' This work was supported by National Science Foundation grant

* Corresponding author; fax 1-405-325-7619. DCB-8916621.

205

with the target membrane. In mammalian cells, a cytosolic ATPase with a subunit molecular mass of 70 kD was able to uncoat clathrin-coated vesicles in vitro (Braell et al., 1984; Schlossman et al., 1984). This uncoating ATPase was shown to be a member of the 70-kD family of heat-shock proteins (HSP70 family) (Chappel et al., 1986). A similar uncoating activity was isolated from yeast cells. On SDS gels, the uncoating activity from yeast showed two peptides in the molecular mass range of 70 kD that were members of the yeast HSP70 family (Gao et al., 1991). After partia1 separation of the two peptides on a Superose-6 fast protein liquid chromatography column, almost a11 of the uncoating activity was associated with the higher molecular mass peptide (Gao et al., 1991). Even though there is a 70-kD family of heat- shock proteins in plants (Neuman et al., 1987), there are no reports on how clathrin-coated vesicles are uncoated. This deficiency prompted the present study of a cytosolic uncoating ATPase in plants.

MATERIALS AND METHODS

Plant Material and Chemicals

Peas (Pisum sativum L. cv Burpeeana) were field grown and harvested 24 to 27 d postanthesis. After the pea seeds had been removed from the pods, peas were frozen with liquid nitrogen and stored at -7OOC. Before use, peas were allowed to thaw at room temperature for 15 min. ATP- agarose was from Sigma, phenyl Sepharose CL-4B, Polybuf- fer Exchanger PBE 94, and Polybuffer 74 were from Phar- macia. Unless indicated otherwise, a11 chemicals were purchased from Sigma. Molecular standards and chemicals for SDS-PAGE were from Bio-Rad.

Purification of Clathrin-Coated Vesicles

A11 operations were carried out at 4OC. Clathrin-coated vesicles from developing pea cotyledons were isolated using three different methods.

Method 1 used a modification of the procedures of Harley and Beevers (1989a) and Pearse (1983). Six hundred grams of peas were ground in portions of 150 g in a Waring Blendor at full speed for 45 s using 200 mL of buffer A (0.1 M Mes- NaOH, pH 6.5, 0.3 M sorbitol, 1 mM EGTA, 0.5 mM MgC12, 0.02% NaN3, 1 m~ DTT, 1 m~ PMSF, 1.2 PM trypsin inhibitor) per 100 g of peas. The homogenate was squeezed through four layers of cheesecloth, and the filtrate was cen-

Abbreviation: IgG, immunoglobulin G.

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206 Kirsch and Beevers Plant Physiol. Vol. 103, 1993

trifuged for 30 min at 13,000 rpm in a GSA rotor. The supernatant was centrifuged for 35 min at 38,000 rpm in a 42.1 rotor (Beckman, L8-70M Ultracentrifuge). The pellet of this centrifugation step was resuspended in buffer B (buffer A without sorbitol), and the suspension was adjusted to a final volume of 60 mL. Sixty milligrams of RNAse A (Sigma) was added and the suspension was incubated at 4OC for 45 min. The suspension was then loaded onto six 20-mL cushions of 40% (v/v) 'H20 in buffer B and centrifuged at 17,000 rpm for 30 min in an SW27 rotor. The supernatants and the cushions were taken off and diluted to 400 mL with buffer B. The vesicles were collected by centrifugation for 35 min at 38,000 rpm in a 42.1 rotor and then resuspended in 20 mL of buffer B. The resuspension was loaded onto two 20-mL linear gradents of 9% (v/v) 'H20:5% (w/v) Ficoll to 90% (v/v) 2H20:25% (w/v) Ficoll in buffer B. After centrifugation for 16 h at 21,000 rpm in an SW27 rotor, the part of the gradient between 13 and 22.5% Suc equivalents (percent w/ w Suc solution that would have the same refractive index as the Ficoll concentration in the gradient) was taken off and diluted 5-fold with buffer B, and the coated vesicles were collected by centrifugation at 38,000 rpm for 35 min in a 42.1 rotor and then resuspended in 0.1 M Mes-NaOH, pH 6.5, 1 m EGTA, 0.5 mM MgCL2 at a concentration of about 1 mg/ mL. The coated vesicles were stored at -7OOC. Before use, the coated vesicles were thawed and centrifuged for 5 min in a microcentrifuge, and the supematant was used for ATP- ase assays.

