ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 296, No. 1, July, pp. 152-160,1992

Subtilisin Cleavage of Tubulin Heterodimers and Polymers’ Sharon

Lobert*

and John J. Correiaip2

*School of Nursing and TDepartment

Received November

l&1991,

of Biochemistry,

and in revised form February

University

Medical Center, Jackson, Mississippi

accessibility mer form.

0 1992 Academic Press, Inc.

39216

6,1992

Native pig brain tubulin in beterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last SO amino acids of the highly variable carboxyl-termini of the CYand /3 subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 M glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, a’ and #V, under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native ar,/3 tubulin to 7-9 for subtilisin-cleaved a’,/3 tubulin. When tubulin heterodimers were cleaved, a single major /3’ isotype was evident; however, when tubulin polymerized in 4 M glycerol was cleaved, two major 8’ isotypes were found. Monoclonal antibodies that recognize a B carboxyl-terminal peptide, residues 410-430, reacted with both major 0’ isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major B’ isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the @ subunit, near residues 430-435, with different

1 This work supported by Research Grant GM 41117(J.J.C). ’ To whom correspondence should be addressed: Department of Biochemistry, University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216. Fax: (601) 984-1501. 152

of Mississippi

to the enzyme

in the heterodimer

and poly-

Structural studies of tubulin by X-ray diffraction have been hampered by the extreme difficulty of obtaining diffraction quality crystals (1). This is primarily due to its high degree of heterogeneity arising from genetic variability as well as post-translational modifications. Similarly, the resolution of fiber diffraction data is limited by the quality of the specimens, although this method has yielded an 18-A structure (2, 3) which is consistent with earlier electron microscopic studies (4, 5). Tubulin is a heterodimer, composed of two polypeptide chains, a! and p, each having about 450 residues and a molecular weight of about 50,000 Da. Much of the variability in the primary structure of the tubulin heterodimers lies in the glutamicacid-rich carboxyl-terminal regions, including approximately the last 50 amino acids of both subunits (6, 7). Immunoassays suggested that a(430-443) and @(412-431) are well exposed when tubulin polymerizes into microtubules (8). Circular dichroism spectra indicated that removal of this region causes a significant change in the (Yhelical content of the heterodimer (9, 10) and nuclear magnetic resonance studies suggested that the carboxyltermini are highly flexible in the monomer and polymer (11). Crosslinking studies demonstrated that the carboxyltermini are not involved in a direct interaction between subunits; however, it has been demonstrated that the amino-terminus of the (Y subunit and the carboxyl-terminus of the p subunit interact within the heterodimer, and that the amino-terminus of the 6 subunit and the carboxyl-terminus of the a! subunit interact as tubulin assembles into microtubules (12,13). The carboxyl-termini of both subunits can be cleaved by limited digestion with subtilisin, removing a peptide of about 2000-4000 Da (10,14) and leaving the partially ooo3-9&x/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form ree.erved.

SUBTILISIN

CLEAVAGE

digested subunits, (Y’ and p’.3 In addition to the subtilisinaccessible region within the carboxyl-terminus, two other regions of subtilisin interaction have been identified. On the (Ysubunit, these regions are also accessible to papain, thermolysin, and V8 protease and/or trypsin and define three subunit domains (13-17). Cleavage of the cusubunit, especially at internal sites, occurs more slowly than cleavage of the p subunit (14). On the /3 subunit three areas, accessible to one or more enzymes (papain, thermolysin, chymotrypsin, and subtilisin), have also been identified, again defining three subunit domains. Attempts at localizing specific sites of subtilisin interaction within the carboxyl-termini have yielded conflicting results. Within the carboxyl region of the (Ysubunit two possible subtilisin-sensitive sites have been found, one near residue 417 and one near residue 443 (10, 15, 18). Within the @ carboxyl-terminus two possible subtilisin sites also have been identified, occurring near residues 407 and 435 (10, 15, 18). Limited proteolysis of the carboxyl-terminus demonstrated that this region is important in controlling assembly of tubulin heterodimers into protofilaments and microtubules. In the absence of microtubule associated proteins or stabilizers such as glycerol, subtilisin-cleaved tubulin assembles at a lower critical concentration than native tubulin, 0.2-0.5 mg/ml and 1.5-1.7 mg/ml, respectively (9,19-21). In addition, only about 30% of subtilisincleaved tubulin depolymerizes under conditions that cause complete disassembly of native tubulin (i.e., at low temperatures or in the presence of CaC&) (9,lO). In fact, the removed carboxyl-terminal peptide is suspected of being the high affinity calcium binding domain (22, 23). It is thought that lateral and hydrophobic interactions are increased by removal of the carboxyl-termini, thereby enhancing polymerization (1, 20). Electron microscopic examination of subtilisin-cleaved tubulin polymers demonstrated that they differ from native microtubules in having more curvature and therefore appear “hooked” (19, 20). In fact ring formation from short polymers of native tubulin, reported to occur most readily in the presence of GDP (24), is enhanced when the carboxyl-termini have been removed (25). Thus it is thought that the highly charged carboxyl-terminus is important in assembly and may direct the proper orientation of heterodimers (9, 20). It has been shown that accessibility of protein regions to enzymatic cleavage provides structural information. s Abbreviations used: a’ and /3’, (Yand /3 subunits cleaved by subtilisin to remove the carboxyl-termini; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; DE-52, diethylaminoethyl cellulose; DMSO, dimethyl sulfoxide; EGTA, [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid; IEF, isoelectric focusing; NBT, nitroblue tetrazolium; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene dilluoride; Pipes, piperazineN,iV’-bis(2-ethanesulfonic acid); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethy1) aminomethane; Triton X-100, t-octylphenoxypolyethoxyethanol.

