THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 253, No. I, Issue of April 10, pp. 2292-2299, 1978

Pm&d in U.S.A.

Purification of an Active Proteolytic Fragment of the Membrane Attachment Site for Human Erythrocyte Spectrin*

(Received for publication, September 20, 1977)

VANN BENNETT+

From the SDepartment of Molecular Biology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, and The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

Controlled digestion with o+chymotrypsin of inside-out vesicles from spectrin-depleted human erythrocyte mem- branes decreases by up to 90% the number of binding sites for [3’P]spectrin, but does not affect the K,) for [3ZPlspectrin of the sites which remain. Water-soluble polypeptides are released from the membranes during partial proteolysis with cY-chymotrypsin which are potent competitive inhibi- tors of binding of [“lP]spectrin to vesicles, and which asso- ciate with spectrin in solution. The inhibition of spectrin binding by the digest results from an interaction with spectrin and not the membrane since vesicles preincubated with the polypeptides and washed prior to assay exhibit no change in binding. DEAE-cellulose chromatography of the a-chymotryptic digest resolves a 72,000-dalton polypeptide, which represents 72% of the Coomassie blue staining mate- rial on sodium dodecyl sulfate gels, and a partially purified 45,000-dalton fragment. The 72,000-dalton fragment, but not other polypeptides in the digest, competitively inhibits bind- ing of [:“Plspectrin to vesicles with a K, of 10m7 M, which is similar to the K,] for association of [:V2Plspectrin with membranes. Inhibitory activity of the fragment and spec- trin-binding activity of vesicles are both destroyed by either dilute acetic acid or N-ethylmaleimide. The 72,000-dalton fragment co-migrates with inhibitory activity on gel filtra- tion with an effective Stokes radius of 39 .&, and during isoelectric focusing which partially resolves two peaks of protein and closely associated activity with apparent isoe- lectric points of 4 and 4.4. The 72,000-dalton polypeptide forms a complex in solution with spectrin, but not heat- denatured spectrin, with a stoichiometry of 0.7 mol of fragment/m01 of spectrin dimer. The fragment constitutes a minimum of 2.4% of the membrane protein, which is close

* This work was initiated in the laboratory of Dr. Daniel Branton at the Biological Laboratories, Harvard University, and was sup- ported in part by Research Grants PCM 7713962 from the National Science Foundation and HL 17411 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by postdoctoral stipends from the National Institutes of Health and the American Cancer Society while at the Biological Laboratories, Harvard University.

9: Present address.

to the value predicted if spectrin binds in a 1:1 ratio to a 72,000-dalton protein.

It is concluded that the 72,000-dalton proteolytic fragment represents the membrane attachment site for spectrin. This fragment is released in equal amounts from vesicles pre- pared from untreated and cY-chymotrypsin-digested eryth- rocytes, even though such external cleavage degrades Band 3 to a 60,000-dalton fragment. The 72,000-dalton fragment therefore cannot originate from Band 3, and is localized exclusively on the inner surface of the membrane. Glyco- phorin and the other sialoglycoproteins can also be excluded as a source of this polypeptide since the periodic acid-Schiff staining profile of vesicles is unchanged by digestion which abolishes binding of [“‘Plspectrin and releases maximal quantities of the fragment.

Interactions between cytoplasmic proteins and the inner surface of the plasma membrane have received increasing attention as key events in complex celluiar phenomena such as cap formation (11, cell motility (21, and attachment of cells to surfaces (3). Details are presently unavailable of the nature of the membrane proteins involved, or of the mechanisms which regulate such interactions. The human erythrocyte provides an experimentally accessible model system to exam- ine protein-membrane associations on a molecular level. The shape and elasticity of red cell membranes (4-6) and the distribution of intramembrane particles (7) and sialoglycopro- teins (8) are controlled by peripheral proteins localized on the inner surface of the membrane. The cytoplasmic surface of the erythrocyte membrane is exposed selectively in inside-out vesicles which are prepared easily and in high yield (9). Spectrin, the major cytoskeletal protein, has been radiolabeled with %lP in intact erythrocytes, and purified in a nonaggre- gated state which can recombine with the membrane (10, 11).

The reassociation of pure [“YP]spectrin with inside-out vesi- cles depleted of spectrin and actin exhibits the same depend- ence on ionic strength, divalent metal ions, and pH as does association of native spectrin with ghost membranes (11). [32PlSpectrin binds with high affinity (K, = I&” to 1t7 M)

to a single class of sites which are localized on the cytoplasmic surface of the membrane, and are acid-labile and rapidly lost with low levels of trypsin (11). This report describes the

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purification of a 72,000.dalton proteolytic fragment, released during controlled digestion of inverted vesicles with n-chymo- trypsin, which associates with spectrin in solution and ex- hibits properties strongly indicating a role as the membrane attachment site for spectrin.

EXPERIMENTAL PROCEDURES

Materials - 1’“PlOrthonhosnhoric acid, 50 mCi/ml, in 0.02 N HCl . . was from New England Nuclear. a-Chymotrypsin, 54 units/mg, was from Worthington Biochemicals, calf intestinal phosphatase, human transferrin, 5’-ATP, adenosine, N-ethylmaleimide, phenylmethyl- sulfonyl fluoride, dithiothreitol, and benzylpenicillin, sodium salt (1625 units/me) were from Sigma. Creatine kinase was purchased from Boehringer Mannhein, -Ultrogel AcA 44 and AcA 34, and amnholines (pH 4 to 6) were from LKB. Acrylamide, N,N’-methyl- enebisacrylamide, ammonium persulfate, N,N,N’,N’-tetramethyl- enediamine, sodium dodecyl sulfate, and Coomassie brilliant blue were from Bio-Rad. DEAE-cellulose (DE52) was from Whatman.