Method 2 was the same as method 1, but the centrifugation on the 'H20 cushion was omitted. In this case, the first high- speed pellet was resuspended in 20 mL of buffer B only, and after digestion with RNAse A was loaded directly onto the 2H20:Ficoll gradient.

Method 3 was according to Harley and Beevers (1989a). In this case, the centrifugation of the crude coated vesicles on the 2Hz0 cushion was replaced by centrifugations on two different Suc gradients, and the digestion with RNAse A was done at 3OoC as described (Harley and Beevers, 1989a).

Clathrin-coated vesicles from bovine brain were isolated using method 1.

Purification of the Uncoating ATPase

Unless indicated otherwise, a11 operations were camed out at 4OC. A11 percentages of ammonium sulfate refer to per- centage of saturation.

The uncoating ATPase from developing peas was isolated according to Gao et al. (1991) with modifications. Eight hundred grams of peas were ground in a Waring Blendor at full speed for 1 min in 1 L of buffer D (40 m imidazole- NaOH, pH 7.0, 75 mM KCl, 5 m magnesium acetate, 1 mM PMSF, 1 mM DTT, 1.2 p~ trypsin inhibitor). The homogenate was squeezed through four layers of cheesecloth and the filtrate was centrifuged at 13,000 rpm for 15 min in a GSA rotor. The supematant was centrifuged at 38,000 rpm for 35 min in a 42.1 rotor. The postmicrosomal supematant was loaded onto a 20-mL ATP-agarose column (Sigma) equilibrated with buffer E (20 mM imidazole-NaOH, pH 7.0, 25 m KCl, 10 m ammonium sulfate, 2 m magnesium acetate, 1 m DTT). When half of the sample had been applied, the

column was washed with 100 mL of buffer F (buffer E containing 500 mM KC1) followed by 100 mL of buffer Ei, and then the rest of the sample was applied. The columrt was subsequently washed with 100 mL each of buffer E, buffer F, buffer E, buffer E containing 1 mM UTP, and buffer E. ATP-binding proteins were eluted from the column with 1 mM ATP in buffer E. The ATP-eluted fraction was either concentrated in a pressure cell (Amicon) and used directly in uncoating assays or further purified by chromatography on phenyl Sepharose (Pharmacia) or chromatofocusing on Poly- buffer Exchanger PBE 94 (Pharmacia). For chromatography on phenyl Sepharose, the ATP-eluted fraction was adjusted to 30% ammonium sulfate saturation and then loaded onto a 5-mL column of phenyl Sepharose that was equilitirated with 30% ammonium sulfate-saturated buffer D. The ccilumn was sequentially washed with 50 mL each of 30% ammcinium sulfate in buffer D, 10% ammonium sulfate in buffer D, buffer D, and finally 80% (w/w) ethylene glycol in buffer D.

When used for chromatofocusing, the ATP-binding proteins were eluted from the ATP-agarose column with l mM ATP in 25 mM imidazole, pH 7.4, and then applied to a chromatofocusing column (1.0 x 20 cm) that was equilibrated with 25 m imidazole-HC1, pH 7.4. Bound protein was eluted by washing the column with 225 mL of a 1:8 diluticm in water of Polybuffer 74 (Pharmacia), pH 4.0. Fractions with eluted protein were concentrated and adjusted to buffer D.

The uncoating ATPase from bovine brain was iscilated according to Greene et al. (1990).

