Eur. J . Biochem. 128, 215-222 (1982) ( - FEBS 1982

Modulation of Some Parameters of Assembly of Microtubules in vitro by Tyrosinolation of Tubulin

Nirbhay KUMAR and Martin FLAVIN

National Heart, Lung, and Blood Institute, National Institutes of Health. Bethesda, Maryland

(Received May 26/August 2, 1982)

Using tyrosinolated and detyrosinolated tubulins, we have compared several parameters of microtubule assembly in vitro. Rates and extents of polymerization were the same under all conditions, but microtubules assembled from detyrosinolated tubulin in the presence of crude microtubule-associated proteins (MAPs) or subsaturating MAP-2 contained a smaller proportion of the MAPs. Preliminary results indicate that this may be a function of the phosphorylation state of MAP-2. Tyrosinolated tubulin assembled into relatively shorter microtubules in the presence of saturating MAP-2. When assembly was induced with substoichiometric concentrations of taxol, in place of MAPs, the rate and extent of assembly were about twice as great with tyrosinolated tubulin.

Tyrosine can be post-translationally added to [l, 21 and removed from [3,4] the C terminus of the a chain of tubulin. Recently it has also been reported that an a-chain mRNA from both chick [5] and rat [6] brain has a C-terminal tyrosine codon. This elaborate machinery suggests some vital function for the C-terminal tyrosine, but as yet there are few clues.

Studies in vitro indicate that the tubulin-tyrosine ligase adds tyrosine to tubulin subunits, and assembled microtubules do not appear to be a substrate [7, 81. In contrast, microtubules are the preferred substrate for the detyrosinolating carboxypeptidase-tubulin [4]. Although this suggests a possible coupling between cycles of tyrosinolation and assembly, ty- rosine turnover studies in vivo, in the presence of drugs that stabilize or dissolve microtubules, have not so far supported any such direct coupling [9]. { ‘Tyrosinolation’ is the correct biochemical nomenclature for the modification we are studying and refers to the same reaction that has been called ‘tyrosy- latioii’ in earlier publications from our laboratory and ‘tyrosi- nation’ by others; see Nomenclature of a-Amino Acids [Eur. J . Biochem (1975) 53, 1 - 14, Section 1.4.4].}

Until now, no parameter of assembly in vitro has been reported to be affected by the presence or absence of C-terminal tyrosine. Using brain tubulin enzymatically modified so as to be maximally tyrosinolated or detyrosinolated, we have now reinvestigated this question. We have focussed especially on the interaction of tubulin with the assembly-promoting microtubule-associated proteins (MAPs), since tyrosinolation modifies the acidic domain [lo] which is the presumed locus of MAP binding. We now report several subtle but significant differences in the behavior of the two tubulin species.

A preliminary report of this work appeared as an abstract [Kumar, N. and Flavin. M. (1980) J . Ce// B i d 91, 324al.

Abbreviations. Tubulin-3xP or tubulin-4xP, microtubule protein puri- fied by three or four cycles of assembly: tubulin-PC, tubulin purified by phosphocellulose chromatography; prefixes T- or D-, maximally tyrosino- lated or completely detyrosinolated tubulin: MAPs, microtubule- associated proteins; PAGE, polyacrylamide gel electrophoresis: Mes, 4-morpholineethanesulfonic acid; buffer A, reassembly buffer; buffer B. phosphocellulose column buffer.

E i i z y ~ e s . Carboxypeptidase A (EC 3.4.1 7.1); tubulin-tyrosine ligase (EC 6.3.2.-).

MATERIALS AND METHODS Preparation of Maximally Tyrosinoluted arid De tyrosinolated Tubulin

The starting material was microtubule protein purified from sheep or bovine brain by three cycles of assembly, without added glycerol, according to the protocol of Asnes and Wilson [ll]. The reassembly buffer (buffer A) was modified to consist of: 100mMKtMes,pH6.7, 1 mMEGTAand0.5mM MgCI,; it was supplemented with 2.5 mM GTP. After the third warm incubation, assembled microtubules were centrifuged (60 min at 30 “C at 50000 x g) through a cushion of 50 ”/, sucrose in buffer A. The pellets, which will be identified as tubulin-3xP, were frozen immediately by immersing the tubes in liquid N2, and were then stored at - 70 “C.