Methods - Erythrocyte ghosts from freshly drawn blood were arenared as described (11). [‘“PJSpectrin (900 to 3,000 cpm/fi.g) was isolated from erythrocytes labeled with :“P, (1 to 3 Cijmmol) and purified to homogeneity by preparative rate zonal sedimentation on linear sucrose gradients (10, 11). Spectrin-depleted inside-out vesi- cles were prepared by incubating erythrocyte ghosts for 30 min at 37” in 0.3 rnM NaPO,, pH 7.5, as described (11). The resulting vesicles contain 1 to 4% of the original spectrin which may be removed by sedimentation through a 10% (w/v) dextran barrier gradient (11). The sedimentation step has been omitted in this report with no detectable change in the properties of [“‘Plspectrin binding. When vesicles were prepared from large quantities of ghosts (>50 ml), phenylmethylsulfonyl fluoride (20 /*g/ml) was included during the 37” incubation to minimize possible proteolysis by contaminating leukocyte products. Binding df [‘“Plspectrin to in&de-out vesicles was measured as described (11). Briefly, [“‘Plspectrin (20 to 110 pg/ ml) was incubated 90 min at 0” in polystyrene tubes (12 x 75 mm) in a 225 ~1 volume containing 50 to 500 pg/ml of membrane protein, 20 rnM KCl, 2 rnM NaPO,, pH 7.5, 0.5 rnM MgCl,, 0.4 rnM dithiothreitol, 4% (w/v) sucrose. In assays of inhibition of binding of [‘“Plspectrin, spectrin, and the proteins were incubated 30 min at 0” before addition of vesicles. Free and membrane-bound YPJspectrin were separated by layering 0.2 ml of the samples over 0.2 ml of 20% (w/v) sucrose in 0.4 ml of polyethylene microfuge tubes followed by centrifugation (20 min, 20,000 x g). The tubes were frozen and the tips containing membrane pellets, and occasionally the tops with free [“2Plspectrin, were placed in glass vials with 5 ml of Bray’s solution and the radioactivity determined. Control samples with heat-denatured (5 min at 55-60”) spectrin were tested at each concentration of IVPlspectrin and with each preparation of vesicles to provide an estimate of the extent of nonspecific binding (11). Such binding was less than 15% of the values for untreated inverted vesicles and was routinely subtracted. The values were determined in duplicate with a range of k-3 to 6%.

Membrane protein was estimated by the method of Lowry et al. (12) with bovine serum albumin as a standard. while spectrin concentrations were determined by absorbance at 280 nm assuming an A;76 ,),,I of 10.1 (13). SDS’ polyacrylamide electrophoresis was performed essentially by the method of Fairbanks et al. (14).

RESULTS

Attempts to Extract Binding Activity with Dissociating

Agents -The spectrin-binding capacity of vesicles is decreased only 30% following extraction with KC1 at concentrations up to 1 M, even though these membranes are depleted of Bands 6, 2.1, and 4.1 (not shown). Exposure of membranes to high levels of salt may abolish binding at temperatures above 20”, but this effect is most likely due to endogenous protease activity since binding is retained in the presence of phenyl- methylsulfonyl fluoride. Preincubation of vesicles with urea (1 M), sodium cholate (25 mM), or Triton X-100 (0.2% v/v) also has little effect on binding of spectrin (not shown). The binding site for spectrin is apparently a tightly bound periph- eral, or possibly an integral, membrane protein. Guanidine or

’ The abbreviation used is: SDS, sodium dodecyl sulfate

KI at 1 M, acetic acid at 0.1 M, or NaOH at 0.1 M abolish binding activity, but the eluted proteins are inactive in competing for binding of spectrin to membranes (see below).

Organic mercurials also destroy binding, but the extracted proteins are inert and no activity is recovered after reduction with dithiothreitol (not shown). Isolation of the spectrin bind- ing site by such extraction procedures is difficult or impossible since the harsh conditions required to remove the binding capacity of membranes are sufficient to denature proteins.

Digestion oflnside-out Vesicles with a-Chymotrypsin -Pro- teolytic cleavage is a method for detaching membrane proteins under mild conditions. Controlled digestion (30 min at 0”) of spectrin depleted inside-out vesicles by 1 pg/ml of cw-chymo- trypsin decreases by about 90% the capacity of these mem- branes to subsequently bind LZL”Plspectrin (Fig. 1). The K,, for 1:“Plspectrin binding to the digested vesicles is about 40 pg/ ml, and is indistinguishable from the value obtained with control membranes (Fig. 1). The cleavage is selective with release of about 14% of the membrane protein. Digested vesicles exhibit loss of Bands 2.1” and 4.1 on SDS-polyacryl- amide gels, while Band 3 is decreased by approximately 60% (Fig. 2). The periodic acid-Schiff staining profile of digested vesicles is unaltered in relative peak heights or electrophoretic mobility (Fig. 2).

The binding capacity of vesicles for [‘“Plspectrin disappears rapidly during proteolysis, and is accompanied by the appear- ance of water-soluble polypeptides released from the mem- branes (Fig. 3). At least five bands are evident on SDS- polyacrylamide gels after digestion for 15 min (Fig. 3). Two of these polypeptides, one at about 72,000 daltons and another at 45,000 daltons, are relatively resistant to cleavage by cy-chy- motrypsin, and are the major bands remaining after digestion for 60 min (Fig. 3). Proteolysis of vesicles on a preparative scale (see below) usually generates these bands predominantly owing to the increased time required in handling large vol- umes of membranes. It is of interest that the amount of the 72,000-dalton fragment released corresponds roughly to the fall in spectrin binding, while the 45,000.dalton fragment continues to increase after binding is reduced maximally (Fig. 3).

Nearly identical polypeptides are released from vesicles prepared from erythrocytes digested on their external surface with 100 pg/ml of a-chymotrypsin for 2V2 h at 37”, even though this treatment reduces Band 3 to a 60,000-dalton fragment (Fig. 4). Binding of L:“Plspectrin is also unchanged in these externally digested vesicles. Thus the polypeptides solubilized during digestion of inverted vesicles and the bind- ing site for spectrin are almost certainly localized exclusively on the inner surface of the membrane.