Assay of the Uncoating Activity

Two hundred to 300 pL of assay mixture contained 20 pg of clathrin-coate< vesicles, 2 mM ATP, 1 unit of creatine phosphokinase, 15 mM phosphocreatine, and a vaiiable amount of uncoating ATPase preparation (depending on the purity of the preparation) in buffer D. In control assays, the uncoating ATPase was replaced by the same volume of buffer D. The mixture was incubated for 45 min at 3OoC and then diluted to 1 mL with buffer D. The diluted mixture was loaded on a 9-mL cushion of 10% Suc in buffer D and centrifuged for 40 min at 40,000 rpm in an SW41 rotor The supematant and the first 400 pL of the Suc cushion were taken off and analyzed by SDS-PAGE. When assays were performed in the absence of ATP, 50 pL of ATPase preparation were preincubated for 30 min at room temperature in the presence of 0.5 units of hexokinase and 5 mM Glc to degrade endogenous ATP.

Elastase Treatment of Clathrin-Coated Vesicles

Elastase treatment of clathrin-coated vesicles to partially digest clathrin light chains was done as described (Lin et al., 1992). Twenty micrograms of clathrin-coated vesicles were incubated in the presence of elastase (final concentration 10 &mL) for 5 min at room temperature. The digestion was stopped by adding PMSF to a final concentration of 5 ITLM.

lmmunoprecipitations

Antibodies against a member of the HSP70 family were obtained from Drs. E. Vierling and A. DeRocher (University



Uncoating ATPase 207

of Arizona). The antibodies were raised in rabbits against anamino-terminal fragment of pea HSP71.2 protein, which isconserved between members of the 70-kD family. The frag-ment was expressed as a fusion protein in Escherichia coli andantigen was purified by preparative SDS-PAGE (DeRocher,1993). IgGs were partially purified from the antiserum (100fiL) by ammonium sulfate fractionation followed by ion-exchange chromatography on DEAE-Sephacel (Sigma) asdescribed (Dunbar et al., 1990).

The purified IgGs were incubated overnight at 4°C with50 ng of ATPase preparation (fraction 1) in 20 mM imidazole-NaOH, pH 7.0, 25 mM KC1, 10 mM ammonium sulfate, and2 mM magnesium acetate. The IgGs with bound antigenswere removed from the mixture with Protein A Sepharose(Sigma), and the residual mixture was assayed for uncoatingactivity. The Protein A Sepharose was washed with 10 mLof 25 mM imidazole-NaOH, pH 7.0, 150 mM NaCl, 1% TritonX-100, and 10 mL of 25 mM imidazole-NaOH, pH 7.0, 150mM NaCl. IgGs and bound antigens were dissociated fromProtein A Sepharose with 1 M acetic acid and then analyzedby SDS-PAGE.

Electrophoresis and Western Blotting

SDS-PAGE was done on gradient gels from 6 to 18% (w/v) acrylamide (Laemmli, 1970), and proteins were visualizedby staining with Coomassie brilliant blue R. For western blotanalysis, proteins were transferred onto nitrocellulose accord-ing to Towbin et al. (1979). Nonspecific interactions wereblocked by incubating the blots for 1 h at room temperaturein 3% (w/v) nonfat dry milk. After incubation with theantibody, immunoreactive products were visualized usinganti-rabbit IgG-coupled alkaline phosphatase (Harlow andLane, 1988).

Protein Determination

Protein was determined using a modification of the methodof Lowry (Larson et al., 1986).

RESULTS

Isolation of an Uncoating Activity from Developing Peas

In mammalian cells, there is a cytosolic ATPase that un-coats clathrin-coated vesicles in vitro. To investigate whetherthere is such an activity in plant cells, a postmicrosomalsupernatant from developing peas was applied onto an ATP-agarose column. Proteins that were retained by the columnwere subsequently eluted with 1 M NaCl, 1 mM UTP, and 1mM ATP. The fractions were assayed for their ability todissociate clathrin from clathrin-coated vesicles by incubatingthem with clathrin-coated vesicles in the presence of 2 mMMg-ATP and an ATP-regenerating system. As a control,clathrin-coated vesicles were incubated with ATP and theATP-regenerating system in the absence of the affinity-purified enzyme. After incubation, residual vesicles wereremoved from the mixture by centrifugation, and then thesupernatant was analyzed by SDS-PAGE for the presence ofdissociated clathrin heavy chain. As shown in Figure 1, asmall amount of clathrin heavy chain was dissociated from

kD 1 2 3 4 S

heavy chain116 > _

66 > _

45> ..