OF TUBULIN

153

Sackett and Wolff (14) suggested that trypsin sites on the (Y subunit and chymotrypsin sites on the @ subunit are accessible only in the heterodimer. Both sites are located in the amino-terminal portion of the subunits. The same studies indicated that the subtilisin sites of the carboxyl-termini remain accessible in both the heterodimers and the polymers. Our experiments described here using isoelectric focusing followed by SDS-PAGE indicate that in fact at least one site on the /3 subunit is more accessible to subtilisin in the polymer. These data suggest that either interactions between subunits in the polymer or a conformational change increases accessibility within the /3 subunit carboxyl-terminus. In addition, internal subtilisin sites generate peptides that copurify with tubulin under all purification schemes used. MATERIALS

AND METHODS

Reagents. Deionized (Nanopure) water was used in all experiments. EGTA, GTP (Type II-S), glycerol, L-histidine, L-glutamate, MgSO,, Nonidet, Pipes, and Triton X-100 were all purchased from Sigma Chemical Co. Sodium phosphate was purchased from J. T. Baker and ammonium sulfate (ultrapure) was from Schwartz/Mann. Tris, 2-mercaptoethanol, and polyacrylamide were from Fisher Biotech. Ampholines were purchased from Pharmacia LKB Biotechnology, Inc. and Serva Inc. and urea (ultrapure) was from Research Organics. The agarose (low melting temperature) was from Bio-Rad and PVDF filters were from Applied Biosystems. Subtilisin (subtilopeptidase A) was purchased from Boehringer Mannheim or Sigma and DE-52 was from Whatman Biosystems Limited. SDS (95%) was purchased from Mallinckrodt. Taxol was provided to us by Dr. Matthew Suffness of the National Cancer Institute. The monoclonal antibody, Tu27B, which reactsagainst/3subunit residues 410-430, was provided by Dr. Anthony Frankfurter, University of Virginia. Tubulin purification. Tubulin, free of MAPS, was purified from pig brain by warm/cold polymerization/depolymerization cycles, followed by phosphocellulose chromatography as described by Williams and Lee (26) and modified by Correia et al. (27). Protein concentrations were determined spectrophometrically (28) (G,,s = 1.23 liters/g-cm) or by the method of Bradford (29) calibrated with tubulin. SDS-PAGE. Slab gels were prepared, stacked at pH 6.8 and resolved at pH 8.8 (30), using impure SDS, which enhanced the separation of (Y and fi tubulin (31). Following electrophoresis, gels were stained with Coomassie brilliant blue R-250. Limited proteolysis. Native tubulin heterodimers or polymers were subjected to limited cleavage of the carboxyl-termini by subtilisin as described by Sackett and Wolff (14). Subtilisin was weighed and dissolved in water at 1 mg/ml or 5 mg/ml. Aliquots were frozen at -7O’C and thawed just prior to use. Tubulin heterodimers at 1.5 or 3 mg/ml were digested at 27°C in 10 mM Pipes pH 6.9, 1 mM MgS04, and 0.1 mM GTP or GDP with 1% (w/w) subtilisin for 13 min and stopped with 1 mM PMSF immediately or after 2 h on ice. In other experiments tubulin at 3 mg/ml was polymerized at 30°C in 100 mM Pipes, pH 6.9, 2 mM EGTA, 1 mM MgS04, and 2 mM GTP for 10 min in 4 M glycerol, or for 30 min in the presence of taxol (taxol:tubulin ratios-l:l, 1.5~1, or 2:l) in the same buffer without glycerol but with 1% DMSO. Following this initial polymerization, tubulin (as microtubules) was digested with 5% (w/w) subtilisin for 2 h at 30°C and the same buffer conditions and then stopped with 1 mM PMSF. In order to establish that differences found by isoelectric focusing experiments were not affected by cleavage solution conditions, tubulin heterodimers at 30°C in 0.1 mM GDP and 1 mM MgS04 were digested in the presence of 10 mM Pipes, pH 6.9, and 4 M glycerol or 1% DMSO or they were digested in 100 mM Pipes, pH 6.9, for 13 min or 2 h. The extent of digestion was analyzed by SDS-