Tubulin-3xP was tyrosinolated for 40 min at 37 ’C in reaction mixtures containing 0.9 ~ 1.1 unit/ml of partially pu- rified [12] tubulin-tyrosine ligase, tubulin at 5 - 6 mg/ml, and l00mM K’Mes, pH6.7, 2.5mM ATP, 0.3mM GTP, 2mM dithiothreitol, 150mM KC1, 5mM MgSO,, 0.25mM L-

tyrosine. Reactions were stopped by lowering the temperature to 0 ‘ C. After 20 min the mixtures were centrifuged for 30 min at 2 C at 40000 x g. The supernatants were equilibrated into buffer A, containing 0.1 mM GTP, by filtration through 10 bed volumes of Sephadex G-50M. Fractions containing protein, identified by the method of Bradford [13], were pooled and incubated at 32 -C for 30 min in the presence of 2.5 mM GTP. Polymerized microtubules (T-tubulin-4xP) were collected by centrifugation for 60min at 100000 x g at 30-35 C, and the pellets were stored at -70 C.

Detyrosinolation was done by incubating tubulin-3xP, at 8 - 10 mg/ml, in buffer A containing 1 mM GTP and 0.25 pg/ml of pancreatic carboxypeptidase A, for 10min at 37 C. The carboxypeptidase was inactivated by incubating an additional 5 - 10 min at 37 “C in the presence of 20 mM dithiothreitol. D- Tubulin-4xP was then isolated as described above. The pro- cedures are illustrated schematically in Fig. 1.

Chromatography of Microtubule Protein oti Phosphocellulose As will be shown, when tubulin-3xP was tyrosinolated, not

only the tubulin, but also the microtubule-associated protein,

216

TUBULIN-3 x P

TYROSINOLATION / \ DETYROSINOLATION

TUBULIN + Mgz+, KC', CARBOXYPEPTIDASE A

Tyrmine, Ligese. Tubulinl 4 10, 3PC

20 mM DlTHlOTHRElTOL 30. 37'C + 10.3PC 1

TUBES CHILLED

SEPHADEX G-50

I TUBULIN P~LYMERIZED

D-TUBULIN-4 x P

3 PHOSPHOCELLULOS~ CHROMATOGRAPHY

1 p x i G q pTzGE-1

TYPICAL DATA ON TYROSINE CONTENT (%I

NON W X I S T I N G CAN ACCEPT SUBSTRATE

Tubulin 3 x P 14 42 44 T-Tubulin-4 x P 44 12 46 D-Tubulin 4 x P 0 En 41

Fig. 1. Schematic protocol for the preparation of maximally tyrosinolared ( T ) and detyrosinolated ( D ) tubulins. At the bottom are shown typical analytical values for T-tubulin-4xP and D-tubulin-4xP and the tubulin-3xP from which they were prepared

was modified; specifically, MAP-2 was phosphorylated. To compare the assembly parameters of the tubulins, it was necessary to remove the MAP fraction and then supplement both tubulins with the same MAP fraction which had not been subjected to modifying conditions. Phosphocellulose (Whatman P-I 1) was first processed according to the manu- facturer's directions. Microtubule protein (8 - 10 mg/ml) was applied to a column (1 g moist cake/2.5 mg protein; width to height ratio 1 : 6) as described by Williams and Detrich [14]. Tubulin was eluted with buffer B (100mM K'Mes, pH 6.8, 2mM dithiothreitol, 2mM EGTA, 0.5mM MgS04, 0.1 mM GTP) at a rate of 10 ml/cm2 x h. As each fraction was collected, it was immediately supplemented with MgSO, and GTP to give final concentrations of 1 mM and 0.5 mM, respectively. Tubulin was detected, in the unbound fractions, by the protein assay of Bradford [13], and was precipitated from the pooled fractions by adding an equal volume of saturated ammonium sulfate in buffer B. The pellets obtained by centrifugation were dissolved in bufferB, and the solution was filtered through 10 bed volumes of Sephadex G-50M equilibrated with buffer B containing 0.5 mM GTP. Fractions containing tubulin-PC were pooled and stored in small aliquots at - 70 "C.