2O *.

Figure 1. SDS-PAGE analysis of the uncoating of clathrin-coatedvesicles by the uncoating ATPase from peas. A poslmicrosomalsupernatant was applied to an ATP-agarose affinity column. Lane 1,Molecular mass standards; lanes 2 through 5, high-speed superna-tants after incubation of clathrin-coated vesicles from bovine brain(20 Mg) in the presence of ATP and an ATP-regenerating systemalone (lane 2) and with protein fractions (50 /ig each) eluted fromthe ATP-agarose column with 1 M NaCl (lane 3), 1 mM UTP (lane4), and 1 mM ATP (lane 5).

the coated vesicles when they were incubated with ATP andthe ATP-regenerating system alone (Fig. 1, lane 2). However,much more clathrin was removed from the vesicles whenthey were incubated in the presence of the protein fractionthat was eluted from the ATP-agarose column with 1 mMATP (fraction 1) (Fig. 1, lane 5). The fraction that was elutedwith 1 mM UTP contained less uncoating activity and thefraction that was eluted with 1 M NaCl did not enhanceuncoating at all (Fig. 1, lanes 4 and 3, respectively).

The uncoating activity contained in fraction 1 bound tophenyl Sepharose in 30% ammonium sulfate. When boundproteins were differentially eluted with 10% ammoniumsulfate (Fig. 2, lane 3), 0% ammonium sulfate (Fig. 2, lane 4),and 80% (w/w) ethylene glycol (Fig. 2, lane 5), the proteinfraction that eluted with 0% ammonium sulfate (fraction 2)contained activity that uncoated both pea and bovine braincoated vesicles (Fig. 2, lanes 6-9), whereas the other twofractions contained little or no uncoating activity (data notshown). When ATP was omitted from the incubation mixture,fraction 2 did not enhance uncoating (Fig. 2, lanes 10 and11) compared with the controls (Fig. 2, lanes 6 and 7). Theuncoating ATPase from bovine brain also uncoated clathrin-coated vesicles from pea (data not shown).

Because in mammalian cells and yeast the uncoating ATP-ase is a member of the HSP70 family, we analyzed fraction2 by western blotting using antibodies against a member ofthe HSP70 family from pea leaves. As documented in Figure3, the antibody recognized the upper member of a doubletof 70-kD peptides. This doublet of peptides was enriched inthe fraction that eluted from ATP-agarose with ATP (Fig. 1)and was further enriched after chromatography on phenylSepharose in the fraction that contained uncoating activity(Fig. 2). Chromatofocusing enriched the upper peptide rela-tive to the lower peptide. The fraction with the upper peptideenriched had uncoating activity (Fig. 4, lanes 3 and 5),




Figure 2. SDS-PACE analysis of the uncoatingactivity after chromatography on phenyl Seph-arose. Protein that was eluted from ATP-aga-rose with 1 ITIM ATP (lane 2) was applied onphenyl Sepharose in 30% ammonium sulfate.Bound proteins were subsequently eluted with10% ammonium sulfate (lane 3), 0% ammo-nium sulfate (lane 4), and 80% (w/w) ethyleneglycol (lane 5). Lanes 6 through 11, High-speedsupernatants after incubation of clathrin-coatedvesicles (ccvs) from peas (p) or bovine brain (b)in the presence (lanes 6-9) or absence (lanes10 and 11) of ATP and an ATP-regeneratingsystem and in the presence (lanes 8-11) orabsence (lanes 6 and 7) of the protein fractioneluted from phenyl Sepharose with 0%ammonium sulfate (fraction 2), specified inthe figure as ATPase. Lane 1, Molecular massstandards.

kD

11

31 > — •

+ + + +-- ATP-- + + + + ATPaseP b P b P b c c v s

ess < 70kD peptides

1 2 34 5 67 8 9 10 11

whereas increase in the concentration of the lower peptideduring the assay did not enhance the uncoating activity (Fig.4, compare lane 3 with lane 4).