Inhibition of LzY2PJSpectrin Binding by Protein,s Solubilized during Proteolysis of Inverted Vesicles -Loss of [:‘“Plspectrin binding following proteolysis of vesicles could be explained in two ways: 1) the attachment site for spectrin is destroyed owing, for example, to a change in conformation or to cleavage through the area of contact with spectrin. 2) The spectrin binding site is released intact into the medium after the binding protein is nicked, or alternatively, an entire protein may dissociate from the membrane if it is itself bound to a polypeptide which is cleaved. To distinguish between these two possibilities, the proteins released during digestion were

examined. A component of the oc-chymotryptic digest inhibits binding of [“‘Plspectrin by 50% at 80 pg/ml, and by 85% at 200

r Nomenclature for human erythrocyte proteins according to Steck (4).

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FREE [32P] SPECTRIN, fig/ml

FIG. 1. Effect of increasing concentrations of [“‘Plspectrin on binding of 13*Plspectrin to control (0) and ar-chymotrypsin-digested (W) inside-out vesicles. Spectrin-depleted inside-out vesicles (1 mgl ml of membrane protein) suspended in 7.5 mM NaPO1, pH 7.5, were incubated for 30 min at 0” in the presence (M) and absence (0) of 0.5 pg/ml of a-chymotrypsin. Phenylmethylsulfonyl fluoride dissolved in dimethylsulfoxide was then added to final concentrations of 125 yglml and 0.25% (v/v), respectively, and the membrane suspensions were centrifuged (15 min, 200,000 x g). The pellets were washed once in 7.5 rnM NaPO,, pH 7.5, 50 pg/ml of phenylmethylsulfonyl fluoride, 0.1% (v/v) dimethylsulfoxide, and resuspended in 20 mM KCl, 2.5 mM NaPO,, pH 7.5. Various concentrations of [“‘Plspectrin (1,020 cpmlyg) was added to the vesicles (111 (0) and 91 (W pg/ml of membrane protein) and binding was determined as described under “Methods.” The K, for spectrin binding can be estimated from a double-reciprocal plot of (lR’P1spectrin bound)-l versus (free f”‘P]spectrin)-’ (inset), and is about 40 pg/ml for control and digested vesicles.

2 4 6 6 IO 2 4 6 8 IO

RELATIVE MOBILITY

FIG. 2. SDS-polyacrylamide electrophoresis of inside-out vesicles before (top panels), and after (bottom panels) limited digestion with a-chymotrypsin. Spectrin-depleted inside-out vesicles were digested with 0.5 pg/ml of c*-chymotrypsin as described in Fig. 1. Ten- microliter aliquots .(30 pg/protein) of control (top left) and digested (bottom left) vesicles were analyzed by electrophoresis on 3-mm-thick 6% polyacrylamide slab gels, and 50-~1 aliquots (80 pg of protein) on 5% polyacrylamide tube gels (right panels). The gels were stained with either Coomassie blue (left panels) or by the periodic acid-Schiff reaction (right panels), and scanned in a densitometer.

pg/ml (Fig. 5A). The extent of inhibition is diminished by increasing concentrations of [“‘Plspectrin, from a nearly 90% fall in binding at 10 pg/ml of spectrin to about 10% at 100 ygl ml (Fig. 5B). Thus the solubilized proteins behave as a competitive antagonist of the binding of spectrin to its mem- brane site.

Several types of experiments indicate the inhibition is specific and not explainable by residual protease activity. The polypeptides in the digest migrate on gel filtration on Sepha- dex G-100 with a Stokes radius corresponding to a molecular weight of about 80,000, and are chromatographed routinely on Sephadex G-75 prior to binding assays. No inhibitory activity is exhibited when the digests are prepared from membranes preincubated with 0.1 M acetic acid, a treatment which abol-

PRE,NC”SAT,ON TIME, MINUTES RELATIVE MORll IT” FIG. 3. Effect of increasing time of digestion of inside-out vesicles

with a-chymotrypsin on binding of [:r2Plspectrin (O), and loss of membrane protein (0) (left panel), and on release of soluble polypep- tides (right panel). Spectrin-depleted inside-out vesicles (1 mg/ml of membrane protein) (“Methods”) were suspended in 7.5 mM NaPO,, pH 7.5, and incubated at 0” for various times with 0.25 pglml of QL- chymotrypsin. The suspensions were treated as described in Fig. 1, and binding of [?‘P]spectrin (1,200 cpm/pg) to the membranes (220 pg/ml) was determined (“Methods”) (left panel). One hundred-mi- croliter aliquots (10 to 18 pg of protein) of the soluble polypeptides released during digestion were electrophoresed on 5% polyacryl- amide tube gels in the presence of 0.2% (w/v) SDS and the gels were stained with Coomassie blue and scanned in a densitometer (right panel). The arrows indicate the position of Bands 3, 4.2, and 6 in scans of gels containing erythrocyte vesicles.

ishes spectrin binding (11) (Fig. 5A). Membranes preincu- bated with the released polypeptides show no change in electrophoretic pattern or in subsequent binding of [32P]spectrin. Other membrane proteins examined show no inhibitory activity, including Bands 2.1, 4.1, and 6 (from KC1 extracts), and Band 5 (eluted from membranes at low ionic strength). The inhibition of binding is not due to dephospho- rylation of [*“P]spectrin since 32P remains bound to spectrin following incubation with the digest and gel filtration on Sephadex G-150.

Binding of [:v2P]spectrin to a Component of the a-Chymo- tryptic Digest -The competitive inhibition of [:rYP]spectrin binding could be explained if polypeptides block spectrin binding sites on the vesicles, or if the polypeptides bind to spectrin in solution and modify the association of spectrin with the membrane. It is unlikely that components in the digest are occupying binding sites on the vesicles since membranes preincubated with the digest and washed once prior to assay exhibit no change in binding of [:r2P]spectrin. Furthermore, [“*P]spectrin binds to agarose beads to which the solubilized

proteins have been covalently coupled (Table I). This provides direct evidence that a component of the digest associates with spectrin in solution. A major portion of the binding to the gel is specific, since adsorption is decreased by about 65% after the beads are reacted with N-ethylmaleimide (Table I). Fur- thermore, binding is saturable since preloading the gel with spectrin also diminishes binding by about 65% (Table I).

These data suggest that the membrane attachment site for spectrin is released intact by digestion. Inhibition of [32P]spectrin binding is not observed with other red cell proteins, and increases, within certain limits, as a linear function of the polypeptides added (Fig. 5A). Binding inhibi- tion thus provides a reliable and quantitative assay to monitor purification of the spectrin-binding polypeptide.

