J Biol Chem, Vol. 274, Issue 34, 24054-24058, August 20, 1999

Tubulin Folding Cofactors as GTPase-activating Proteins
GTP HYDROLYSIS AND THE ASSEMBLY OF THE /-TUBULIN HETERODIMER^*

Guoling Tian, Arunashree Bhamidipati, Nicholas J. Cowan, and Sally A. Lewis^§

From the Department of Biochemistry, New York University Medical Center, New York, New York 10016

	ABSTRACT

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In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized - and -subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. - and -tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A-E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by -tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein -tubulin to hydrolyze its GTP.

	INTRODUCTION

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Tubulin is an / heterodimer. Both - and -tubulins are GTP-binding proteins; -tubulin binds GTP nonexchangeably, whereas the GTP-binding site on -tubulin is exchangeable (1). GTP hydrolysis by -tubulin is a key element in determining the dynamic behavior of microtubules; the hydrolysis of GTP is coupled to tubulin polymerization such that variation in the size of the "cap" of GTP-bound subunits on a given microtubule determines whether it will continue to grow or undergo transition to a rapidly depolymerizing phase (2-4).

GTP hydrolysis is also essential for the biogenesis of native tubulin. Recent analysis of the tubulin folding pathway has shown that the  and  polypeptides must interact with a series of chaperone proteins before reaching the native state (reviewed in Ref. 5). The first step in this pathway is the binding of newly synthesized (or denatured) tubulin molecules to cytosolic chaperonin (6) (also referred to as TRiC (7) and CCT (8)). Following one or more rounds of ATP hydrolysis by cytosolic chaperonin, the resulting quasi-native tubulin intermediates interact with tubulin-specific chaperones named cofactors A-E (9-11). Native tubulin is released from a supercomplex that contains both - and -tubulin and cofactors C, D, and E and that hydrolyzes GTP as part of this reaction (5, 11, 12).

We sought to determine the nature of the GTP hydrolysis reaction performed by the tubulin-cofactor supercomplex and its role in producing native tubulin. Here we show that both in the folding reaction and in the related reaction of cofactors with native tubulin, the cofactors act as GTPase-activating proteins (GAPs),¹ stimulating many-fold the negligible intrinsic GTPase activity of the tubulin to which they are bound.

	EXPERIMENTAL PROCEDURES

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Purification of Cofactors and Labeled Tubulin-- To generate purified tubulin ³⁵S-labeled in the - or -subunit, full-length tubulin cDNAs encoding mouse 2 (13) or mouse 3 (14) were ³⁵S-labeled by coupled transcription/translation in rabbit reticulocyte lysate (Promega Inc., Madison, WI) and purified by anion exchange chromatography on DEAE-Sephacel (11). Tubulin folding cofactors A, B, C, and D were either purified from bovine testis tissue or from Escherichia coli (as cloned recombinant proteins) as described (9-11). Tubulin folding cofactor E (which generates insoluble inclusion bodies upon expression in E. coli) was purified following expression as a biologically active protein in insect Sf21 cells using the BacPAK baculovirus expression system (CLONTECH Inc., Palo Alto, CA). For experiments using ribose-modified GTPs, native tubulin was exchanged with the corresponding nucleotide by anion exchange chromatography of twice-cycled bovine brain microtubules on DEAE-Sephacel (10) using buffer containing 50 µM dGTP or ddGTP.

Subunit Exchange Experiments-- Microtubules were purified from calf brain by the method of Shelanski et al. (15). Subtilisin-truncated tubulin (S-tubulin) and tubulin free from associated proteins were prepared as described (16, 17). Chicken erythrocyte tubulin (E-tubulin) was purified from heparinized chicken blood (Pelfreeze Inc., Rogers, AK) by the procedure of Murphy and Wallis (18).

In experiments to determine the potential for spontaneous tubulin subunit exchange, DEAE-Sephacel-purified bovine brain tubulin and S-tubulin (2 µM each) were incubated at 30 °C for 1 h in folding buffer (20 mM MES, pH 6.8, 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 1 mM GTP). To assess the role of cofactors in the exchange reaction, tubulin ³⁵S-labeled in its -subunit (1 µM) was incubated under the same conditions with various concentrations of E-tubulin or with 0.9 or 1.4 µM S-tubulin in the presence or absence of cofactors C, D, and E (each present at 1 µM) together with either 1 mM GTP or 1 mM GTPS. Reaction products were analyzed by nondenaturing polyacrylamide gel electrophoresis as described (6, 19).