Different Clathrin-Coated Vesicle Fractions from Peas AreUncoated to a Different Extent

During equilibrium density centrifugation on a 2H2O:Ficollgradient, clathrin-coated vesicles sediment to a broad rangeof equilibrium densities (10-22% [w/w] Sue equivalents),with the highest concentration of clathrin-coated vesiclesbeing around 15% (w/w) Sue equivalents (data not shown).Therefore, it is possible that there are different populationsof clathrin-coated vesicles having different equilibrium den-sities analogous to the situation encountered in mammaliansystems (Bomsel et al., 1988). We were interested to seewhether there was differential uncoating of vesicles of dif-ferent densities by the uncoating ATPase. After equilibrium

1 2 3 4

density centrifugation of a crude preparation of clathrin-coated vesicles on a 2H2O:Ficoll gradient, five fractions ofvesicles were taken that sedimented to different densities inthe gradient, and the fractions were adjusted to approxi-mately the same concentration of clathrin heavy chain. Figure5, lanes 2 through 6, show that 200 pL of each fractioncontained about the same amount of clathrin heavy chain.When 200 nL of each fraction was incubated with the sameamount of uncoating activity, the amount of clathrin releasedfrom the vesicles decreased with increasing density of thevesicles (Fig. 5, lanes 12-16), with the vesicles of lowestdensity releasing almost all of their clathrin (Fig. 5, compare

k D 1 2 3 4200r

6 7 8

kO200> —

97> —

66> —

45> m 14r "* —

2O —

Figure 3. Immunoblot analysis of fraction 2 using an antibodyagainst a member of the HSP70 family from pea leaves. Lanes 1and 2, Coomassie stain; lanes 3 and 4, western blot. Lane 1,Molecular mass standards; lanes 2 and 3, fraction 2; lane 4, emptylane.

Figure 4. Composite SDS-PACE analysis of the uncoating activityafter chromatofocusing of fraction 1. Lane 1, Molecular mass stand-ards; lanes 6 and 8, proteins (20 and 4 ^g/ respectively) elutedbetween pH 5.5 and 5.0; lane 7, proteins (20 Mg) eluted betweenpH 5.0 and 4.5; lanes 2 through 5, high-speed supernatants afterincubation of clathrin-coated vesicles from bovine brain with ATPand an ATP-regenerating system alone (lane 2) and with protein (20and 4 ̂ g, respectively) eluted between pH 5.5 and 5.0 (lanes 3 and5) or with protein (20 /ig) eluted between pH 5.0 and 4.5 (lane 4).



Uncoating ATPase 209

lane 6 with lane 16). When the vesicles were incubatedwithout uncoating activity, only a small amount of clathrinwas released, and the amount of clathrin heavy chain disso-ciated was about the same for vesicles of different densities(Fig. 5, lanes 7-11).

To investigate whether there is also a variability in theamount of clathrin released from different preparations ofvesicles, we isolated clathrin-coated vesicles by three differ-ent methods and tested the vesicle preparations for theirability to be uncoated by the uncoating ATPase from peas.Method 1 involved a centrifugation on a 2H2O cushion fol-lowed by a centrifugation on a 2H2O:Ficoll gradient. Method3 involved two Sue gradients followed by a 2H2O:Ficollgradient, and method 2 involved just a 2H2O:Ficoll gradient(see "Materials and Methods"). All the vesicles used for thisexperiment were taken out of Ficoll gradients around 15%(w/w) Sue equivalents, and the fractions were adjusted tothe same concentration of clathrin heavy chain as shown inFigure 6, lanes 8 through 10. When 30 /uL of each adjustedfraction was incubated with uncoating activity, the amountof clathrin dissociated from the coated vesicles was differentfor the respective preparations.

The most clathrin was dissociated from vesicles preparedby method 1 (Fig. 6, lane 5), less clathrin was dissociatedfrom vesicles prepared by method 2 (Fig. 6, lane 7), and theleast amount of clathrin was removed from vesicles preparedby method 3 (Fig. 6, lane 3).

