The Binding Sites of Cytochalasin D. I . EVIDENCE THATTHEY MAY BE PERIPHERAL MEMBRANE PROTEINS JANET TANNENBAUM,' STUART W. TANENBAUM AND GABRIEL C. GODMAN Departments of Microbiology and Pathology, Columbia Unioersity, New York, New York 10032

ABSTRACT Binding sites for tritiated cytochalasin D (3H-CD) on the isolated plasma membrane from HEp-2 cells were reversibly inactivated, but not dissociated from the membrane, by dialysis in 0.6 M KCl. Activity was restored by subsequent dialysis in 0.06 MKCLTreatment with 0.2 mM ATP at low ionic strength also inactivated these sites, apparently irreversibly. Extraction of the membrane with 6% Triton X-100 removed 75% of its protein, resulting in a two-fold increase in specific binding activity for 3H-CD. Both high and low affinity binding sites were retained by the detergent-extracted membrane; at least 60% of the high affinity sites were resistant to this treatment. Evidence is presented for the attachment to the HEp-2 plasma membrane of both actin and myosin. The results support the tentative conclusion that plasma membrane binding sites for 3H-CD are peripheral proteins on the cytoplasmic face of the membrane. They are consistent with the hypothesis that myosin may be the location of the high affinity binding site and actomyosin may be the low affinity site. Comparison of these observations with those reported for the congeneric drug, cytochalasin B, suggests that CD binding sites differ from the high affinity site for cytochalasin B.

The cytochalasins are a group of fungal metabolites which influence locomotion, contractility, and a variety of related processes in mammalian cells (Wessells et al., '71; Carter, '72; Miranda et al., '74a; Pollard and Weihing, '74; Rathke et al., '75). To elucidate the mechanism of action of these compounds, we are investigating the location and nature of their cellular binding sites. We have previously reported (Tannenbaum et al., '75b) that 3H-CD binds to the isolated plasma membrane and microsomal fractions of HEp-2 cells. The plasma membrane fraction, like intact cells, exhibited binding activity of both high and low affinity, while the microscomes appeared to contain only low affinity sites. Since protease drastically decreased the binding capacity for 3H-CD of isolated plasma membrane, but not that of intact cells, we concluded that proteins exposed on the cytoplasmic face comprised binding sites for J. CELL. PHYSIOL.. 91: 225-238.

CD. Additional evidence that binding sites for 3H-CD are intracellular has been obtained by comparison of the activation energies for binding of 3H-CD to intact cells and to isolated plasma membranes (Tannenbaum et al., '75a). In this communication we report that binding sites for CD on the plasma membrane are resistant to extraction with nonionic detergent and, therefore, are most likely contained on peripheral proteins. Evidence is also presented that actin and (putatively) myosin are attached to the plasma membrane of the HEp-2 cell. These data, together with our ancillary observations, are consistent with the hypothesis that membrane-bound myosin comprises Received May 24, '76. Accepted Sept. 22, '76. Present address: Laboratories of Virology, St. Jude Children's Research Hospital, P.O. Box 318, Memphis, Tennessee 38101, U.S.A. Present address: School of Biology, Chemistry and Ecology, SUNY College of Environmental Science and Forestry, Syracuse, N.Y. 13210, U.S.A.

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1. TANNENRAUM, S. W. TANENBAUM AND 6. C:. GODMAN

the high affinity site and that actomyosin is a low affinity site for CD in such preparations. MATERIALS AND METHODS

Cytochalasin D was isolated in this laboratory from cultures of Zygosporium m a sonii by a procedure slightly modified from that of Minato and Matsumato ('70). The purified recrystallized cytochalasin exhibited all of the physical, chromatographic and analytical properties of authentic CD.3 Radioactive CD, prepared from this compound by the generalized relatively nondestructive procedure of Hembree et al. ('73), was purified to constant specific activity by preparative thin-layer chromatography; this tritiated CD migrated coincident with the authentic compound in several analytical thin layer chromatography systems3 The biological activity of both the radiolabeled and unlabeled CD, including comparison with inactive derivatives of CD, were entirely comparable, and the validity of the 3H-CD as a probe was confirmed (Tannenbaum et al., '75b). Hep-2 cells were grown as monolayers in Eagle's minimal essential medium supplemented with 10% newborn calf serum, 60 U/ml penicillin and 60 pg/ml streptomycin (Grand Island Biological Co.). The plasma membrane fraction was isolated (Atkinson and Summers, '71) without the use of sodium iodacetate or sodium azide (Tannenbaum et al., '7%). Tritiated cytochalasin D (3H-CD) (Tannenbaum et al., '75b) was dissolved in DMSO and diluted with SMT buffer (0.25 M sucrose-1 mM MgCl2-10mM Tris HCI, pH 7.0) before use. Samples of freshly isolated Hep-2 plasma membrane (Atkinson and Summers, '71) were dialyzed at 0-4°C with stirring in the solutions detailed in the legends to tables 1 and 2. ATP (disodium salt) was obtained from P. L. Biochemicals, Inc., and dithiothreitol (DTT) from Calbiochem. For treatment with KI, the membrane was first dialyzed overnight against 0.06 M KC1 in 10 mM Tris HCl, pH 8.0, then collected by centrifugation and incubated for ten minutes at 37°C in 10 mM Tris HCl, pH 8.0,