Synthesis of ³²P-Labeled Guanine Nucleotides-- Unlabeled ddGDP was prepared by incubation of 5 mM ddGTP, 0.05 mM ADP, 1 unit of nucleoside diphosphate kinase (bovine liver), and 1 unit of hexokinase (bakers' yeast) (both from Sigma) in 20 mM Tris-HCl buffer, pH 7.6, containing 20 mM glucose and 1 mM MgCl₂ at 30 °C for 16 h. Reaction products were purified by extraction with phenol and chloroform. ADP was converted to ATP by the addition of 1 mM ddGTP and 1 unit of nucleoside diphosphate kinase and further incubation at 30 °C for 1 h. ddGDP was purified from other nucleotides by anion exchange chromatography on a column (5/5 15Q; Amersham Pharmacia Biotech) developed with a linear gradient (0.02-1.0 M) of ammonium bicarbonate and checked by its mobility on TLC.

[-³²P]dGTP and [-³²P]ddGTP were prepared in reactions containing 0.2 mCi of [-³²P]ATP (specific activity, 3,000 Ci/mmol) and either 50 µM dGDP or ddGDP in 5 µl of 20 mM Tricine buffer, pH 7.2, containing 1 mM MgCl₂ and 1 unit of bovine liver nucleoside diphosphate kinase. After incubation at 30 °C for 30 min, reaction products were isolated by extraction with phenol and chloroform. Remaining [-³²P]ATP was converted to ADP by addition of unlabeled ATP to 50 mM together with 10 mM glucose and 1 unit of hexokinase and a further incubation at 30 °C for 10 min. Following a further extraction with phenol and chloroform, the final reaction products were purified by anion exchange chromatography as described above.

[-³²P]GDP was generated by incubating [-³²P]GTP (specific activity, 8 Ci/mmol) and GDP (each at 0.1 mM) in a 50-µl reaction in the presence of 1 unit of nucleoside diphosphate kinase for 10 min at 30 °C, followed by extraction with phenol and chloroform and purification by anion exchange chromatography as described above.

GTP Hydrolysis Experiments-- To measure the rates of hydrolysis of cofactors C, D, and E at different tubulin concentrations, D (0.32 µM), E (0.67 µM), and C (1.1 µM) were incubated at 30 °C in folding buffer with [-³²P]GTP (22.5 µM) and various concentrations of DEAE-purified tubulin. Aliquots (2 µl) were withdrawn from the 12-µl reaction into 1 M perchloric acid at 1, 2, 3, 4, and 5 min, and the amount of phosphate released was quantitated (20). In all cases less than 10% of the input GTP was hydrolyzed. The hydrolysis of [-³²P]GTP, [-³²P]dGTP, and [-³²P]ddGTP by the tubulin-chaperone supercomplex was measured in the same way using 0.5 µM bovine brain tubulin, 0.1 µM each cofactors C, D, and E, and 20 µM nucleotide. In some experiments (see "Results"), the tubulin was preincubated with podophyllotoxin (20 µM) for 10 min at 30 °C.

Nucleotide Content of Cofactor D--Tubulin Complexes-- ³²P-Labeled cofactor D--tubulin complexes resolved on nondenaturing gels (10) were located by autoradiography of the wet gels and excised. Nucleotide was extracted by maceration in 50% aqueous methanol. Gel fragments were removed by centrifugation, and the supernatants were dried under vacuum. The residue was dissolved in H₂O and spotted onto phosphoethyleneimine TLC plates. The plates were developed with 1.2 M LiCl (1).