DEAE-cellulose Chromatography of Polypeptides Released during Digestion of Vesicles-Nearly all of the protein re- leased during digestion of inverted vesicles by a-chymotrypsin

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TABLE I

2.1

3 4.1 4.2’

A BC DEF FIG. 4. SDS-polyacrylamide electrophoresis of inside-out vesicles

prepared from untreated cells (A) or from cells digested externally with ol-chymotrypsin (D), cu-chymotrypsin-digested inside-out vesi- cles from untreated cells (B) or from externally digested cells (E), and polypeptides released by digestion of inside-out vesicles from untreated cells (C) and from externally digested cells (F). Four milliliters of erythrocytes were incubated for 150 min at 37” in 10 ml of Krebs-Ringer bicarbonate, pH 7.4, in the presence and absence of 100 pglml of oi-chymotrypsin. The cells were then washed three times and incubated 14 h at 24” in 40 ml of Krebs-Ringer bicarbonate, 10 rnM glucose, 200 units/ml of penicillin G, 100 pglml of phenyl- methylsulfonyl fluoride, 0.05% (v/v) dimethylsulfoxide. The cells were washed three times, and spectrin-depleted inside-out vesicles were prepared (“Methods”). The membranes from control (A) and digested (0) cells (3.5 mg/ml of membrane protein) were digested with 1 pg/ml of cu-chymotrypsin as described in Fig. 1, and centri- fuged 30 min at 48,000 x g. The supernatants (C and F) were collected, and the pellets (i?, E) washed once and resuspended in the original volume. Fifteen-microliter portions of the membrane sam- ples (A, B, D, E) (50 to 55 Kg of protein) and 25-/Al aliquots of the supernatants (C, F) (10 to 15 pg of protein) were electrophoresed on 3-mm-thick 6% polyacrylamide slab gels in the presence of 0.2% SDS, and the gels were stained with Coomassie blue.

Specific binding of [YYLPlspectrin to a-chymotryptic digest immobilized on agarose beads

Column pretreatment kyJPISpectrin bound”

None” 24.4 N-Ethylmaleimide’ 8.3 Spectrin” 8.4

(1 Expressed as micrograms [“lplspectrin bound per ml of packed agarose gel. [:“PISpectrin (27 pg, 2,388 cpmlyg) was added to columns prepared as described above, and the unbound protein eluted with 3 column volumes of buffer.

b Polypeptides solubilized by cr-chymotryptic digestion of vesicles (Fig. 5) were coupled to Sepharose (Cl) 4B by the benzoquinone procedure (15) to a concentration of about 0.2 mg of protein/ml of packed gel. The beads were washed extensively with glycine (0.1 M,

pH 7.8), packed into columns containing 0.6 ml of gel, and equili- brated at 0” with 20 mM KCl, 5 mM sodium phosphate buffer, pH 7.5, 0.5 rnM MgCl,, 0.5 rnM dithiothreitol.

c N-Ethylmaleimide (10 mM) in 10 mM sodium phosphate buffer, pH 7.5, was added to columns prepared as described above and allowed to react for 1 h at 24”. The gel was then washed with 5 column volumes of equilibration buffer.

d Eighty micrograms of spectrin was added to the prepared columns followed by 5 column volumes of buffer.

TABLE II

Summary of isolation of 72,000-dalton fragment Per cent

m&T

Vesicles 520 (100) (2.4Y Solubilized polypeptides” 74 (14.2) 1.7e 17 Protein adsorbed to DEAE- 63 (12.1)

cellulose” DEAE-Peak 1” 14 (2.7) 4.2 64 Rechromatographed Peak 1” 6 (1.1) 6.2 72 DEAE-Peak 2” 25 (4.8) 40.1 <I

” Expressed as per cent inhibition per pg per ml of protein of binding of [“‘Plspectrin (23 pg/ml) to vesicles.

h Calculated from densitometer scans of SDS-polyacrylamide gels stained with Coomassie blue (Fig. 7).

c Minimum estimate calculated on the basis that the fragment represented 17% of the solubilized polypeptides which were 14.2% of the total membrane protein.

FIG. 5. Inhibition of [:‘“Plspectrin binding by components solubi- lized during digestion of spectrin-depleted inside-out vesicles with ol-chymotrypsin. A, effect on binding of increasing concentrations of protein released from control (0) and acid-denatured (m) vesicles. B, effect of increasing concentrations of [:“Plspectrin on binding in the presence of 0 (O), 59 (A) and 110 (W) pglml of protein released from control vesicles. Spectrin-depleted inside-out vesicles (“Meth- ads”) were extracted (30 min, 0”) with 6 volumes of 0.4 M KC1 to deplete the membranes of Bands 2.1 and 6, and divided into two portions. One (0) was untreated, and the other (M) was washed once in 0.1 M acetic acid. The membranes were digested with ol-chymo- trypsin (see Fig. l), and the solubilized proteins were lyophilized, resuspended at a concentration of 4 mg/ml of protein, and 1 ml of chromatographed on Sephadex G-75 columns (0.5 x 20 cm) equili- brated with 20 rnM KCl, 2.5 mM NaPO,, pH 7.5. The protein

d Prepared as described in Fig. 6. e The solubilized protein was chromatographed on Sephadex G-

75, to remove protease (see Fig. 5). Approximately 80% of the protein was recovered in the excluded volume, and the SDS-polyacrylamide gel pattern was identical with that of the starting material.

is adsorbed to a slurry of DEAE-cellulose (Table II). The batchwise adsorption of protein permits rapid concentration of material in large volumes and a concomitant removal of cy- chymotrypsin, a basic protein which is not bound to DEAE- cellulose at pH 7.5. Two peaks of protein are resolved by eluting the DEAE-cellulose with a discontinuous gradient of KC1 (Fig. 6A 1. Inhibition of [:‘LP]spectrin binding is obtained

appearing in the void volume was pooled, and various concentrations were added at 0” to tubes containing [“‘Plspectrin (29 pg/ml, 800 cpmlpg); after 45 min, vesicles (390 pg/ml of protein) were added and binding was determined (“Methods”) (A). In another assay (B), binding to vesicles (250 pg/ml of protein) was measured with various concentrations of [:‘“Plspectrin with either no additions (O), 59 ygl ml (A), or 110 pg/ml (W) of protein released from cu-chymotrypsin- digested vesicles.