with either 0.06 hi KI or 0.6 M KI. All solutions containing KI were prepared immediately before use. The treated membrane samples were collected by centrifugation (7,000 X g for ten minutes at 0-4"), suspended in chilled SMT, and assayed for binding activity with 3H-CD; those exposed to DTT or KI were washed in SMT before resuspension. In some cases, samples of freshly isolated plasma membranes were assayed for binding activity without further treatment (untreated controls). In experiments to test the reversibility of the KCI effects, replicate samples of membrane were dialyzed in 0.6 M KCl and then transferred to 0.06 MKCI(all in 30 mM Tris HCl, pH 6.8) for further dialysis, either in their original dialysis sac (table 2A) or after centrifugation to separate the membrane pellet from materials released into the supernatant (table 2B). In similar manner, reversibility of the ATP effect was examined either by dialysis of membrane against 0.2 mM ATP-1 mM MgC12-1mM Tris, pH 7.0, followed by 0.06 M KC1-30 mM Tris, pH 6.8, or by dialysis in ATP-DTT solution followed by 0.06 M KC1-5 mM DTT-1 mM MgCl2-30mM Tris HC1, pH 7.0. Membrane pellets were then resuspended in SMT buffer. Detergent extraction was performed by incubating freshly isolated HEp-2 plasma membrane for 15 minutes at 37°C in 6% Triton X-100 dissolved in phosphate buffered saline (Dulbecco's PBS, calciummagnesium-free, pH 7.2-7.4; Grand Island Biological Co.). The treated membrane was then washed in chilled SMT and resuspended in this buffer. Samples of membrane which had been dialyzed in ATP as described above were extracted with detergent by the identical method. Binding activity for 3H-CD was assayed by incubating samples suspended in SMT The crystallized unlabeled C D tested by: elemental analysis; melting point and mixed melting point; 60 and 100 MHz NMR spectra; mass spectrum, including parental ion and breakdown pattern of fragments, infrared spectrum, preparation of a t least three chemical derivatives; and thin layer chromatography in six solvent systems with different supports, conformed to the criteria hitherto published by Aldrich and Turner ('69). Minato and Matsumoto ('70).Minato et al. ('73) and Lehet and Tamm ('74) for the pure compound.

BINDING SITES OF CYTOCHALASIN

227

L)

TABLE 1

Effect of salt or ATP extractions on binding of 3H-CD to HEp-2 plasma membrane Specific binding activity (dpm 'Hipg protein)

Treatment

137 25,26 88%15 38 94

0.060 M KC1/24 hour dialysis 0.600 M KC1/24 hour dialysis 0.035 M KC1/24 h o u r dialysis 0.2 mM ATP-1 mM Tris/24 hour dialysis Untreated (control) 0.2 mM ATP-1 mM T r i s - 5 mM DTT/40 hour

21

dialysis U n t r e a t e d (control) ~~~

~~

118 ~~

~

~~

Sainples of HEp-2 plasma rnemhrane were dialyzed at 0 - 4 T with stirring as indicated. The KCI solutions were prepared in 30 mM Tris HCI, pH 6.8, the ATP solution was adjusted to pH 7.0, and the ATP-DTT solution tu p H 6.8 before use. Membrane pellets were collected from dialyzed samples by centrifugation and aasayed for binding of 'HCD. Untreated samples of HEp-2 plasma membrane (from different preparations) had a specific binding activity in the range of about 80-100dpin ?H/pg protein. Within each extraction procedure ( i e , KCI, ATP, or ATP-DTT) replicate vaniples of membrane from a single preparation were used. Recovery of protein in memhrane pellets after dialysis was about 95% for 0.06 M KCI treatment and about 75%for the ATP. 0.6 hl KCI, or 0.035 11 KCI treatments. TAHLE 2

Reoersibility of effect of KC1 on binding of "-CD to HEp-2 plasma membrane Treatment First dialysis

Specific binding activity (dpm 'H/LLKprotein)

Second dialysis

~~

A. 0 . 0 6 ~ K C l 0.60 M KCI 0.60 M KCI B. 0.60MKCI 0.60 M KCI

0.06 M KCI None 0.06 M KCl 0.06 M KCI 0.06 M KCl (minus supernatant)

129k21 25,26 ' 118511 97?2 120.t2

Data from table I First dialysis: Replicate samples of HEp-2 plasma membrane were dialyzed overnight at 0-4"C,against 30 mM Tris HCI, pH 6.8, containing the indicated concentrations of KCI. Second dialysis: A. Samples, still in their respective dialysis bags, were transferred to a fresh solution of 30 mM KCI and were dialyzed for 20 hours. Membrane pellets were then ohtained by centrifugation and Tris, pH 6.8-0.06M assayed for hinding of 3H-C13.B. Samples were centrifuged to obtain membrane pellets. One pellet was resuspended in its own supernatant, the other ("minus supernatant") in fresh 30 mM Tris, pH 6.8-0.06M KCI. Both were dialyzed against this same solution for 23 hours, then centrifuged and assayed as in A. Untreated samples (from different preparations) had a specific binding activity in the range of80-100 dpm nH/pg protein. Recovery of protein in the membrane pellets was about 95% after the double dialysis in 0.06 M KCI and about 75% for all other samples listed.