	RESULTS

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The Tubulin Folding Supercomplex Is a Dimer-making Machine-- Native - and -tubulins reportedly exist in free equilibrium with the heterodimer, with an apparent dissociation constant of about 1 µM (21-24). However, the fact that tubulin-specific chaperones bring together - and -tubulin (Fig. 1A) is puzzling if the subunits can freely exchange. To examine the dissociation of tubulin, we used S-tubulin (25, 26), which migrates at a position distinct from that of untreated tubulin heterodimers upon native gel electrophoresis (Fig. 1B). If subunit exchange among heterodimers is indeed spontaneous, then co-incubation of untreated tubulin and S-tubulin might be expected to generate a population of hybrid heterodimer molecules consisting of full-length - and truncated -tubulin or truncated - and full-length -tubulin having a mobility intermediate between that of native brain tubulin and S-tubulin. We detected no such hybrid heterodimers (Fig. 1B, lanes 1 and 2). However, when the same reaction was repeated using brain tubulin ³⁵S-labeled in its -subunit in the presence of the three tubulin-folding cofactors (C, D, and E) that are common to the - and -tubulin folding pathways (Fig. 1A), a shift of label to an electrophoretic position between tubulin and S-tubulin was observed (Fig. 1C, lanes 3 and 4). This shift was inhibited by the inclusion of the slowly hydrolyzable analog GTPS (Fig. 1C, lanes 5 and 6). (In these reactions, most of the counts are found in the slowly migrating tubulin-cofactor supercomplex). These results imply that exchange of subunits between tubulin and S-tubulin requires the action of cofactors and the hydrolysis of GTP.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Subunits in the /-tubulin heterodimer can only exchange via transit through the supercomplex in the tubulin folding pathway. A, summary of tubulin-specific chaperones (cofactors) and their interactions in the tubulin folding pathway. For a more detailed account of the generation of tubulin-cofactor complexes and their interactions, see Refs. 5 and 11. B, no hybrid dimers are formed between full-length and truncated brain tubulin upon coincubation. S-tubulin, undigested tubulin, or equimolar amounts of both were incubated in buffer containing GTP at 30 °C, and the reaction products were visualized by Coomassie staining following resolution on a nondenaturing gel. Upper and lower arrows indicate the location of S-tubulin and undigested tubulin, respectively. C, cofactors C, D, and E together promote the formation of hybrid dimers between tubulin and S-tubulin. 1.4 µM (lanes 1, 3, and 5) or 0.9 µM (lanes 2, 4, and 6) unlabeled S-tubulin was coincubated with tubulin 35S-labeled in its -subunit either alone (lanes 1 and 2) or with cofactors C, D, and E in the presence of GTP (lanes 3 and 4), or with cofactors C, D, and E in the presence of GTPS (lanes 5 and 6). Reaction products were analyzed by native gel electrophoresis followed by autoradiography. Arrows (bottom to top) indicate the positions of tubulin dimers, hybrid dimers, S-tubulin, and the tubulin-cofactor supercomplex (arrested with GTPS). D, native bovine brain (lane 1, lower arrow) and E-tubulin (lane 2, upper arrow) migrate with different mobilities upon native gel electrophoresis. E, subunit exchange between tubulin and E-tubulin also depends on tubulin-specific chaperones and GTP hydrolysis. Autoradiograph of a nondenaturing gel in which purified brain tubulin 35S-labeled in its -subunit was incubated with a 3-fold molar excess of E-tubulin containing either 1 mM GTP or GTPS in the absence or presence of a stoichiometric equivalent of cofactors C, D, and E. Upper and lower arrows indicate the positions of E-tubulin and bovine brain tubulin, respectively.

Because it is possible that treatment of tubulin with subtilisin significantly alters its exchange properties, we repeated this experiment using E-tubulin. Native brain tubulin and E-tubulin (27) migrate with different mobilities upon native gel electrophoresis (Fig. 1D) because of sequence differences in the -subunits; however, the sequence of the -subunit of E-tubulin (28) is 99% identical to that of the mouse -tubulin, which we labeled to test for subunit exchange. If free exchange of subunits among heterodimers can indeed occur, then incubation of brain tubulin labeled in its -subunit with unlabeled E-tubulin should result in a shift of the label to the electrophoretic position characteristic of the E-tubulin dimer. Once again, no such shift was observed in this experiment (Fig. 1E, lane 1); the same result was obtained over a range (0.6-9.6 µM) of tubulin concentrations (data not shown). However, when the same reaction was repeated in the presence of cofactors C, D, and E, a shift of label to the electrophoretic position of E-tubulin was observed (Fig. 1E, lane 2), showing that in this case -subunit exchange had occurred between the two types of tubulin. The generation of hybrid heterodimers in this experiment was also dependent upon the hydrolysis of GTP, because the reaction was inhibited by GTPS (Fig. 1E, lane 3). Thus, the subunits of the /-tubulin heterodimer can only exchange by transit through the supercomplex, which is a part of the de novo tubulin folding pathway.