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FRACTION NUMBER FRACTION NUMBER

7

L-J

FIG. 6 (left). DEAE-cellulose chromatography of soluble polypep- tides released after digestion of inside-out vesicles with cu-chymo- trypsin. A, elution of protein (0) and inhibition of [32Plspectrin binding (0) with a discontinuous gradient of KCl. B, rechromatog- raphy of Peak 1 (A) with a continuous gradient of KCl. Spectrin- depleted inside-out vesicles were prepared from 200 ml of erythrocyte ghosts (“Methods”) and suspended in a 220-ml volume containing 7.5 mM NaPO,, pH 7.5, 2.6 mg/ml of membrane protein, and 1 Kg/ml of a-chymotrypsin. After 30 min at 0”, phenylmethylsulfonyl fluoride dissolved in dimethylsulfoxide was added at final concentrations of 50 wg/ml and 0.5% (v/v), respectively, and the membrane suspension was centrifuged (45 min, 100,000 x g) in a SW 27 rotor. The supernatant (370 &ml of protein) was combined with 20 ml of packed DE52-beaded cellulose equilibrated with 7.5 mM NaPO,, pH 7.5, and mixed gently for 30 min at 0”. The DE52 cellulose was packed into a column (2 x 10 cm), and the supernatant (57 @g/ml of protein) was discarded. The column was washed with 250 ml of 7.5 mM NaPO,, pH 7.5, and then eluted with 0.2 M and 0.4 M KC1 dissolved in 7.5 mM NaPO,, pH 7.5, as indicated in A. The fractions (3 ml) were monitored for protein (A,,,) (0) and for inhibition of binding of F*Plspectrin (23 @g/ml, 3000 cpm/pg) to inside-out vesi-

almost entirely in Peak 1 (Fig. 6A), and the specific activity of this fraction is increased about 2.5fold over that of the starting material (Table II). l&chromatography of Peak 1 on DEAE-cellulose with a linear gradient of KC1 yields one major peak of protein and coinciding inhibitory activity (Fig. 6B). The specific activity of this material is about 3.6-fold greater than that of the digest (Table II).

Analysis of the fractions by SDS-acrylamide electrophoresis (Fig. 7) indicates that Peak 1 consists of a 72,000-dalton polypeptide with a small peak of about 68,000 daltons which is most likely a degradation product of the 72,000-dalton frag- ment. Rechromatographed Peak 1 exhibits a similar profile except that polypeptides in the low molecular weight range are diminished (not shown). Seventy-two per cent of the Coomassie blue-staining material in this preparation is pres- ent as the 72,000-dalton fragment, which represents about a 4- fold enrichment over the amount of this polypeptide present in the whole digest (Table II). Peak 2 (Fig. 6A ) contains primarily a 45,000-dalton fragment, which has been observed previously in proteolytic digests of erythrocyte ghosts, and was attributed to the portion of Band 3 accessible from the cytoplasmic surface of the membrane (16).

The isolated 72,000-dalton fragment inhibits binding of [32P1spectrin (15 pglml) to vesicles (72 pg/ml) by 50% at 12 pg/

300

25 % I 6 200 --

3 100 7 ’

2 4 6 8 IO

RELATIVE MOBILITY

cles (191 pg/ml of membrane protein) (“Methods”) (0). The peak of inhibitory activity (1, A) was dialyzed against 7.5 mM NaPO,, pH 7.5, with applied to a 1 cm x 5 cm DE52 cellulose column which was eluted with a linear gradient of KC1 (0 to 0.3 M) dissolved in 7.5 mM NaPO,, pH 7.5, with 3-ml fractions (B). Protein (0) and inhibition activity (0) were determined as before, and the peak fractions (19 to 26) were pooled. This preparation is referred to in the remainder of this report as the 72,000-dalton fragment (see Fig. 7).

FIG. 7 (right). SDS-polyacrylamide electrophoresis of inside-out vesicles (A), soluble polypeptides released after digestion of inside- out vesicles with a-chymotrypsin (B), and polypeptides in Peak 1 (C) and Peak 2 (0) from DEAE-cellulose chromatography of the material in B. Spectrin-depleted inside-out vesicles were treated with cr- chymotrypsin, and the polypeptides released during the digestion were separated by DEAE-cellulose chromatography (see Fig. 6). Ten microliters of vesicles (21 kg of membrane protein) (A), 25 pl of the supernatant after centrifugation of the digested vesicles (7.4 pg) (B), 25 ~1 of Peak 1 (8.4 gg) (C), and 10 ~1 of Peak 2 (13.8 Kg) (0) were electrophoresed on 3-mm-thick 6% polyacrylamide slab gels. The gels were stained with Coomassie blue and scanned with a densitom- eter.

ml and almost completely abolishes binding at 70 pg/ml (Fig. 8). In contrast, comparable concentrations of fractions contain- ing the 45,000-dalton polypeptide do not inhibit and actually slightly stimulate binding (Fig. 8).

Properties of 72,000-dalton Fragment -Spectrin and the 72,000-dalton fragment associate to form a complex which can be separated from unbound fragment by gel filtration on Ultrogel AcA 34 (Fig. 9). The binding represents a selective interaction since no fragment is bound to heat-denatured spectrin (Fig. 9). The relative peak areas of Coomassie blue- stained gels of the complex are 1 spectrin:0.12 fragment (Fig. 9) which corresponds to 0.7 mol of fragment bound/m01 of cup- spectrin dimer (Bands 1 and 2),3 assuming molecular weights of 72,000 and 460,000, respectively, and that Coomassie blue stains these polypeptides with equal intensity. The latter assumption is supported by studies of binding of Coomassie Blue G to proteins (18) which indicate that most polypeptides adsorb nearly equivalent amounts of dye on a weight basis.