154

LOBERT

AND

PAGE of native and digested tubulin. The times for limited digestion of heterodimers or polymers were selected in order to achieve consistent extents of digestion of /3 tubulin under the required conditions. In order to purify the cleaved product, digested tubulin was cycled through warm/ cold polymerization/depolymerization, subjected to ammonium sulfate precipitation, or chromatographed through a DE-52 anion-exchange column (9). This partially purified tubulin was used in the IEF experiments described below. Polymerization-depolymerization. Digested tubulin was purified by one cycle of polymerization and depolymerization. Immediately following digestion, polymers were pelleted or heterodimer solutions were made 4 M glycerol and 2 mM GTP and warmed at 37°C for 30 min and then pelleted. Warm pellets were obtained by centrifugation for 15 min at 100,000 rpm at 30°C in a Beckman TLlOO centrifuge. Pellets were resuspended in 100 mM Pipes, pH 6.9, 2 mM EGTA, 1 mM MgSO, and left on ice for 20-30 min. Resuspended pellets were then centrifuged at 100,000 rpm for 5 min at 4°C. The cold supernatant was used immediately or frozen at -70°C.

CORREIA out in the second dimension after equilibrating the tubes in stacking buffer, pH 6.8, with 5% 2-mercaptoethanol and 6% glycerol for 1 h and embedding with 0.75% agarose at the top of the slab gel as described by Correia et al. (27). IEF was also carried out in vertical slab gels (14 X 16 X 0.15 cm) as described in Detrich and Overton (33). Briefly, 4.3% polyacrylamide slab gels were prepared with 9.16 M urea, 2.5% ampholine (LKB pH 4.5-5.4, Serva pH 5.0-5.5, and LKB pH 5.0-7.0 at a ratio of 5:3:2) and 2% Nonidet. Overlay (25 nl/lane) prepared with 1.2% ampholines (at the same ratio), 9.16 M urea, and 1% Nonidet was used for prefocusing at 16’C for 1 h at 8 W constant power. Protein samples of SO-100 pg, prepared as above, were electrophoresed at 8 W for 5 h and then 10 W for 1 h. The pH gradients of the IEF gels were determined either from a tube or a vertical strip, 1.4 cm wide, adjacent to the protein lanes. These were cut into 0.5- or l-cm sections and left in 1 ml of water overnight before the pH was read. Gels were stained with Coomassie brilliant blue R-250.

Ammonium sulfate precipitation. Ammonium sulfate precipitation was used as an alternate method for (Y’ and 0 purification. The digested tubulin was brought to 32% saturation (0.177 g/ml), left 30 min on ice, and centrifuged at 76,000 rpm for 1 min at 4’C in a Beckman TLlOO centrifuge. The supernatant was brought to 43% saturation with ammonium sulfate, left on ice 30 min, and then centrifuged at 100,000 rpm for 2 min at 4°C. The second pellet was resuspended, equilibrated, and used immediately or frozen at -7O’C.

Immunoassay. Western blot transfers (34) onto PVDF filters were carried out and the filters were reacted overnight with mouse monoclonal antibodies, Tu27B, that react against a synthetic p subunit peptide (residues 410-430 conjugated to BSA) (35, 36). In order to visualize the interaction, a second reaction with goat antimouse alkaline-phosphataseconjugated antibodies was carried out and developed with BCIP and NBT.

DE-52 ion-exchange chromatography. Digested tubulin was loaded onto a 1 X 13-cm DE-52 column preequilibrated with the heterodimer or polymer buffer (9). For heterodimers 10 mM Pipes, pH 6.9, 1 mM MgS04, and 0.1 mM GTP or 10 mM sodium phosphate, pH 7, and 1 mM MgCl* was used. For polymers 100 mM Pipes, pH 6.9, 1 mM MgS04, 2 mM EGTA, and 1 mM GTP was used. The elution gradient was established with 15 ml or 30 ml of the same buffer and 15 ml or 30 ml of 0.8 M NaCl in buffer. Protein was eluted at a rate of about 0.5 ml/min. In some experiments 0.1% Triton X-100, or 0.2% Triton X-100 and 0.05% SDS, was added to the buffer. The addition of detergent necessitated a change in the maximum ionic strength required for elution from 0.8 M NaCl to 1.6 M NaCl.