Cofactors as -Tubulin GTPase-activating Proteins-- We have previously shown that when tubulin and either cofactors C and D or cofactors C, D and E are mixed, GTP hydrolysis ensues (11). To understand the nature of this reaction, we measured the rate of GTP hydrolysis as a function of the concentration of added tubulin (Fig. 2A). We found that the cofactors behave with Michaelis-Menton kinetics, with tubulin-GTP as substrate and P_i as product. The cofactors have a K_m for tubulin of 0.1 µM and a turnover number of about 1.6 min¹ (6 pmol P_i/min/3.8 pmol cofactor D monomer, where D is the limiting cofactor). Because the K_m is about 200-fold lower than the critical concentration for tubulin polymerization and because the rate of GTP hydrolysis is saturable with increasing tubulin, it is clear that this hydrolysis is not the result of the cofactors acting as microtubule-associated proteins and promoting microtubule polymerization with its concomitant GTP hydrolysis. Rather, GTP hydrolysis is the result of the interaction of cofactors with tubulin dimers themselves. To underscore this point, we found that these two types of GTP hydrolysis are additive (Fig. 2B). At 9 µM tubulin but not at 3 µM tubulin, some polymerization-dependent GTP hydrolysis is observed. At the former concentration there is no net polymerization of microtubules, but there is hydrolysis because of tubulin-tubulin interactions (29). The addition of cofactors C, D, and E to tubulin at these two concentrations results in approximately the same increase in the rate of hydrolysis in each case, showing that the two types of hydrolysis are proceeding independently of each other. We conclude that the cofactors are acting on the G-protein tubulin as GAPs. In the absence of cofactor E, the combination of cofactors C and D have GAP activity; C and D stimulate GTP hydrolysis by tubulin, although they have a higher K_m for tubulin (0.19 µM) and a 6-fold lower turnover number (0.3/min) (Fig. 2A).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   The tubulin-folding cofactors behave as GTPase-activating proteins. A, rate of hydrolysis of GTP as a function of tubulin concentration. GTP hydrolysis rates were measured at different tubulin concentrations in the presence of cofactors C, D, and E (left-hand panel) or C and D (right-hand panel). 1/rate (pmol Pi released/min) is plotted versus 1/tubulin concentration (µM). B, rates of GTP hydrolysis resulting from tubulin-tubulin interactions and from interaction of dimer with cofactors C, D, and E is additive. Hydrolysis reactions were assembled with [32P]GTP and different amounts of cofactors and tubulin (see "Experimental Procedures"). The release of Pi is plotted as a function of time. Closed circles, cofactors C, D, and E and 9 µM tubulin; open circles, cofactors C, D, and E and 3 µM tubulin; closed squares, 9 µM tubulin alone; open squares, 3 µM tubulin alone; closed triangles, cofactors C, D, and E alone. Rates of hydrolysis are 10.5, 9.3, 0.9, 0.1, and 0.1 pmol/min, respectively. C, GTP can be replaced by dGTP or ddGTP in the tubulin folding reaction. 35S-Labeled native tubulin (present as a marker) is shown in lane 1. This material containing bound GTP, dGTP, or ddGTP (see "Experimental Procedures") was incubated with cofactor D in the presence of 10 µM GTP (lane 2), dGTP (lane 4), or ddGTP (lane 6). The resulting cofactor D--tubulin complex was discharged to native dimer by the addition of cofactors C and E and (to provide the -subunit) unlabeled native tubulin containing the corresponding nucleotide (lanes 3, 5, and 7). Upper and lower bands are the cofactor D--tubulin complex and tubulin dimer, respectively. D, GTP, dGTP, and ddGTP hydrolysis by the supercomplex and inhibition of hydrolysis by the microtubule poison podophyllotoxin. Rates of hydrolysis of GTP, dGTP, and ddGTP in the presence native tubulin (0.5 µM) with or without added tubulin-folding cofactors C, D, and E. Diamonds, cofactors + tubulin; triangles, cofactors + tubulin + podophyllotoxin; open circles, tubulin alone; stars, tubulin + podophyllotoxin.