Uncoating of Plant Coated Vesicles Requires IntactClathrin Light Chains

Recently, four peptides, of 50, 46, 40, and 31 kD, fromclathrin-coated vesicles isolated from developing peas wereshown to have light chain-like properties (Lin et al., 1992).Antibodies prepared against the 46-kD peptide recognizedthe other light chain peptides, as shown by western blotting(see Fig. 7, lane 10). We used these antibodies to investigatewhether these light chain-like peptides are also released bythe uncoating ATPase and whether these peptides are im-portant for uncoating. Because light chain-like peptides aresensitive to the protease elastase, we treated clathrin-coatedvesicles with elastase and then tested their susceptibility to

method

kD200> — .

3 3 1 1 2 2 3 1 2

1 2 3 4 5 6 7 8 9 1 0

Figure 6. SDS-PACE analysis of the uncoating by uncoating ATPaseof different vesicle preparations. Clathrin-coated vesicles preparedby three different methods (see "Materials and Methods") wereadjusted to about the same concentration of clathrin heavy chain.Lanes 8 through 10 show 10 ^L of each adjusted fraction. Lanes 2through 7, High-speed supernatants after incubation of 30 til ofeach vesicle preparation in the presence (lanes 3, 5, and 7) andabsence (lanes 2, 4, and 6) of uncoating ATPase. Lane 1, Molecularmass standards.

the uncoating ATPase. Lanes 6 and 7 of Figure 7 compareprotein patterns of elastase-treated (lane 7) and untreated(lane 6) vesicles. The intact light chain peptides present inthe untreated vesicles (Fig. 7, lane 10) were partially digestedin the elastase-treated vesicles (Fig. 7, lane 11). When elas-tase-treated vesicles and untreated vesicles were incubatedwith the uncoating ATPase, clathrin heavy chain was re-leased from the untreated vesicles (Fig. 7, lane 4), whereasthere was no clathrin heavy chain released from elastase-treated vesicles (Fig. 7, lane 5) when compared with thecontrol (Fig. 7, lane 3). This shows that even though elastase-treated vesicles and untreated vesicles contain about the sameamount of clathrin heavy chain (Fig. 7, lanes 6 and 7), onlythe clathrin of the untreated vesicles can be dissociated.Figure 7, lane 8, shows that the uncoating ATPase also

c v fractions A B C D E A B C D E A B C D E

1 2 3 4 5 6 7 8 910 11 121314 15 16

Figure 5. SDS-PACE analysis of the uncoatingof different populations of clathrin-coated ves-icles. After centrifugation of a crude prepara-tion of clathrin-coated vesicles on a 2H2O:Ficollgradient, five fractions of vesicles were col-lected from the gradient and adjusted to thesame concentration of clathrin heavy chain.Lanes 2 through 6 show 200 ^L of each adjustedfraction. Lanes 7 through 16, High-speed su-pernatants after incubation of 200 ^L of clath-rin-coated vesicles in the presence (lanes 12-16) and absence (lanes 7-11) of uncoating ac-tivity. Fraction A, Vesicles at 22%; fraction B,vesicles at 18.6%; fraction C, vesicles at 15.6%;fraction D, vesicles at 13.2%; and fraction E,vesicles at 10.2% (w/w) Sue equivalents. Lane1, Molecular mass standards.




elastase

kD200>

s s s s c c s s

1 2 3 4 5 6 7 8 9 10 11

Figure 7. SDS-PACE and western blot analysis of the uncoating ofelastase-treated vesicles. Lanes 6, 7, 10, and 11, Treated and un-treated coated vesicles (c); lanes 2 through 5, 8, and 9, High-speedsupernatants (s) after incubation of elastase-treated and untreatedcoated vesicles in the presence (lanes 4, 5, 8, and 9) and absence(lanes 2 and 3) of uncoating ATPase. Lane 1, Molecular massstandards. Lanes 1 through 7, Coomassie stain; lanes 8 through 11,western blot using antibodies against the 46-kD light chain peptide.

dissociates the clathrin light chain peptides from the un-treated vesicles, whereas no light chains are released fromthe elastase-treated vesicles (Fig. 7, lane 9) even thoughfragments of the light chain peptides were still present (Fig.7, lane 11). These results suggest that the light chain peptidesmust be intact for the uncoating of plant coated vesicles bythe uncoating ATPase from peas.