RESULTS

Isoelectric focusing and two-dimensional SDS-PAGE. Isoelectric focusing (IEF) in 25-cm tube gels was carried out as described by George et al. (32) and modified by Correia et al. (27). Tube gels of 4.3% polya&amide with 2.5% ampholine (LKB pH 4.0-6.5 and Serva pH 5.05.5 at a ratio of 1:1),4 9.16 M urea, and 2% Nonidet were prefocused at 16°C with 50 ~1 of overlay containing 9.16 M urea, 1.2% ampholines (same ratio as above), and 1% Nonidet at 300 V for 1 h, 400 V for 1 h, 500 V for 30 min, 600 V for 30 min, and 700 V for 30 min. Protein samples (50-200 pg) prepared in the same concentrations of urea and Nonidet with the addition of 5% P-mercaptoethanol and 100 mM NaCl were loaded and electrophoresed at 16°C for 72 h at 700 V constant voltage. The anode and cathode buffers were 10 mM L-glutamate, pH 3.3, and 10 mM L-histidine, pH 7.7, respectively. SDS-PAGE was carried

4 Early results were obtained with 2% ampholines (LKB, pH 4.0-6.0 and 5.0-7.0 at ratios 3:l or 1:l) in the tube gels and overlay. During the course of this work subsequent lots of this product failed to produce satisfactory results in terms of both isotype separation and solubility of the protein. In some experiments the pH gradients extended only to pH 5.5 or 5.8. Protein tended to precipitate at the tops of the gels. We found that by adding 100 mM NaCl to protein samples the solubility increased significantly in the presence or absence of ampholines. We tried the vertical slab gels described in this paper, but found that none produced adequate resolution of 01’ and 0’ by two-dimensional experiments. Thus we explored mixtures of ampholines in 25cm tube gels. The recipes given here resulted in both sharp bands and about a 4-cm spread of the more basic o and most acidic 6 isotypes.

Purification

of Subtilisin-Cleaved

Tub&n

When tubulin heterodimers or polymers were digested with subtilisin, 8% polyacrylamide slab gels showed 34to 40-kDa peptides in addition to cleaved (Y’ and /3’ subunits. It was not possible to remove these peptides with any of the three methods of purification used in these experiments (Fig. 1). Digested product was cycled through warm polymerization and cold depolymerization, or precipitated with ammonium sulfate or eluted from a DE-52 (anionic) column with nearly identical results. This is in contrast to Kanazawa and Timasheff (9) who report pure (Y’ and p’ upon elution from DE-52. This could be due to differences in buffer conditions and source of enzyme. However, varying the buffer (10 mM Pipes or 10 mM sodium phosphate), magnesium salt (MgSO, or MgClJ, or subtilisin source (Boehringer Mannheim or Sigma) in the DE-52 elution experiments of heterodimers did not result in pure CX’,/3’. The amount of low-molecular-weight contaminant is dependent upon the time of digestion under each condition (see below). We have attempted to maximize the conversion of @to /3’. The variability observed in the amount of /-I and p’ in Fig. 1 reflects nearly complete conversion. Longer times of digestion only produce more 34- to 40-kDa peptides with a loss of 0’. However, longer times of digestion do facilitate complete conversion of (Y to (Y’.~Attempts to solubilize the peptides with a nonionic detergent or a combination of nonionic and ionic detergents with the hope of separating them from cy’ and 0’ ’ Data on the cleavage of a and fl tubulin by subtilisin in the presence of various cations will be presented elsewhere (S. Lobert and J. J. Correia, in preparation).

SUBTILISIN

12

-

3

-____r

4

5

6

CLEAVAGE

789

------m-m

FIG. 1. Purification of subtilisin-cleaved heterodimeric and polymeric tubulin. Lanes 1 and 9 show low-molecular-weight markers bovine serum albumin, MW 66,200, ovalbumin, MW 45,000, and carbonic anhydrase, MW 31,000. In lane 2 is native tubulin, lanes 3-5 subtilisin-cleaved tubulin polymerized in 4 M glycerol, and lanes 6-8 subtilisin-cleaved heterodimers. Tubulin in lanes 3 and 6 was carried through one cycle of warm polymerization and cold depolymerization; lanes 4 and 7 show tubulin purified by ammonium sulfate precipitation; and lanes 5 and 8 show tubulin purified by DE-52 chromatography.

OF TUBULIN

1

155

2

3

4

FIG. 2. IEF of native and subtilisin-cleaved tubulin polymers and heterodimers. Tubulin was purified by warm polymerization and cold depolymerization. The basic and acidic ends of the gel are at the top and bottom, respectively. The arrows indicate the most prominent isotypes, pZs 5.56 and 5.51 (from top to bottom), identified as /3’ in Fig. 3. Lane 1, native tubulin; lane 2, tubulin polymerized in 4 M glycerol prior to digestion by subtilisin; lane 3, tubulin heterodimers subjected to limited subtilisin cleavage; lane 4, tubulin polymerized with taxol (stoichiometry 1:l) prior to subtilisin digestion. Note that lane 4 is from a separate gel and that this is a composite figure.