To gather more direct evidence that the GTP hydrolysis step in the tubulin heterodimerization reaction is performed by the -tubulin subunit and not by one of the cofactor proteins contained in the supercomplex, we looked at the reaction of cofactors with tubulin in the presence of ribose-modified forms of GTP; both dGTP and ddGTP support microtubule growth in vitro and are hydrolyzed as efficiently as GTP by the -tubulin subunit upon polymerization (30). This property is diagnostic of the tubulin GTPase; polymerases and transferases, for example, utilize deoxy- and dideoxyribonucleotides very poorly. We found that dGTP and ddGTP also support the formation of tubulin-cofactor D complexes and can be used in the reaction that generates the heterodimer from these complexes by the addition of cofactors C and E and tubulin dimer (as a source of the -subunit) (Fig. 2C). This result is independent of the concentration of nucleotide in the range 10-300 µM (data not shown). Furthermore, both dGTP and ddGTP are hydrolyzed as rapidly or slightly more rapidly than is GTP by the supercomplex (Fig. 2D). This unusual behavior therefore supports the idea that it is the -tubulin in the supercomplex that performs the hydrolysis step leading to heterodimer release. We also examined the effect of microtubule poisons on the rate of hydrolysis by the supercomplex. Podophyllotoxin, which inhibits tubulin-dependent GTP hydrolysis in microtubule polymerization reactions (31) and suppresses hydrolysis-dependent dynamic instability (32), also slows GTP hydrolysis by the supercomplex (Fig. 2D) to a remarkably similar extent (33). These data provide further evidence that it is the tubulin in the supercomplex that hydrolyzes GTP when stimulated by cofactors.

GTP Hydrolysis Acts as a Switch for the Release of Folded Tubulin from Chaperone Complexes-- The data presented in Fig. 1 show that GTP hydrolysis is necessary for the reaction in which cofactors act on native tubulin to scramble the heterodimers. We have previously shown that GTP is likewise necessary in the tubulin folding reaction in which cofactors bring together newly folded - and -tubulin subunits (11). These two reactions share the GTP hydrolysis-dependent release of heterodimer from the supercomplex (Fig. 1). We hypothesized that GTP hydrolysis may directly lead to release of dimer if cofactors have a much lower affinity for GDP-tubulin than GTP-tubulin. To test this idea, we incubated tubulin with cofactors in the presence of either [-²P]GTP or [-²P]GDP. Tubulin-cofactor D complexes form very inefficiently when only GDP is present (Fig. 3A). To confirm this result, we mixed tubulin and cofactor D with equal quantities of [-³²P]GTP and [-³²P]GDP and analyzed by TLC the nucleotide content of the complexes thus formed. Only GTP was found in such complexes (Fig. 3B). These experiments show that cofactor D has a much lower affinity for GDP--tubulin than for GTP--tubulin. We infer that hydrolysis of GTP by -tubulin in the supercomplex leads directly to its release from that complex (in the form of tubulin heterodimer).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   GTP hydrolysis acts as a switch for the release of dimer from the supercomplex. A, tubulin-cofactor D complexes form very inefficiently from GDP-tubulin. Cofactor D was incubated with native tubulin in the presence of either 20 µM [32P]GTP or [32P]GDP and the reaction products analyzed on a nondenaturing gel; the arrow marks the position of cofactor D--tubulin complex. B, cofactor D preferentially interacts with GTP-tubulin. Autoradiograph of analysis by TLC of the nucleotide content of reactions in which cofactor D was incubated with tubulin containing either [32P]GTP (lanes 1 and 3) or an equimolar mixture of [32P]GTP and [32P]GDP (lanes 2 and 4); the content of labeled GTP and GDP in the entire reaction (lanes 1 and 2) or in the resulting purified cofactor D--tubulin complexes (lanes 3 and 4) is shown.