with 0.52 pg/ml 3H-CD (1KM), or other concentrations as indicated in the figure legends, for 15 minutes at 37"C, then centrifuging to collect the pellets (7,000 X g for 10 minutes at 0-4").We have hitherto shown (Tannenbaum et al., '75b) that binding of 3H-CD reaches a maximum level within 15 minutes at 37°C. Since only about 1-2% of the 3H-CD is bound to pellets in these assay conditions, the concentration of "free" (unbound) 3H-CDis virtually identi-

cal to the total concentration of 3H-CDoriginally added. After the membrane preparations were washed in chilled SMT, again centrifuged, and resuspended in distilled water, samples were taken for determination of protein (Lowry et al., '51) and tritium. Background counts were determined by similarly processing samples containing 3H-CD and SMT without membrane material. For scintillation counting the samples in distilled water were digested with Solu-

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Fig. 1 HEp-2 plasma membrane ( X 31,320). a Freshly isolated membrane fraction. Note microfilaments apparently attached to the membrane. b Membrane was dialyzed against 0.2 mM ATP-1 mM Tris, pH 7.0. Microfilaments are greatly depleted.

ene- 100 (Packard Instruments) and mixed with Omnifluor-toluene cocktail (4 g/l, New England Nuclear). Efficiency was determined by internal standardization. The gel filtration assay for binding of 3HCD to the supernatant of ATP-dialyzed membrane was performed on a Sephadex G-25 column as previously described for binding assays of the post-granular supernatant fraction (Tannenbaum et al., '75b). Pellets of membrane were fixed for electron microscopy with 2.5% glutaraldehyde in Millonig's phosphate buffer, pH 7.2, then washed in buffer, post-fixed in 1% osmium tetroxide in Millonig's buffer, washed again, and stained overnight in 0.25% aqueous uranyl acetate. The pellets were washed, dehydrated in a graded series of methanols and propylene oxide, and embedded in Durcopan (Fluka, A. G.). Thin sections were prepared and examined by standard techniques. SDS-disc gel electrophoresis was performed by the method of Fairbanks et al.

('71), except that 1%mercaptoethanol was substituted for DTT in the solvent used to dissolve the samples. Pellets of Hep-2 plasma membrane were suspended in this solvent and incubated at 37°C for 30-45 minutes before application to the gels. Soluble samples, i.e., the supernatant solutions from various dialysis procedures, were subjected to overnight dialysis against 40 mM Tris HC1-4 mM EDTA, pH 8.0, to remove interfering ions. They were then concentrated to a volume of about 0.05ml in an Amincon Minicon 815 concentrating chamber and combined with sample buffer (Fairbanks et al., '71) for electrophoretic examination. Standards consisted of rabbit striated muscle myosin re ared as described by Richards et al. 67 and muscle actin (donated by Doctor A. Penn). The processed samples were applied in 0.1 ml portions to 5.6% acrylamide gels: the myosin standard contained about 5 p g protein and the actin standard about

FP

229

BINDING SITES OF CYTOCHALASIN D

25 pg; samples from untreated membrane were applied in quantities of 100 and 180pg protein, and samples of treated preparations derived from 150 pg protein (KCl or ATP dialysis) or 400pg protein (detergent extractions) of original membrane. Electrophoresis was performed at a constant current of 8 mA/tube at room temperature. Gels were stained with Coomassie brilliant blue and scanned at 550nm with a 0.05 mm slit in a Beckman spectrophotometer equipped with a Gilford linear transport accessory. Since the bands on some gels did not exhibit enough contrast to be clearly visible in photographs or on densitometric scans, gel staining patterns were routinely copied in scale-model drawings; R f values were calculated from these drawings. Estimation of the proportion of the total bound 3H-CD which is present at high affinity sites was performed by graphical analysis. The linear component of the binding curves, which is seen at high concentrations of 3H-CD for untreated plasma membrane (Tannenbaum et al., ’75b) or detergent-extracted membrane (fig. 4), was subtracted from the total binding to yield the binding curves for the high affinity sites (fig. 5). RESULTS

lonic manipulations As shown in table 1, dialysis of isolated Hep-2 plasma membrane with 0.6 M KC1 greatly decreased the binding activity for 3H-CD.Comparable treatment with 0.06 M KCl enhanced binding while 0.035 M KCl decreased it but slightly. Treatment with 0.06 M KI or 0.6 M KI solutions gave results similar to those for 0.06 M KC1 and 0.6 M KCl, respectively (data not shown). The effect of dialysis against 0.6 M KCI could be completely reversed by subseuent dialysis of the plasma membrane still in its original dialysis sac) in 0.06 M KCI (table 2). This result suggested the possibility that 0.6 M KC1 did not cause dissociation of binding sites from the plasma membrane. When membrane that had