bulin (32,37) to 7-9 for digested tubulin6 In addition the (Y’and ,B’subunits were more basic than the native tubulin prior to DE 52 chromatography were unsuccessful. The subunits, having isoelectric points ranging from 5.46 to interactions holding these peptides together and allowing 5.75 compared to native tubulin where pls ranged from copurification with tubulin subunits are unknown; how5.22 to 5.45. Table I gives the isoelectric points for native ever, since native behavior in terms of polymerization/ and subtilisin-cleaved tubulin isotypes. For the cleaved depolymerization and solubility is maintained in subtilheterodimer, a prominent band with a pl near 5.51 was isin-cleaved tubulin, noncovalent interactions with infound. A second less prominent band of pI near 5.56 was ternally cleaved peptides are suspected (27). In all of our also evident. When glycerol-induced polymers were diexperiments, SDS gels were overloaded (312 pg/lane) to gested with subtilisin, both isotypes, pI 5.51 and 5.56, establish the purity of the cleaved tubulin. Overloading were equally prominent. It is important to note that when caused the (Y’ band to smear slightly. The extent of (Y glycerol is used to induce polymerization, the solution cleavage is most evident in the IEF results below. We contains a mixture of heterodimers and polymers (38,39); always observed the 34- to 40-kDa peptides, apparently therefore this “digested polymer” pattern actually repderived from cleavage at the internal subtilisin sites. Thus resents both heterodimer and polymer cleavage. This difin all experiments described below these peptides were ference is evident in all samples regardless of the method present. These results suggest all subtilisin-treated tuof purification. bulin is contaminated with internal site digestion prodSince polymer digestion required 2 h to obtain nearly ucts. The influence of these contaminants on polymerthe same extent of cleavage as the 13 min digestion of ization is unknown. Isoelectric

Focusing

Isoelectric focusing of native tubulin or subtilisincleaved heterodimers or polymers resulted in very different isotype patterns (Fig. 2). The number of isotypes was reduced by subtilisin cleavage from 17-22 for native tu-

6 These numbers are derived from a combination of our best slab and tube IEF experiments and are most clearly seen in the data presented in Fig. 3. Both systems result in protein precipitation at the tops of the gels. The slab gels tend to preferentially exclude (~,a’. In Figs. 2 and 3 less protein is found in the more basic regions of the gels. The tube gels exhibit streaking toward the top of the gels as can be seen in the twodimensional experiments (Fig. 3).

LOBERT AND CORREIA

156 TABLE

I

Isoelectric Points of Tubulin Isotypes” Source Native tubulin Subtilisin-cleaved Heterodimer

Glycerol

a or a’

Bor 8’

5.30-5.45

5.22-5.33

5.75 5.58 5.53

5.56 5.53 5.51b 5.46

5.75’ 5.58 5.53

5.70’ 5.56b 5.53 5.516 5.46

5.75’ 5.58 5.53

5.70 5.56b 5.53 5.51 5.46

tubulin

polymerized

Taxol polymerized

a For clarity only the most prominent isotypes are listed. ’ Indicates the most prominent j3’ isotypes. ’ Evident in the tube gel but not in the two-dimensional limited incorporation.

gel due to

the heterodimer (Fig. l), 2-h heterodimer digestions were carried out in order to ascertain whether the differences in the heterodimer and polymer isotype patterns were due simply to the length of digestion. In addition, solution conditions were varied for 13-min and 2-h digestions. Tubulin heterodimers were subjected to subtilisin cleavage at 27°C for 13 min and then placed on ice for 2 h. In other experiments, heterodimers were digested for 13 min or 2 h at 27°C in 100 mM Pipes, pH 6.9, or in 10 mM Pipes, pH 6.9, and 4 M glycerol or 1% DMSO. The IEF and two-dimensional isotype patterns for the 13-min digestions were identical to the patterns from heterodimer cleavage experiments under the usual conditions (data not shown). When heterodimers were digested for 2 h, much of the /3 tubulin was reduced to smaller peptides (34-40 kDa or less). In order to maximize the amount of /3’ present, we selected heterodimer digestion conditions that limited the extent of (Yand p digestion.5 Therefore the appropriate controls for these experiments were limited digestions that produced the same extent of conversion of fl to p’ under different conditions as determined by SDS-PAGE. Digestion of heterodimers in the cold or in the presence of glycerol, DMSO, 10 mM Pipes, or 100 mM Pipes resulted in patterns identical to that in lane 3 of Fig. 2. These results indicated that the second major p’ isotype, occurring when the glycerol-induced polymers were cleaved, was not simply produced by a longer period of digestion or polymer solution conditions. In order to determine if the isotype differences found in these experiments were truly associated with heterodimer or polymer forms of tubulin, taxol was used to sta-