DISCUSSION

The present study demonstrates that in plant cells there isa cytosolic ATP-dependent enzyme activity that uncoatsclathrin-coated vesicles in vitro. The fact that the uncoatingATPases from both bovine brain and peas were able to uncoatclathrin-coated vesicles from peas and that the uncoatingATPase from peas also uncoated clathrin-coated vesicles frombovine brain shows that the mechanism of uncoating issimilar in brain and peas. In brain, uncoating of clathrin-coated vesicles is dependent on clathrin light chains LCa andLCb (Rothman and Schmid, 1986). According to DeLuca-Flaherty et al. (1990), the interaction of the uncoating ATPasewith LCa is most important for the uncoating process. WithinLCa, a stretch of 26 amino acids was suggested as the bindingsite for the uncoating ATPase. This binding site is conservedbetween different mammalian species (DeLuca-Flaherty etal., 1990).

The identification of clathrin light chains in plant systemshas been elusive (Coleman et al., 1988). We have recentlyidentified four peptides (50, 46, 40, and 31 kD) with lightchain characteristics in highly purified clathrin-coated vesi-cles from developing pea cotyledons (Lin et al., 1992). Al-though they have different molecular masses than mamma-lian light chains, it would appear that since the plantuncoating ATPase is equally effective with bovine brain and

plant clathrin-coated vesicles, there must be similar bindingsites involved in the uncoating process in the light chaincomponents. Our data show that in the uncoating reaction,these light chains are released together with the clathrinheavy chain and that a partial digestion of the light chainpeptides by elastase is accompanied by the failure of theuncoating ATPase to uncoat these treated vesicles. This sug-gests that in plants, as in mammalian systems, intact lightchains are required for uncoating. However, it cannot beexcluded that elastase also impairs the clathrin heavy chainor other yet uncharacterized coat proteins that might beessential for uncoating.

Vesicles prepared by different methods were uncoated todifferent degrees. The least amount of clathrin was releasedfrom vesicles whose preparation involved Sue gradient cen-trifugation. It has been shown that prolonged exposure toconcentrated Sue solutions destabilizes clathrin-coated vesi-cles (Nandi et al., 1982). It is also possible that the digestionwith ribonuclease at 30°C instead of 4°C involved in thepreparation of vesicles for Sue density centrifugation mighthave impaired labile light chain domains required for thebinding of uncoating ATPase. When the three different ves-icle preparations were analyzed by western blotting using theanti-light chain serum, the light chain patterns of the threepreparations showed only minor differences (data notshown). However, it cannot be excluded that there weremore drastic differences in the conformations of the lightchain peptides.

During centrifugation of a crude preparation of clathrin-coated vesicles, the vesicles sedimented to a broad range ofdensities (between 10 and 22% [w/w] Sue equivalents) withthe maximum quantity of coated vesicles around 15% (w/w)Sue equivalents. Vesicles of lower densities were better sub-strates for the uncoating ATPase than were vesicles of higherdensities. In ultrastructural studies of uncoating of crudeclathrin-coated vesicles from a variety of mammalian tissue,it was consistently observed that despite the presence ofexcess uncoating ATPase, 20 to 30% of the vesicles retainedtheir coats (Paddenberg et al., 1990). This observation sug-gests varying degrees of susceptibility to uncoating in thepopulation of crude vesicles. The variability in uncoating ofclathrin-coated vesicles of different densities suggests thatthere might be differences in the light chains of the differentvesicle populations. Western blot analysis of the light chainpeptides did not show any significant differences betweenthe different vesicle populations (data not shown). However,this does not exclude the possibility that there may be differ-ences in the folding pattern of the light chains or in the waythey are associated with other coat proteins. On the otherhand, differential uncoating could be a consequence of dif-ferences in the coat structure due to the presence/absence ofminor, yet uncharacterized peptides. There is also a possibilitythat the fractions of lower density are more enriched inclathrin baskets and that these baskets could be dissociatedmore easily than the coats of intact vesicles.