;r‘

21

2

A

B

C

-3

D

Fig. 2 Selective extractions of HEp-2 plasma membrane: SDS-Disc Gel Electrophoretic Patterns. Samples were applied to 5.6% acrylamide gels and subjected to SDS-disc gel electrophoresis. Protein bands were visualized by Coomassie brilliant blue staining. In these sketches, arrows indicate position of tracking dye and dashed lines designate minor bands which were barely visible. Actin standards migrated to a position corresponding to band 20 (R f, 0.50) and myosin heavy chain standards (R f, 0.08) to a position slightly ahead of band 4. A HEp-2 plasma membrane after dialysis against 0 . 0 6 ~ KC1-30 mM Tris, pH 6.8; untreated plasma membrane gives an indistinguishable pattern. Note that band 4 consists of at least three fine bands which are not resolved in this sketch. B HEp-2 plasma membrane after extraction in 6% Triton X-100-PBS for 15 minutes at 37°C. Extraction has removed many polypeptides from the residual membrane pellet. C HEp-2 plasma membrane dialyzed in 0.2 mM ATP-1 mM Tris, pH 7.0, for 48 hours and then, after centrifugation to separate the membrane pellet from its supernatant, extracted with 6%Triton X-100 as in B. Note that band 20, corresponding to actin, has been completely removed from the membrane; band 3, putatively myosin, is also absent (Cf. B). D “Supernatant” of C, containing material released from plasma membrane during dialysis in ATP. Bands 20 and 3 are the only polypeptides extracted under these conditions.

230

J. TANNENBAUM, S. W. TANE.NBAUM AND G. C . GODMAK

1.01

01

I

I

Fig. 3 A portion of densitometric scan of HEp-2 plasma membrane samples. Replicate samples (about 150 p g protein) of HEp-2 plasma membrane were dialyzed against 0 . 0 6 ~KC1 in 30 mM Tris, pH 6.8; 0.2 mM ATP-1 mM Tris, pH 7.0; or 0.6 M KCI-30 mM Tris, pH 6.8, for about 20 hours at 0-4°C. Membrane pellets were then processed for SDS-disc gel electrophoresis. Gels were stained with Coomassie brilliant blue and scanned at 550 nm. Curves for membrane dialyzed with 0.06 M KCI (solid line) or 0.6 M KCI (not shown) superimposed. The curve for ATP-dialyzed membrane (dotted line) shows a considerable loss of material from one peak, which co-migrates with the actin standard (arrow). Direction of migration is from left to right. Ordinate: A550in arbitrary units. The higher molecular weight region, including hands 1-5 is not shown in this tracing.

been dialyzed in 0.6 M KCI was centrifuged to separate any components which might have been released, and then was dialyzed against 0.06 M KC1, binding activity was completely restored (table 2B). Dialysis with ATP at low ionic strength lowered the specific binding activity of HEp-2 plasma membrane to a value similar to that obtained by dialysis with 0.6 M KCl (table 1).Unlike the KCl effect, however, this decrease could not be reversed by subsequent dialysis of the membrane (with its supernatant) against 0.06 MKCI.Similar dialysis procedures with ATP in the presence of 5 mM DTT also resulted in irreversible loss of binding activity, even when the membrane was subsequently dialyzed in 0.06 M KCI with DTT plus 1 mM MgClz (table 1; reversibility data not shown). Change of reaction conditions to a lower concentration of DTT, 0.1 mM, gave simi-

lar results (data not shown). Binding activity was not detectable in the supernatant by gel filtration, probably because the amount of protein released by ATP dialysis was too minute to be assayed by this method. Electron micrographs of HEp-2 plasma membrane invariably showed microfilaments apparently attached to the membrane (fig. la) (Tannenbaum et al., '75b). Dialysis with ATP at low ionic strength dissociated these microfilaments from the membrane (fig. Ib). Examination of the supernatant from the dialyzed membrane by SDS disc gel electrophoresis (fig. 2D) demonstrated that this procedure had removed from the membrane both a protein (band no. 20) which comigrated with muscle actin, and a high molecular weight protein (band no. 3) which approximately comigrated with the heavy chain of striated muscle myosin. In contrast, microfilaments were not removed from membranes dialyzed against 0.06 M KCl or 0.6 M KC1 (results not shown). Supernatants of membranes dialyzed against these KCl solutions did not show any detectable bands of protein when examined by SDS disc gel electrophoresis (data not shown). Disc gel electrophoresis of freshly isolated plasma membrane and plasma membrane dialyzed against KCI or ATP gave the banding pattern seen in figure 2A. Densitometric tracings of the stained gels from membrane dialyzed against 0.06 M KCI or 0.6 M KCl were superimposable, but dialysis with ATP markedly decreased the peak corresponding to actin (fig. 3 ) . Only the lower weight portion of the densitometric scan is shown, because the multiple, high molecular weight bands (no. 1-5) were not distinctly resolved by the densitometer. Consequently, evidence for extraction of the high molecular weight band (no. 3) was based solely on its demonstrated presence in the extract (fig. 2D).

Extraction with non-ionic detergent Extraction of isolated HEp-2 plasma membrane with 6% Triton X-100 in PBS at 37°C for 15minutes removed about 75% of the protein from the membrane. The re-

231

BINDING SITES OF CYTOCHALASIN D

EFFECT OF CONCENTRATION OF 3H-CD ON BINDING DETERGENT-EXTRACTED PLASMA MEMBRANE

TO

300 -

Fig. 4 Binding of‘ “-CD to detergent-extracted plasma membrane. HEp-2 plasma membrane was extracted with 6% Triton X-100 in PBS as described in MATERIALS AND METHODS. The washed membrane was then assayed for binding of’ 3H-CDat the concentrations of drug indicated. The deflection of the curve at about 0.2 wg/ml and the apparent non-saturability up to 1.04 pg/ml are the same as previously observed in non-extracted membrane (Tannenbaum et al., ’75b).