Dixon plots of inhibition of [mP]spectrin binding by the fragment (Fig. 10) confirm that the inhibition is competitive

3 The evidence that spectrin is composed of an c@ dimeric unit of Bands 1 + 2 is provided primarily by cross-linking studies (13, 17), although higher polymers of (1 + 2)N or (1 + 2), Q (1 + 2),~+, transitions also are consistent with this data.

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;w L 3 5

PROTEIN, #g/ml RELATIVE MOBILITY

FIG. 8 (left). Effect of increasing concentrations of the 72,000- dalton fragment (0) and the partially purified 45,000-dalton frag- ment (m) on binding of L3*Plspectrin to inverted vesicles. Binding of [32Plspectrin (15 wg/ml, 3,000 cpmlpg) to inside-out vesicles (72 +g/ ml of membrane protein) was measured (“Methods”) in the presence of various concentrations of either the 72,000-dalton fragment (0) or the partially purified 45,000-dalton fragment (D), which were ob- tained and analyzed as described in Figs. 6 and 7.

I I I I It I I I I I I I I I

8 0 16 24 32

FRAGMENT, yg/ml

FIG. 9 (rig&). SDS-polyacrylamide electrophoresis of the ex- cluded volume of an ultrogel AcA 34 column loaded with spectrin alone (A) the 72,000-dalton fragment alone (B), the 72,000 dalton fragment and either untreated spectrin (C), or heat-denatured spectrin (0). Spectrin and the 72,000 dalton fragment were incubated for 30 min at 0” in sucrose (lo%, w/v), 0.2 mM MgCl,, 20 pM ATP, 0.4 mM dithiothreitol 20 mM KCl, 2.5 mM NaPO,, pH 7.5 with either spectrin alone (157 pg/ml) (A), fragment alone (210 pg/ml) (B), spectrin and fragment (157 and 210 kg/ml, respectively) (C), or heat- denatured (5 min, 60”) spectrin and fragment (157 and 210 pglml, respectively) (0). Samples (0.4 ml) of these solutions were chromat- ographed on a Ultrogel AcA 34 column (0.9 x 30 cm) equilibrated with 50 mM KCl, 0.25 mM MgCl,, 0.5 mM dithiothreitol, 5 rnM NaPO,, pH 7.5, flow rate 10 ml/h, and 0.5-ml fractions collected. The excluded volumes were pooled, dialyzed against water, and lyophi- lized. The samples were electrophoresed on a 6% polyacrylamide slab gel which was stained with Coomassie blue and scanned in a densitometer. The relative peak areas in C are spectrin 1, fragment 0.12 which corresponds to a ratio of 0.7 mol of fragment/m01 of spectrin assuming molecular weights of 72,000 and 460,000, respec- tively.

FIG. 10. Dixon plot of inhibition of binding of L3*Plspectrin to inverted vesicles in the presence of increasing concentrations of the 72,000-dalton fragment with 16 (O), 31 (A), and 47 (W) pg/ml of [32P]spectrin. Binding of [32Plspectrin (2,050 cpm/pg) at concentra- tions of 16 (O), 31 (A), and 47 (m) pg/ml to inside-out vesicles (55 pg/ml) was determined (“Methods”) in the presence of various concentrations of the 72,000-dalton fragment isolated as described in Fig. 6.

FRAGMENT, fig/ml MEMBRANE PROTEIN, fig

FIG. 11. Effect of pretreatment of the 72,000-dalton fragment (left

with respect to spectrin. The K, is about 7 pg/ml, or 1O-7 M,

and is quite similar to the K,, of 40 pg/ml (9 x 1Oms M) for association of [32P]spectrin with inverted vesicles (Fig. 1). Thus, the binding of the fragment to spectrin occurs with a similar affinity as for binding of spectrin to the membrane.

The inhibitory activity of the fragment is abolished by prior treatment with dilute acetic acid or N-ethylmaleimide (Fig. 11). The binding capacity of inside-out vesicles for [3’P]spectrin is also destroyed by these agents under identical conditions (Fig. 11). The loss of binding is not explainable by dissociation from the membrane of a soluble, active component since the supernatants of vesicle suspensions incubated with acetic acid or N-ethylmaleimide are without effect on binding of spectrin (not shown).

panel) and inside-out vesicles (right panel) with no additions (O), dilute acetic acid (HAc) (A) or N-ethylmaleimide (NEM) (m) on inhibition of binding of [3*Plspectrin to inverted vesicles (left panel), and on binding of P2Plspectrin to inverted vesicles (right panel). Aliquots of the 72,000-dalton fragment and of inside-out vesicles were incubated for 30 min at 24” in 7.5 mM NaPO,, pH 7.5, with no additions (O), 0.17 M acetic acid (A), or with 2.5 mM N-ethylmaleim- ide (m). The solutions containing the fragment were dialyzed against 7.5 mM NaPO,, pH 7.5, and the vesicles were washed three times in this buffer. Binding of [32Plspectrin (19 pg/ml, 1,020 cpm/pg) to inside-out vesicles (110 Kg/ml of membrane protein) was determined (“Methods”) in the presence of various concentrations of 72,000- dalton fragment which was untreated (0) exposed to acetic acid (A) or to N-ethylmaleimide (D) (left panel). Binding of [32Plspectrin (16 pg/ml, 2,000 cpm/pg) was also measured with various concentra- tions of inside-out vesicles which were untreated (O), exposed to acetic acid (A), or to N-ethylmaleimide (D) (right panel).