bilize the polymeric state prior to subtilisin digestion, thereby increasing the relative amount of polymer in solution. As can be seen in Fig. 2, the more acidic isotype (~15.51) of the polymer pattern was reduced in the cleaved taxol-treated polymers. The most prominent isotype was found to have a pl near 5.56. The remaining material at or near ~15.51 may represent heterodimer product derived from unpolymerized tubulin. Experiments conducted at taxobtubulin ratios l:l, 1.5:1, and 2:l all gave similar results (lane 4, Fig. 2 and Fig. 3d). Microtubules formed in the presence of 4 M glycerol or taxol are known to have reduced dynamics. In addition, subtilisin-digested tubulin forms a polymer that is more stable and less prone to dynamic instability. Thus, the distinct isoforms are most likely derived from noninterconverting or slowly interconverting heterodimer and microtubule polymer pools. We cannot exclude additional isotype differences in these patterns due to p tubulin class differences (see Discussion section). Nor can we exclude the possibility of an equilibrium between conformational forms that is only slightly shifted by forming microtubules and thus always produces a mixture of subtilisin digestion products. Polymeric tubulin is primarily GDP containing tubulin; the GTP cap is believed to be very small. To establish that this IEF pattern difference is not just due to a difference in the conformations of GTP- and GDP-tubulin, heterodimers were digested in the presence of GTP and focused in slab gels as in Fig. 2. GTP-tubulin and GDPtubulin digested heterodimer patterns were identical (data not shown). These results suggest that the isotype pattern differences were most likely due to heterodimer and polymer differences. In addition, these results suggest that GDP-tubulin heterodimers and GDP microtubules are in different conformations. Manganese Q-band EPR experiments on GDP-tubulin heterodimers and taxol-stabilized GDP microtubules also reveal a dramatic difference in conformation, at least at the metallocenter (J. J. Correia and A. H. Beth, in preparation). Two-Dimensional Electrophresis In order to identify which isotypes form the (Y, (Y’, p, and 0 subunits, two-dimensional electrophoresis was carried out (Fig. 3). Native tubulin 1yisotypes, pls 5.30 to 5.45, were consistently more basic than /3tubulin isotypes, pls 5.22-5.33 (Fig. 3a). It is clear from these experiments that the differences in IEF patterns of subtilisin-cleaved heterodimers and polymers were primarily due to relative amounts of 6’ isotypes (Figs. 3b and 3~). The three major CY’isotypes, pls 5.75, 5.58, 5.53, were identical in the digested polymer and heterodimer. When glycerol-induced polymers were digested, two major 0’ isotypes were present with pls 5.56 and 5.51; however, the digestion of tubulin heterodimers resulted in only one major 0’ isotype, pl 5.51. It should be noted that, by SDS-PAGE criteria, the isotype with a pl 5.56 had a slightly lower molecular

157

FIG. 3. Two-dimensional IEF-SDS-PAGE of native and subtilisin-cleaved tub&n. Tubulin was purified by warm polymerization and cold depolymerization. Following isoelectric focusing, tube gels (25 cm) were equilibrated, cropped, and embedded in agarose above 8% polyacrylamide gels, pH 8.8, and electrophoresed in the second dimension in order to identify the subunits. The basic and acidic ends of the tube gels are at the left and right, respectively. (a) Native tubulin, (b) subtilisin-cleaved heterodimers, (c) subtilisin-cleaved tubulin polymerized in 4 M glycerol, and (d) subtilisin-cleaved tubulin polymerized in taxol. The arrows indicate the most prominent /3’ isotypes for subtilisin-cleaved tubulin. From left to right the pls are 5.56 and 5.51. Note that uncleaved (Y and much smaller amounts of uncleaved p are present in these gels. For example, (Y is present in b, c, and d beginning at pl near 5.45 and extending toward the acidic region of the gel.

weight (or was less negatively charged, and thus bound more SDS, increasing its mobility) than the isotype with a pI of 5.51. The two-dimensional gel pattern of taxoltreated subtilisin-cleaved tubulin showed a single prominent 0’ isotype with a pI 5.56, significantly more basic than the single major heterodimeric @’ isotype and identical to one of the two major ,6’ isotypes found in the cleaved glycerol-polymerized tubulin. An additional /?’ isotype, pl 5.53, occurs in heterodimers and polymers. However, another minor p’ isotype, pI 5.70, occurs only in polymers. These data together suggest that the differences were indeed due to heterodimeric and polymeric differences in accessibility to subtilisin within or near the 6 carboxyl-terminus. Western Blots In order to localize the site of subtilisin cleavage on the p subunit, a Western blot of the two-dimensional SDS gel from the subtilisin digestion of glycerol-induced polymers was reacted with mouse monoclonal antibodies that recognize a synthetic peptide composed of @residues 410430 (data not shown). Both major isotypes, ~15.56 and 5.51, reacted with these antibodies, indicating that the subtilisin cleavage sites most likely lie within the last 20 amino acids. The small pI and apparent molecular weight differences suggest that these cleavage differences may correspond to the removal of only one or two additional