+

sulting membrane pellet showed a two-fold increase in specific binding activity for 3HCD compared to a replicate sample of freshly isolated membrane “extracted” with PBS (259 dpm 3H/pg protein for detergent extracted membrane versus 127 dpm 3H/pg protein for PBS “extracted” membrane). The binding curve for detergent-extracted plasma membrane (fig. 4) was determined in order to see if both high and low affinity sites for 3H-CDwere resistant to this treatment. We have previously shown that high affinity sites were saturable at a 3H-CD concentration of about 0.2pg/ml, but low affinity sites were unsaturable for concentrations as high as 1.04 pglml (Tannenbaum et al., ’7Sb). As shown in figure 4,the curve for detergentextracted membrane changes slope at about 0.26 pg/ml, but also does not reach saturation at < 1.04 pg/ml 3H-CD.It therefore a pears that both saturable (high affinity and apparently unsaturable (low affinity) binding sites for 3H-CD are retained after extraction of the plasma membrane with Triton X-100. The binding curves for the high affinity sites of plasma

P

membrane (Tannenbaum et al., ’75b) and detergent-extracted membrane (fig. 4) were calculated by subtracting the amount of 3H-CD associated with low affinity sites from the total bound drug (fig. 5 ) . Comparison of the binding curves calculated for the high affinity sites before and after extraction with detergent (fig. 5) demonstrated an apparent decrease in the affinity of these sites in the extracted membrane. Saturation of the high affinity binding component appeared to occur at a 3H-CD concentration of about 0.15 pg/ml (0.3pM) for untreated membrane, and about 0.30 pg/ ml (0.6 p M ) for detergent-extracted membrane. The reason for this decrease is not known, but may have been caused by association of residual Triton X-100with 3HCD or with the binding sites for 3H-CD. From the amount of 3H-CD bound at saturating concentrations (fig. S ) , it can be calculated that at least 60% of the high affinity binding sites are recovered, still attached to the membrane, after extraction with 6% Triton X-100. At saturation of high affinity sites, detergent-extracted membrane binds a maximum of about 190 dpm

232

J. TANNENBAUM, S. W. TANENBAUM AND G . C . GODMAN HEp-2 PLASMA MEMBRANE: HIGH AFFINITY BINDING BEFORE AND AFTER DETERGENT EXTRACTION

-.E 0

c

200-

r

c

0

3H-CD/pg protein and untreated membrane about 80 dpm/pg protein at their respective high affinity sites (fig. 5). Since extraction with Triton X-100 removes approximately 75% of the protein from the membrane, 1 p g of membrane protein theoretically yields 0.25 pug of extracted membrane protein. If no high affinity binding sites are dissociated from the membrane by detergent, the specific binding activity should be 80 dpm 3H-CD/0.25pg protein of extracted membrane (or 320 dpm 3H-CD/pg protein). The observed value of 190 dpm 3H-CD/pgprotein is 60% of this value. Thus, Triton X-100 removes (or inactivates) a maximum of 40% of the high affinity binding sites. The loss of high affinity sites might actually be less than 40%; the value used for retention of protein after extraction (25%) is likely to be an underestimate since it is subject to the error of loss due to random failure of some membrane “ghosts” to sediment in the viscous 6% Triton X-100 solution. If, for example, the extraction procedure had dissolved only 67% of the membrane protein and the additional apparent 8% loss represented nonsedimenting membrane “ghosts,” the calculated recovery of high affinity binding sites would be 80%. Since detergent-extracted membrane exhibited some binding activity which was not saturable at concentrations of 3H-CD up to 1.04 pglml (fig. 4), it appeared that at

,)

1

least some low affinity binding sites were also resistant to Triton X-100. Because these sites were apparently not saturable, the only way to quantitate them is to assay the amount of drug bound at some standard concentration of 3H-CD before and after extraction. However, as stated above, the binding affinity of membrane was decreased after extraction with detergent. Therefore, the amount of 3H-CD bound to these sites at any given concentration would automatically be lower than the value for untreated membrane, and would result in an underestimate of the retention of low affinity sites after extraction. Ignoring this error, it can be estimated that at least 40% of the low affinity sites were resistant to Triton X-100, as follows. At the concentration of 3H-CDused in the binding assay (0.52pg/ml), about half of the total bound drug is at high affinity sites. Since we know that at least 60% of the high affinity sites were recovered after extraction, then some 30% out of the total 50% normally bound to membrane was bound to high affinity sites of extracted membrane (60% X 50% normally bound to high affinity sites). At 0.52pg/ml of 3H-CD, extracted plasma membrane retained 25% of its protein and 50% of its total binding activity (therefore the specific binding activity, dpm 3H/pg protein, doubled). Of this 50% recovered, since 30% was at high affinity sites, the difference (20%) must have been