The fragment migrates on gel filtration as a single major peak with an effective Stokes radius of 39 A (Fig. 12). A small

Isoelectric focusing of the fragment in a sucrose gradient (Fig. 13) partially resolves two populations of protein with

peak of protein appears in the excluded volume, but is not apparent isoelectric points of 4 and 4.4. These values are only associated with inhibitory activity (Fig. 12), and may repre- approximate since this preparation precipitates at acid pH. sent aggregated or denatured material. Binding inhibition activity correlates closely with the distri-

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2298 Membrane Attachment Site for Spectrin

FRACTION NUMBER

FIG. 12. Ultrogel AcA 44 chromatography of the 72,000-dalton fragment. One-half milliliter (AA,,, 1.3) of the 72.000-dalton fragment isolated as described in Fig. 6 wasapplied to a column (1 x 60cm) of Ultrogel AcA 44 equilibrated with 50 mM NaPO,, 0.5 mM NaN,, pH 7.5, and 0.7-ml fractions were collected. Protein (A*“,,) (0) and inhibition of binding of [“‘Plspectrin (22 fig/ml, 2,400 cpm/& to inside-out vesicles (i20 pg/mi) were mon&red. The column-was calibrated (inset) with calf intestinal phosphatase (46.1 A; .a,,,, 6.4, it4 = 138,000, V 0.725 (19)) rabbit muscle creatine kinase (36.2 A: s,, lL’ 5.3, M, = 82,600, V 0.735 (20)), and human hemoglobin (31.6 A; sin’,,, 4.5, M, = 64,500, V 0.749 (21)). where the Stokes radius was calculated assuming a maximum contribution of hydration. The arrow (inset) indicates the effective Stokes radius of the fragment.

! I / I / I

0.3

I g 0.2

Ls 4”

0. I

FRACTION NUMBER

FIG. 13. Isoelectric focusing of the 72.000-dalton fragment. Six milliliters (ApHO 0.6) of 72,000-Dalton fragment isolated as described in Fig. 5 was mixed in a llO-ml linear gradient (0 to 50%) of sucrose containing 1% (w/v) ampholines (pH range 4 to 6) in a LKB isoelectric focusing column. Focusing was begun with 8 watts and terminated after 18 h at 4”. Fractions (4.5 ml) were collected and the pH adjusted to 7.5. Protein (A& (0) and inhibition of binding of 13”Plspectrin (20 @g/ml, 2,500 cpm/pg) to inside-out vesicles (130 pgl ml of membrane protein) (“Methods”) (0) were monitored.

bution of protein (Fig. 13). Isoelectric focusing of the fragment in polyacrylamide gels (not shown) reveals considerable mi- croheterogeneity with at least five bands of protein within a range of about 0.7 pH unit.

DISCUSSION

Several lines of evidence presented in this report strongly support the view that the 72,000-dalton proteolytic fragment purified from c-u-chymotryptic digests of inside-out’ vesicles is the membrane attachment site for spectrin. This polypeptide is released from the cytoplasmic surface of membranes (Fig. 4) under conditions which remove about 90% of the binding capacity for [R2Plspectrin, but which have no effect on the K, of the sites which remain (Fig. 1). The fragment, but not other polypeptides in the digest (Figs. 6 and 8) or other erythrocyte

proteins, competitively inhibits binding of spectrin to inside- out vesicles with a K, of about 10. 7 M (Fig. 10) which is similar to the K,, for association of [:“PJspectrin with membranes (Fig. 1). Inhibitory activity of the fragment and binding activity of vesicles exhibit a similar sensitivity to acetic acid and N- ethylmaleimide (Fig. 11). Enrichment of the fragment (4.1- fold) corresponds closely to the purification of inhibitory activ- ity (3.6-fold) (Table II). Inhibitory activity co-migrates with the 72,000-dalton fragment on DEAE-cellulose chromatogra- phy (Fig. 6), gel filtration (Fig. 12), and isoelectric focusing (Fig. 13). The fragment forms a complex in solution with spectrin, but not heat-denatured spectrin, with a stoichiome- try of 0.7 mol of fragment/m01 of spectrin calculated on the basis of Coomassie blue staining intensity (Fig. 9). Finally, the fragment constitutes a minimum of 2.4% of the membrane protein (Table II, Fig. 7), which is in fair agreement with the value of 3.9% predicted if a a/3-spectrin dimer,” M, 460,000, which is present as about 20% of membrane protein, binds to a 72,000-dalton protein in a 1:l ratio.

It thus appears that spectrin binds in a stoichiometry of 1:l to a membrane protein with a molecular weight of at least 72,000. It is likely that only a portion of this protein or of the spectrin molecule is involved in this complex, and additional functions of these proteins remain to be elucidated. This is the

first report of isolation of a membrane attachment site for a major cytoskeletal protein. The same approach could, in principle, be applied to other proteins such as cY-actinin and tubulin provided a membrane preparation could be obtained in which the cytoplasmic surface was accessible.

The membrane protein which is the source of the fragment is not known, although all of the polypeptides with molecular weights less than 72,000 daltons on SDS-polyacrylamide gels can be excluded. The 72,000-dalton polypeptide is released in equal amounts from vesicles prepared from untreated and (Y- chymotrypsin-digested erythrocytes, even though external cleavage degrades Band 3 almost completely to a 60,000-dalton band (Fig. 4). Thus the major polypeptide in the Band 3 region is almost certainly not the origin of the fragment, and conse- quently is also not the attachment site for spectrin. This conclusion is supported further by the fact that the 45,000- dalton polypeptide which also is released by cu-chymotrypsin and is thought to be a portion of Band 3 (161, causes no inhibition of spectrin binding (Figs. 6 and 8).

Glycophorin and other sialoglycoproteins can also be ruled out as a source of the fragment since the periodic acid-Schiff staining profile is unaltered by limited digestion of vesicles with ar-chymotrypsin (Fig. 2), and, moreover, the molecular weight of the fragment is much larger than that of any of these proteins. Band 4.2 is unlikely since this polypeptide remains in the digested membranes (Fig. 2). Bands 2.1 and 4.1 are also unlikely candidates since these proteins can be extracted from vesicles with high concentrations of KC1 which do not affect binding of spectrin, and the solubilized proteins do not inhibit binding of spectrin (data not shown). The possibility must be seriously considered that the attachment protein stains poorly with Coomassie blue, perhaps owing to extensive glycosylation of a portion of the molecule. It is pertinent in this regard that numerous glycoproteins are apparent after treatment of erythrocyte membranes with galactose oxidase and NaB[“Hl,, which are not observed by conventional methods (22). The parent polypeptide of the spectrin-binding fragment can be identified unequivocally once antisera against the fragment are available.