acidic residues near residue 435. In addition, all ,!3and ,8’ species more acidic than the isotype of pI 5.56 reacted. No (Y or (Y’ reaction occurred. DISCUSSION Our initial interest in this work was derived from a desire to isolate a more homogeneous tubulin heterodimer from limited subtilisin treatment. The heterogeneity of tubulin arises from genetic variability, primarily in the carboxyl-termini, and post-translational modifications, polyglutamylation and phosphorylation, also in the carboxyl-termini (40, 41). This is believed in part to have hampered attempts to produce diffraction quality crystals for three-dimensional structural analysis. Structural information has been obtained by limited proteolysis using several enzymes to explore solvent accessible regions of the tubulin subunits in the heterodimer and polymer (1317). Three domains were identified, consistent with electron microscopic and fiber diffraction studies (15). One of these domains includes the carboxyl-terminus and was described by circular dichroism and NMR as highly flexible in both the heterodimer and the polymer and as having some a-helical content (g-11,42). Limited proteolysis using subtilisin, which cleaves the carboxyl-termini, demonstrated the importance of this region in assembly of microtubules (9, 10, 20). We initially produced subtilisin-cleaved tubulin for solubility and preliminary crys-

158

LOBERT

AND

tallization studies (1). Using two-dimensional IEF-SDSPAGE experiments we have verified the reduction in charge heterogeneity of tubulin (Fig. 2). By IEF criteria tubulin heterogeneity was reduced after cleavage of the carboxyl-termini, decreasing the number of isotypes from 17-22 for native tubulin to 7-9 for subtilisin-cleaved heterodimers or polymers; however, additional heterogeneity due to the presence of smaller cleaved peptides persisted. These results agree with the work presented by Lee et al. (41), demonstrating that subtilisin cleavage reduced the overall charge heterogeneity of rat brain tubulin. In addition our data suggest a structural difference in the carboxyl-terminus of the p subunit in heterodimers and polymers. In our experiments, we were unable to separate the 34to 40-kDa subtilisin-cleavage products from the cleaved (Y’and /3’ heterodimer. These smaller peptides, which are probably produced by digestion at internal sites on CYand/ or 0 (15), were consistently found on overloaded SDS gels regardless of the method used to purify a’$’ (cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography). It has been reported that limited digestion of calf brain tubulin, followed by DE-52 chromatography, produces pure (Y’and 6’ with no associated smaller peptides (9). Using pig brain tubulin in our experiments, we were not able to obtain this result, regardless of the buffer conditions or subtilisin source used. Excluding the trivial explanation that we overload our SDS gels (212 pg/lane) and those authors did not [see Ref. (9), Fig. 1, 8 pg/lane], it is possible that cleavage of the isotypes in pig brain tubulin results in a product that differs from calf brain tubulin in terms of the interaction of smaller peptides with (Y’and 0’. However, in an effort to test this possibility, our time course experiments with phosphocellulose-purified calf brain tubulin revealed that similar 34- to 40-kDa peptides appeared in significant quantities as early as 5 min after beginning digestion with 1% subtilisin (w/w) at 30°C. Thus in our experiments with heterodimers or polymers, the 34- to 40-kDa peptides were present to varying extents.7 The companion bands (assuming a single cut mechanism) of lo-16 kDa appeared as stainable material in the front. IEF followed by two-dimensional SDS-PAGE demonstrated a difference in subtilisin-cleaved heterodimers and polymers. It should be noted that after 5 min digestion of heterodimers we observed an increase in turbidity 7 Since the two-dimensional gels in Fig. 3 were cropped to focus on the a’ and fl’ regions for the sake of clarity, the 34- to 40-kDa peptides are not shown. The influence of these smaller cleavage products on microtubule assembly is not known. In addition, minor products caused by chemical proteolysis of tubulin occurred, especially in the 72-h tube gels. This complicates the interpretation of the origin of the minor subtilisin peptides. This chemical cleavage is strongly time, temperature and pH dependent and will be discussed elsewhere (J. J. Correia, L. Libscomb, and S. Lobert, in preparation).