233

BINDING SITES OF CYTOCHALASIN D

Triton X-100 (fig. 5). Since somewhat less drastic conditions of extraction with this detergent were reported to remove all integral proteins and glyco roteins from red cell ghosts (Yu et al., '73 , we anticipated that our extraction procedure would remove all integral proteins from the HEp-2 plasma membrane. Although resistance to extraction with Triton X-100 of binding activity for 3H-CD suggests that the binding sites might be peripheral membrane proteins, it could be argued that they were very tightly bound integral proteins which could not be solubilized by Triton X-100. The loss of binding activity after dialysis with ATP at low ionic strength (table 1) is consistent with the behavior expected of a binding site contained on a eripheral protein (Singer, '74; Steck, '747, which would be dissociated from the membrane by this treatment. However, conclusive proof would require demonstration of active binding sites for 3H-CD in the supernatant of the dialyzed membrane. Since the latter has not been achieved, identification of binding sites for 3H-CDas peripheral membrane proteins must be tentative. Nevertheless, the data so far obtained suggest that peripheral proteins are involved. These results contrast sharply with the behavior reported for high affinity binding DISCUSSION sites of the congeneric 3H-CBin red blood The HEp-2 plasma membrane appears to cell ghosts. Lin and Spudich found that contain high as well as low affinity sites for extraction with Triton X-100, using milder 3H-CD, and the binding of the drug conditions than those employed here, comrequires proteins which are present on the pletely abolished the activity of the high cytoplasmic face of the membrane (Tan- affinity binding site for 3H-CB (Lin and nenbaum et al., '75b). These binding sites, Spudich, '74b). These authors have premoreover, are firmly attached; dialysis sented further chemical evidence that this against 0.06 Mor 0.6 MKCIdoes not dissoci- binding site is an integral protein which ate them from the membranes (tables 1,2). spans the red blood cell membrane (Lin Our data suggest that at least a large frac- and Spudich, '74a). Conversely, extraction tion of the binding sites for 3H-CDmay be of the red blood cell ghosts with EDTA at peripheral components of the membrane. low ionic strength, a procedure analogous The strongest evidence for this conclusion to the ATP dialysis described above, did is the observation that binding activity of not remove high affinity binding activity the isolated plasma membrane was resis- for 3H-CB from the membrane (Lin and tant to extraction with Triton X-100 at Spudich, '74b). Thus, this high affinity bind37°C. At least 60%of the high affinity bind- ing site for 3H-CB appears to differ from ing activity was recovered, still attached to the binding sites for 3H-CD. the membrane, after extraction with 6% The removal of membrane-attached mi-

bound at low affinity sites. Thus, the Tritonextracted low affinity sites still bound 20% of their normal ca acity of 50%, which is a recovery of 40% 20%+-So%). Therefore, a significant portion of low affinity binding sites for 3H-CD are also resistant to Triton x-100. Disc gel electrophoresis of the detergent-extracted plasma membrane (fig. 2B) demonstrated that detergent had released all proteins (detectable by staining with Coomassie brilliant blue) except the high molecular weight bands (no. 1, 3, 4), and actin (no. 20). Very small amounts of two other protein species (dashed lines in fig. 2B) were present as barely visible bands. Both bands 3 and 4 (Rp of 0.06 and 0.07 respectively) approximately co-migrated with muscle myosin heavy chain (Rf 0.08). When HEp-2 plasma membrane was dialyzed against ATP at low ionic strength and then extracted with Triton X-100, actin (band no. 20) was totally removed from the membrane (fig. 2C). As observed for the ATP dialysis alone, band 3 was also removed. The triplet of bands 1 and 2 seen in figure 2C was probably resolved from the single band (no. 1 in gel B), because gel C was subjected to a longer electrophoretic run.

7

P

234

J. TANNENBAUM, S. W. TANENBAUM AND G. C. CODMAN

crofilaments b dial sis against ATP at low ion strength Jig. ly, which dissociated a protein comigrating with actin on SDS-gel electrophoresis (figs. 2, 3), show that filament actin is linked to the HEp-2 plasma membrane. The association of actin filaments with the plasma membrane is of general occurrence in animal cells (Pollard and Weihing, '74; Pollard, '75). Assuming that the band which co-migrates with muscle actin is composed entirely of actin-like protein, it appears that dialysis with ATP removed only part of the membrane-associated actin (fig. 3). The extraction of the remaining actin with Triton X-100 suggests that this protein is linked to the membrane by hydrophobic bonds, representing either direct insertion of actin into the lipid bilayer or attachment of the actin to other molecules which insert into the membrane. Others have also reported that extraction with Triton X-100 could remove only part of the membraneassociated actin (Spudich, '74; Taylor et al., '76). Similarly, ionic conditions known to depolymerize actin filaments reportedly dissociated only some of the actin from plasma membrane (Gruenstein et al., '75; but see Korn and Wright, '73 for a possible exception). Sequential extraction of Dictyostelium membrane with Triton X-100 and then 0.6 M KI removed all of the membrane-associated actin (Spudich, '74). Operationally, at least, there would then appear to be two classes of actin in association with the plasma membrane. The evidence is consistent with the speculation that detergent-dissociable actin represents at least those microfilaments whose attachment to the membrane depends on a hydrophobic linkage site. However, this is probably not the only means by which actin filaments can associate with the membrane, as detergent treatment of itself does not remove all membrane-bound actin. Failure of 0.6 M KCl to remove actin or visualizable microfilaments from HEp-2 plasma membrane would indicate that an actin-binding protein like that described by Hartwig and Stossel ('75) does not link these actin filaments to the HEp-2 cell