The lack of direct involvement of Band 3 and glycophorin in

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Membrane Attachment Site for Spectrin 2299

attachment of spectrin to the membrane is in accord with Motility (Cold Spring Harbor Conferences on Cell Prolifera-

previous studies (23) in which the detergent-solubilized pro- tion) (Goldman, R., Pollard, T., and Rosenbaum, J., eds) Vol.

teins did not associate with spectrin, although binding was 3, pp. 651-664, Cold Spring Harbor Laboratory, Cold Spring

observed under identical conditions between Band 3 and Harbor, N. Y.

7. Elgsaeter, A., and Branton, D. (1974) J. Cell Bcol. 63, 1018-1036 Bands 6 and 4.2. Spectrin, possibly together with erythrocyte 8. Nicolson, G. L., and Painter, R. G. (1973) J. Cell Bml. 59, 395-

actin, has been shown to control the topography of sialoglyco- 406

proteins (B), and intramembrane particles (7), structures 9. Steck, T. L., and Kant, J. A. (1974) Methods EnzymoZ. 31, 172-

lull which are thought to contain sialoglycoproteins (24-26) as well as Band 3 (27, 28). A direct association has been proposed between spectrin and portions of the particles expressed on the inner surface of the membrane (27-29). It is possible that the spectrin attachment site is itself associated with Band 3 or glycophorin, or that this protein is a heretofore unrecognized component of the particles. It may be possible by morphologi- cal methods to define the relationship between the attachment site and particles with ferritin-labeled antibodies directed against the fragment.

Erythrocyte membranes exhibit properties indicating the presence of mechanisms for long range interactions and regu- lation. Lateral movement of proteins in these membranes is slow compared to the rates of diffusion determined for the other integral membrane proteins (30, 311, and depends on the metabolic state of the cell (31). Furthermore, various hor- mones and cyclic nucleotides induce generalized alterations in the physical properties of erythrocyte membrane (32-34). The role of spectrin in these phenomena is difficult to evaluate experimentally since removal of spectrin from the membrane

by extraction at low ionic strength reduces ghosts to small vesicles. The fragment may be useful in this regard, since it should compete with the membrane for the binding site of spectrin and induce a controlled dissociation of spectrin from the membrane.

IY”

10. Bennett, V. (1977) ,Qfe Sci. 21, 433-440 11. Bennett, V., and Branton, D. (1977) J. BioZ. Chem. 252, 2753-

2763 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

(1951) J. Bzol. Chem. 193, 265-275 13. Clarke, M. (1971) Biochem. Biophys. Res. Commun. 45, 1063-

1070 14. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971)

Biochemistry 10. 2606-2617 15. Brandt, J., And&son, L.-O., and Porath, J. (1975) Biochim.

Bzophys. Acta 386, 196-202 16. Steck, T. L., Ramos, B., and Strapazon, E. (1976) Biochemistry

15, 1154-1161 17. Wang, K., and Richards, F. M. (1974) J. BioZ. Chem. 249, 8005-

8018 18. Bradford, M. (1976) Anal. Blochem. 72, 248-254 19. Fosset, M., Chappelet-Tordo, D., and Lazdunski, M. (1974)

Biochemzstry 13, 1783-1788 20. Sober. H. A.. ed. (1968) Handbook for Biochemistrv. D. G38. The

Chemical kubber Co. Press, Cleheland, Ohio I 21. Altman, P. L., and Dittmer, D. S., eds. (1972) Biology Data Book

2nd Ed, Vol. 1, pp. 370-385, Federation of American Societies for Experimental Biology, Bethesda, Md.

22. Gahmberg, C. G. (1976) J. BioZ. Chem. 251, 510-515 23. Yu, J., and Steck, T. L. (1975) J. BioZ. Chem. 250, 9176-9184 24. Tillack. T. W.. Scott. R. E.. and Marchesi. V. T. (1972) J. Em.

Med.‘135, liO9-12i7 25. Marchesi, V. T., Tillack, T. W., Jackson, R. L., Segrest, J. P.,

and Scott, R. E. (1972) Proc. N&Z. Acad. Sci. l?. S. A. 69. 1445-1449

26. Grant. C. W. M.. and McConnell. H. M. (1974)Proc. Natl. Acad.

Acknowledgment -Valuable discussions with Daniel Bran- ton are gratefully acknowledged.

Sci.‘lJ. S. A. il, 4653-4657. ’ 27. Pinto da Silva, P., and Nicolson, G. L. (1974) Biochim. Biophys.

Acta 363, 311-319

REFERENCES 28. Yu, J., and Branton, D. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3891-3895

1. Edelman, G. M. (1976) Science 192, 218-226 29. Elgsaeter, A., Shotton, D. M., and Branton, D. (1976) Biochim. 2. Kern, E. D. (1976) in Cell Motility (Cold Spring Harbor Confer- Biophys. Acta 126, 101-122

ences on Cell Proliferation), (Goldman, R., Pollard, T., and 30. Peters, R., Peters, J., Tews, K. H., and BBhr, W. (1974) Blochim. Rosenbaum, J., eds) Vol 3, pp. 623-630, Cold Spring Harbor Bio~hys. Acta 367. 282-294 Laboratory, Cold Spring Harbor, N. Y. 31. Fowl&,“V., and Branton, D. (1977) Nature 268, 23-26

3. Rees, D. A., Lloyd, C. W., and Thorn, D. (1977) Nature 267, 124- 32. Kury, P. G., and McConnell, H. M. (1975)Biochemistry 13, 2798- 128 2803

4. Steck, T. L. (1974) J. CeZZ BioZ. 62, l-19 33. Rasmussen, H., Lake, W., Gasie, G., and Allen, J. (1975) in 5. LaCelle, P. L., and Kirkpatrick, F. H. (1975) in Erythrocyte Erythrocyte Structure and Fun&on (Brewer, G. J., ed) pp.

Structure and Function (Brewer, G. J., ed) pp. 535-557, Alan 467-490, Alan R. Liss, Inc., New York R. Liss, Inc., New York 34. Sonenberg, M. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 1051-

6. Sheetz, M. P., Painter, R. G., and Singer, S. J. (19703 in Cell 1055

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V Bennettfor human erythrocyte spectrin.

Purification of an active proteolytic fragment of the membrane attachment site

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