The mechanisms controlling susceptibility of clathrin-coated vesicles to uncoating have not been resolved. Clathrin-coated vesicles are formed during endocytosis from coatedpits on the plasma membrane and during intracellular trans-port on the cytoplasmic surface of the trans-Golgi. During



Uncoating ATPase 21 1

their formation and during their existence in the cytoplasm, the clathrin-coated vesicles are presumably exposed to the cytoplasmic uncoating activity, yet they remain intact. How- ever, since they must be uncoated prior to fusion with the target organelle, a regulated change in their coat structure is required that makes the coat susceptible to the uncoating ATPase. The presence of clathrin-coated vesicles of different densities might reflect the existence of coated vesicles with different functions or of a different degree of maturity along their way from the trans-Golgi network to an endosomal compartment. The vesicles of lower density that are more susceptible to uncoating might be more closely related to an endosome-like compartment, whereas the vesicles of higher density that are more resistant to uncoating might be younger vesicles that just pinched off the trans-Golgi network. In animal systems, different populations of clathrin-coated vesicles with differences in size, density, function, and protein composition have been reported (Bomsel et al., 1988).

A problem that was encountered with the uncoating assay was that vesicles would release some clathrin even in the absence of uncoating activity and that this amount of clathrin was different for vesicles from different preparations. This was especially true for vesicles isolated from bovine brain. This instability has been observed in mammalian systems, particularly at elevated pH (Paddenberg et al., 1990). It has also been shown that the uncoating ATPase is a constituent of highly purified clathrin-coated vesicles (Paddenberg et al., 1990). Although this fact could account for some of the uncoating observed in the absence of added ATPase, it appears that since some uncoating occurred in the absence of added ATP and enzyme, other mechanisms are also operat- ing. The uncoating of clathrin-coated vesicles in control assays was minimized either by using freshly prepared vesicles or by pelleting and resuspending the vesicles immediately before the uncoating assay.

In mammalian and yeast cells, the uncoating activity is a member of the HSP70 family (Chappel et al., 1986; Coa et al., 1991). Antibodies against a member of the HSP70 family from pea leaves recognized mainly the upper member of a doublet of 70-kD peptides contained in a fraction with uncoating activity isolated from developing peas (fraction 2). Indications that the immunoreactive peptide also contains uncoating ATPase activity is provided by chromatofocusing experiments. Fractions from the chromatofocusing column in which the upper immunoreactive peptide was enriched rel- ative to the lower member of the doublet had enhanced uncoating activity (Fig. 4, lane 3). However, other peptides were also enriched in this fraction and, therefore, it cannot be excluded that one of these other peptides is the uncoating ATPase. Using the antibodies against HSP71.2, we investi- gated whether immunospecific remova1 of the upper 70-kD peptide would result in loss of uncoating activity. Unfortu- nately, the antibody failed to interact with native 70-kD peptide, nor did it effect the uncoating activity (data not shown).

The identification of coat proteins of clathrin-coated vesicles from plants is only at its start. The characterization of additional coat components and the sequence analysis of coat proteins will allow a more detailed investigation of the uncoating process. Uncoating experiments in vitro using baskets

lacking one or other coat component should reveal proteins that are crucial to uncoating. This will open up new possibil- ities for the investigation of the mechanism of uncoating in vivo and its role and importance for intracellular protein transport.

ACKNOWLEDCMENTS

We thank Drs. Elizabeth Vierling and Amy DeRocher for the antibodies against HSP70 and Dr. Hongbo Lin for the antibodies against the clathrin light chains.

Received February 12, 1993; accepted May 26, 1993. Copyright Clearance Center: 0032-0889/93/l03/0205/08.

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