membrane, because actin is dissociated from "actin-binding protein" by this concentration of KCl (Hartwig and Stossel, '75; Stossel and Hartwig, '75). The membrane-attached actin that is not removed by detergent may well be disposed as a planar cytoskeletal meshwork on the cytoplasmic face of the lipid bilayer, analogous to the model hitherto proposed for the detergent-resistant submembranous network of spectrin and actin of the eyrthrocyte membrane (Steck, '74; Yu et al., '73) and for plasma membrane of the amoebae of DictyosteZium (Spudich and Clarke, '74; Clarke et al., '75). We propose that bands 3 and/or 4 may represent myosin heavy chains on the basis of their apparent similarity in molecular weight to striated muscle myosin, as judged by SDS-disc gel electrophoresis. Definitive identification of these bands will require their biochemical characterization; at this point we merely wish to underscore the probability that myosin is directly or indirectly linked to the HEp-2 plasma membrane. There is ample evidence for the association of myosin with plasma membrane in other systems (Spudich, '74; Spudich and Clarke; '74; Gwynn et al., '74; Willingham et al., '74; Olden et al., '76; Painter et al., '75; Brandon, '75). Band 3 might also be related to the "actinbinding protein" isolated from macrophages (Hartwig and Stossel, '75; Stossel and Hartwig, '75, '76;): the two proteins have similar molecular weights, and the reported solubility properties of "actin-binding protein" (Hartwig and Stossel, '75; Stossel and Hartwig, '75), are also similar to band 3 protein from our HEp-2 membrane; however, band 4 could not represent such a protein. Yet another intracellular protein of high molecular weight (250,000 dalton) recently described, called filamin (Wang et al., '75), should be considered in further study of the identity of the high molecular weight bands. Definitive identification of these bands will obviously require further characterization. The observations that myosin comprises a high affinity binding site for 3H-CD in

-_ - ,.---LIlNUlNL >I 1 L> U k

-‘-i

platelets {Puszkin et al., ’73) and the relationship of actin-like microfilaments (presumably as part of an actomyosin system) to many of the cell functions influenced by cytochalasins (Wessells et al., ’71; Miranda et al., ’74a,b; Godman et al., ’75) readily conduce to the hypothesis that the contractile proteins, actin and myosin, may comprise the cellular binding sites for 3H-CD. This hypothesis is plausible for the HEp-2 plasma membrane in view of the findings that actin appears to be anchored to the plasma membrane, and the evidences that myosin is also present. The results of dialysis of the plasma membrane against KCl (table 1)are consistent with a role for myosin in the binding of 3H-CD,but tend to exclude actin as a major binding site. Although definite myosin filaments are not seen in non-muscle cells, it is thought that short polymers may be present in association with actin filaments (Pollard and Weihing, ’74; Pollard, ’75). Potassium chloride (0.6 M) would be expected to depolymerize myosin polymers into monomeric units (Pollard and Weihing, ’74); all other concentrations of KC1 employed allow myosin to remain polymerized. In contrast, actin is filamentous (i.e., polymerized) in all three concentrations of KC1 used, but is “soluble” in 0.6 M and 0.035 MKCI(Barany et a]., ’61; Pollard and Weihing, ’74). Puszkin et al. (’73) reported that platelet myosin in its polymerized form (0.06 M KC1) bound 3H-CD at high affinity, whereas depolymerized myosin (0.6 M K C ~did ) not bind the drug. The most marked loss of HEp-2 membrane binding activity for 3H-CD was observed after dialysis with 0.6 M KCI, thus resembling the pattern found for platelet myosin. If this phenomenon solely reflected a change in actin filaments as they became “soluble,” the same change should have occurred , such after treatment with 0.035 M K C ~but conditions depressed drug binding only slightly. This observation indicates that the dramatic effect of 0.6 MKCIwas not the result of an influence on actin. Only the treatment which depolymerized myosin (0.6 M KC1) was able to decrease binding activity

CYTOCHALASIN D

235

for 3H-CD. It should be noted that KCl solutions did not inhibit binding of 3H-CB to the high affinity binding sites of red blood cell ghosts (Lin and Spudich, ’74b), suggesting that the pattern observed in our study is not a totally non-specific effect of salt on the association of the hydrophobic cytochalasin molecule with intramembranous binding sites. On the basis of these results and our previous observation (Tannenbaum e t al., ’75b) of the similarity in average intrinsic association constants for high affinity binding of 3H-CDto cells and to muscle myosin (Puszkin et al., ’73), we tentatively conclude that myosin contains the high affinity binding site for 3H-CD in the HEp-2 cell and plasma membrane. This conclusion also accords with the observed loss of binding capacity of the membrane fraction after dialysis against ATP at low ionic strength (table l),because myosin is depolymerized by this treatment (Brahms and Brezner, ’61; Harrington and Himmelfarb, ’72), hence unable to bind 3H-CD4. Identification of the low affinity sites on the plasma membrane is more difficult. The influence of 0.6 M KCl on the plasma membrane clearly must include this subclass as well, because binding activity is decreased by more than 50% (table 1) and high affinity sites could account for no more than about half the total binding observed at the concentration of 3H-CD employed. Thus, myosin may be involved in some low a 6 n ity sites as well. It is unlikely that myosin itself has a low affinity binding site for 3HCD, since Puszkin et al. found only one (high affinity) site per myosin molecule. However, myosin associated with actin (i.e., actomyosin) might form a low affinity binding site for 3H-CD. Much of the membrane-associated myosin is probabl in the form of actomyosin. Puszkin et al. f73) reported that CD inhibited the superprecipitation of actomyosin; this suggests that CD ‘It should be noted that if our band 3 were “actin-binding protein” it could not be a binding site for ‘H-CD. Dialysis in 0.6 M KCI would dissociate “actin-binding protein” from actin (and thus from the membrane); we have shown that this procedure did not release binding sites for ’H-CD from the membrane.