	DISCUSSION

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The simple and direct data presented here showing that - and -tubulin subunits do not exchange in the absence of tubulin-folding cofactors imply either that the subunits are very tightly associated in the heterodimer or that they denature as they dissociate. The latter explanation cannot be true, however, because this would imply that tubulin is highly unstable, which it is not, even at concentrations below the reported dissociation constant of the heterodimer, long thought to be in the micromolar range (21-24). The conclusion is inescapable that the subunits of the tubulin heterodimer are tightly bound to each other and are not in free equilibrium. There are compelling recent data that support this surprising finding; when tubulin is bound to a column via antibody specific to the -subunit, the -subunit cannot be dissociated unless it is exposed to Tris buffer at high pH (34). How then can we explain the corpus of results that have been interpreted to reflect a micromolar dissociation constant for the heterodimer? If tubulin undergoes some concentration-dependent conformational change (but does not dissociate), this would account for many of these experimental results.

Our data imply that the process whereby tubulin heterodimers are assembled includes a quality control step. We know that only dimer, not individual subunits, can be released from the supercomplex, because dimers can only exchange subunits via the supercomplex (Fig. 1). Furthermore, dimer is released only when it has demonstrated its ability to hydrolyze GTP (Fig. 1, C and E). In support of this interpretation, tubulin molecules with mutations that are predicted to abolish GTP hydrolysis are not assembled into microtubules when expressed in vivo and remain complexed with cofactors when made in vitro (35). Additionally, in a genetic screen for viable yeast harboring -tubulin mutations that altered rates of GTP hydrolysis, only mutations that increased the rate of hydrolysis were found (36). Many toxins bind to tubulin and interfere with its polymerization, some by changing the GTPase activity of tubulin (reviewed in Ref. 31). These toxins can poison microtubules substoichiometrically; if poisoned subunits are added to the growing end of a microtubule, the growth and stability of the whole tubule is affected (32, 37). By analogy, the quality control of tubulin by cofactors in vivo may be important for protecting the microtubules of the cell from poisoning by misfolded, non-GTP-hydrolyzing subunits. In addition to acting in the de novo folding pathway (10, 11), cofactors can interact with native tubulin dimer to convert GTP-tubulin to GDP-tubulin (Fig. 2); this process could function in the quality control of the pool of tubulin in the cell and/or in regulating tubulin polymerization.

Many GTP-binding proteins, including the heterotrimeric G proteins and the Ras-related small GTP-binding proteins, hydrolyze GTP at a very low intrinsic rate that can be increased enormously by the binding of specific GAPs. These proteins appear to contribute an "arginine finger" to the catalytic site of the GTP-binding protein and in doing so activate it (38). Although the GTP-binding site of tubulin and the related bacterial protein FtsZ have a different fold from that of all other GTP-binding proteins (39, 40), the experiments described here show that, like other GTP-binding proteins, a specific GAP (in this case tubulin-folding cofactors) stimulates the negligible intrinsic GTP hydrolysis rate of tubulin by orders of magnitude. In this sense, -tubulin also behaves like a GAP when it interacts with the GTP-binding domain of an adjacent -subunit during microtubule polymerization (39, 41). One of the tubulin-folding cofactors and -tubulin may therefore share some structural homology that allows each to contribute to the catalytic site of -tubulin polypeptides in tubulin-cofactor supercomplexes and in microtubules, respectively.

	ACKNOWLEDGEMENTS

We thank N. Kallenbach and E. Hamel for helpful discussions.

	FOOTNOTES

^* This work was supported by grants (to N. J. C.) from the National Institutes of Health and the Department of Defense Breast Cancer Research Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Postdoctoral Fellowship from the American Cancer Society.

^§ To whom corresponding should be addressed: Dept. of Biochemistry, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-5138; Fax: 212-263-8166; E-mail: LewisS01@mcrcr.med.nyu.edu.

	ABBREVIATIONS

The abbreviations used are: GAP, GTPase-activating protein; dGTP, deoxy GTP; ddGTP, dideoxy GTP; S-tubulin, subtilisin-truncated tubulin; E-tubulin, chicken erythrocyte tubulin; MES, 4-morpholineethanesulfonic acid; GTPS, guanosine 5'-3-O-(thio)triphosphate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

	REFERENCES

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