CORREIA

monitored at 350 nm that plateaus near 13 min. This result is consistent with reports that subtilisin-cleaved GDP-tubulin readily forms rings and ring aggregates (1, 25). By the end of our digestions we have a mixture of heterodimers, rings, and ring aggregates. Thus subtilisin cleavage of heterodimers and rings may contribute to the results seen in our heterodimer IEF experiments. The solubility of tubulin in the IEF experiments was found to be variable. Consistent with previous reports, tubulin tended to precipitate at the tops of the gels (32). In fact the amount of (Y,(Y’found in slab gels was consistently less than expected, although identical LY’patterns occurred in heterodimer and polymer two-dimensional gels (Fig. 3, Table I). In Fig. 3 (lanes b, c, d), undigested (Y,which does not appear in Fig. 2 (lanes 2-4), is evident. This solubility problem may mask some of the actual differences. For example, one minor 6’ isotype, ~15.70, appeared in tube gels of both cleaved glycerol-induced and taxol-induced polymers but only in the cleaved taxol-induced polymer two-dimensional gels (Fig. 3). This isotype did not appear in either cleaved heterodimer tube gels or two-dimensional gels, suggesting an additional heterodimer, polymer differential subtilisin interaction. For this discussion we have focused on p’ differences in major isotypes because (i) the p chain is digested first and (ii) these isotypes demonstrate more consistent solubility. The isoelectric points of the major /3’ isotypes occurring in the heterodimers and taxol-induced polymers were found to be 5.51 and 5.56, respectively. This difference is not due to nucleotide differences at the exchangeable site. Heterodimer patterns were identical in the presence of GDP or GTP. Furthermore, it is not due to glycerol in the polymer samples, because taxol-induced microtubules, in the absence of glycerol, also gave a polymer pattern. Control digestions of heterodimers in the presence of glycerol, DMSO, or variable Pipes concentrations or at various temperatures reveal no influence of these variables on the heterodimer subtilisin digestion pattern. In interpreting this result it should be noted that previous attempts to localize the sites of subtilisin cleavage have yielded conflicting results. Maccioni et al. (10) found, by peptide analysis of subtilisin-cleaved heterodimers, one major and one minor subtilisin-accessible site within the carboxyl-terminus of the /3 subunit, Glu 407Phe 408 and Glu 435-Phe 436, respectively. The proteolytic studies of Sackett and Wolff (14) suggested that subtilisin sites were equally accessible in the heterodimer and polymer. De la Vina et al. (15) found, using monoclonal antibodies raised against a @subunit peptide, 412-431, that subtilisin cleaves heterodimers primarily within the last 20 amino acids. Our experiments demonstrate that there are at least two subtilisin sites within the last 20 residues of the carboxyl-terminus of the p subunit. We propose that subtilisin cleaves near Glu 435 in both the heterodimer and the polymer. In the polymer at least one additional acidic group is removed compared to the het-

SUBTILISIN

CLEAVAGE

erodimer, producing the more basic and slightly smaller isotype that predominates when taxol is used to polymerize tubulin. Calculated isoelectric points using the porcine tubulin sequence of Krauhs et al. (43) indicated that the removal of only one or two of the nine acidic residues near 435 will account for the observed pI difference of 0.05. Our data suggest that a conformational change occurs in the carboxyl-terminus of the p subunit in tubulin polymers during assembly, producing a subtilisin-accessible region that either is not present in the heterodimer or has more limited accessibility. Our data, however, do not exclude the possibility of differential cleavage of /3 tubulin classes. We found that at least two p’ bands, pls 5.53 and 5.46, are identical in the heterodimer and polymer (Fig. 3). This result (consistent with the antibody experiment described above) indicated that subtilisin does not cleave between p residues 410 and 430, since this region is conserved among all classes (I, II, III, and IV) of brain tubulin and, therefore, would produce identical changes in all classes of tubulin (6). Isotype classes are identified through antibody interactions with the last 20 amino acids of the subunit (44). Since in our experiments we deliberately removed this region, we are unable to establish whether differential cleavage of specific tubulin classes in heterodimers or polymers could account for the differences we identified. It should be noted that these data do not exclude differential cleavage of heterodimers and polymers in a classindependent manner, i.e., all classes cleaved at the same residue. For example, we calculated (IBI Pustell Sequence Analysis Software) that cleaving /3 tubulin classes I, III, and IV [see Ref. (6), Fig. I] at residue 433 would generate peptides that are more basic by the amount we observed in these experiments, 0.05. However, if class II tubuiin was cleaved at residue 433, the pl would not change. Our experiments would give identical results whether or not class II tubulin was cleaved at residue 433. Similar arguments could be made for other positions or combinations of other positions. Thus this differential subtilisin effect could be occurring at all fl subunits, but only select classes (classes I, III, and IV if residue 433 is removed in microtubules) would show an IEF shift. Lee et al. (41) determined in two-dimensional IEF-SDS-PAGE similar to ours, that a single spot corresponded to a single isotype (flII1). Thus it seems possible that the major isotypes we identified (~1s 5.51 and 5.56) correspond to cleavage of the major classes of tubulin present in vertebrate brain (I, II, and IV) (44). ACKNOWLEDGMENTS We thank Dr. Susan Wellman for critical reading of this manuscript and Ms. Bettye Sue Hennington for assistance with the immunoassays and for helpful discussions throughout these experiments and preparation of this manuscript. We are grateful to Mr. Tim Vickmark for photographic assistance. We also thank Dr. Anthony Frankfurter for helpful discussions during this work and for kindly providing’the mono-

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159

clonal antibodies and Dr. Matthew Suffness of the National Cancer Institute for supplying taxol for the polymerization experiments.

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