236

J. TANNENRAUM, S. W. TANENBAUM AND G. C. GODMAN

does, in fact, bind to this complex. We randa for assistance with electron microsinterpret the results of the reconstitution copy and to Doctor Arline D. Deitch for experiments of Puszkin et al. (’73) to mean help in obtaining figure 2. We thank Doctor that the binding of actin to myosin lowered Michael Flashner for the myosin standard. the affinity of the myosin binding site for LITERATURE CITED 3H-CD;however, additional data about the nature of the actomyosin binding sites for Aldridge, D., and W. Turner 1969 Structures ofcytochasins C and D. J. Chem. Soc., (C): 923-928. 3H-CD are needed to permit a definitive Atkinson, P., and D. Summers 1971 Purification and conclusion. If the activity of an actomyosin properties of HeLa cell plasma membranes. J. Biol. binding site for 3H-CD were to depend on Chem., 246: 5162-5175. the behavior of its myosin component, we Barany, M., B. Nagy, F. Finkelman and A. Chrambach 1961 Studies on the removal of the bound nucleocould explain the influence of 0.6 M KCl on tide of actin. J. Biol. Chem., 236: 2917-2925. low affinity sites of the HEp-2 membrane. Brahms, J., and J. Brezner 1961 Interaction of myosin Myosin polymers associated with memA with ions. Arch. Biochem. Biophys., 95: 219-228. brane-bound actin should become mono- Brandon, D. L. 1975 Myosin-like polypeptides in plasma membrane preparations. FEBS Lett., 58: meric, but remain attached to actin, in the 349-352. presence of 0.6 M KCI. These monomers of Carter, S. 1972 The cytochalasins as research tools myosin, like “free” myosin monomers, in cytology. Endeavour, 31: 77-82. would be unable to bind 3H-CD, thus re- Clarke, M., G. Schatten, D. Mazia and J. Spudich 1975 Visualization of actin fibers associated with sulting in loss of low affinity binding activthe cell membrane in amoeba of Dictyostelium disity on the HEp-2 plasma membrane. coideum. Proc. Nat. Acad. Sci., 72: 1758-1762. Our results therefore support the hy- Fairbanks, G., T. Steck and D. Wallach 1971 Elecpothesis that CD may trigger a hypertrophoretic analysis of the major polypeptides of contraction of the cell (Miranda et al., the erythrocyte membrane. Biochemistry, 10: 26062617. ’74a,b; Godman et al., ’75) by binding to sites on the subplasmalemmal actomyosin Godman, G. C., A. F. Miranda, A. D. Deitch and S. W. Tanenbaum 1975 Action of cytochalasin D o n cells network attached to the cytoplasmic face of established lines. 111. Zeiosis and movements at of the plasma membrane. The resistance of the cell surface. J. Cell Biol., 64: 644-667. these binding sites for CD to extraction Gruenstein, E., A. Rich and R. Weihing 1975 Actin with non-ionic detergent suggests that they associated with membranes from 3T3 mouse fibroblast and HeLa cells. J. Cell Biol., 64: 223-234. differ from the high affinity sites for CB, which have been reported to be inacti- Gwynn, I., R. Kemp, B. Jones and U. Groschel-Stewart 1974 Ultrastructural evidence for myosin of the vated by similar treatment (Lin and smooth muscle type at the surface of trypsin-disSpudich, ’74b). In the accompanying sociated embryonic chick cells. J. Cell Sci., 15: 279manuscript we present evidence that the 289. high affinity site for CB, which is related to Harrington, W. and S. Himmelfarb 1972 Effect of adenosine di and triphosphates on the stability of the hexose transport system (Lin and synthetic myosin filaments. Biochemistry, 1 1 : 2945Spudich, ’74a,b), does not bind CD, but 2952. that these two drug congeners do share a Hartwig, J. H., and T. P. Stossel 1975 Isolation and common, low affinity binding site. properties of actin, myosin, and a new actin-binding ACKNOWLEDGMENTS

This work was submitted by J. Tannenbaum in partial fulfillment of the requirements for the Ph.D. Degree in the Faculty of Pure Science, Columbia University. The research was supported by Grants AI-11902, CA20836-01 and CA-13835-03 from the National Institutes of Health and VC-76 from the American Cancer Society. We are grateful to Doctor Armand F. Mi-

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BINDING SITES OF CYTOCHALASIN D

red cell membrane in relation to glucose transport.

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