When unmodified microtubule protein was fractionated on phosphocellulose, crude MAPS fraction was also isolated by eluting the column with buffer B containing 1 M KC1. Protein- containing fractions were concentrated by pressure filtration through an Amicon PM-30 membrane, equilibrated into buffer B by filtration through Sephadex G-50m, and stored at

Protein in the concentrated fractions was determined by the method of Lowry et al. [IS] using bovine serum albumin as a standard.

- 70 "C.

Preparation qf M A P - 2 and Laheled Phosphorylated M A P - 2 MAP-2 was prepared from tubulin-3xP by a heat step

followed by elution from Biogel A 1.5 m, by the procedure of Kim et al. [16] with minor modifications. The final fraction was concentrated by pressure filtration through an Amicon XM- lOOA filter.

[32P]MAP-2 was prepared as described by Sloboda et al. [17] and Vallee [18]. Typically, tubulin-3xP at a concentra- tion of 5 mg/ml was incubated with 50pM [p3'P]ATP (500 Ci/mol) and 50 mM K'Mes, pH 6.7, 10 mM MgSO,, 1 mM EGTA, 1 mM dithiothreitol, 10pM CAMP. Reactions were started by adding the ATP to the prewarmed mixture. After 15min at 30°C the tubes were chilled, solid NaCl was added to a final concentration of 0.75 M, and the tubes were placed in a boiling water bath for 5min. Protein was pre- cipitated from the supernatant obtained after centrifugation at 2°C by adding an equal volume of saturated ammonium sulfate in buffer B. After centrifugation the pellet was dissolved in, and filtered through, Sephadex G-50M with bufferB. Aliquots were stored at - 70 "C. The second purification step [18], elution from BiogelA 1.5 m, was omitted in this case.

Assays f o r Tubulin a n d ~ f o r I t s S ta te of Tyrosinolation

Native tubulin was determined by the colchicine binding assay of Garland and Teller [19]. Approximately 10-pg and 25- pg aliquots of protein were incubated for 90 min at 37 'C in 150 pl of solution containing 10 mM sodium phosphate, pH 6.9,s mM MgSO,, 240 mM sucrose, 0.1 mM GTP, 12 pM [3H]colchicine (100 Ci/mol). A 100-p1 aliquot was transfer- red onto a 0.5-ml bed of DEAE-cellulose (DE-23, in a Bio-Rad polypropylene Econo column 731-1 11 0) pre-equilibrated by washing with 15 ml of 10 mM phosphate/5 mM MgSO, buffer (pH 6.9). Tubulin is retained by this DEAE-cellulose. Columns were washed with 1.5 ml of the same buffer, and the DEAE- cellulose was transferred quantitatively to a scintillation vial for counting in Aquasol (New England Nuclear). Under these conditions native tubulin was found to bind 0.7 mol colchi- cine/mol.

The proportion of tubulin with C-terminal tyrosine, and the additional proportion which could accept tyrosine, were de- termined as described by Nath and Flavin [20]. The proce- dure involves removal of tyrosine by carboxypeptidase A (0.25 pg/rnl) and addition of tyrosine by tubulin-tyrosine ligase [12]. Typical values for unmodified, tyrosinolated and de- tyrosinolated tubulin are shown in Fig. 1, expressed as a percentage of native tubulin (determined as above). The properties of the 'non-substrate' moiety shown in Fig. 1 are described by Nath and Flavin [20].

Miscellaneous Procedures

Microtubule assembly was monitored at 37 'C, using a Cary 16 spectrophotometer. Absorbance was monitored at 350 nm. The temperature in the cuvette holder was maintained using a circulating water bath. Samples were prepared in 300 pi of buffer A (containing 1 mM GTP unless otherwise specified) at 0-4°C in cuvettes with 10-mm light path and 4-mm width (Precision Cells, type 18 M) and polymerization was initiated by placing the chilled cuvettes in the prewarmed cuvctte holder. Time to reach 37 "C in the cuvettes was 50 - 60 s. Absorbance was recorded for up to five samples simultaneously using a Cary recorder.

217

The rate of exchange of tubulin subunits into microtubules at steady state was determined as described by Margolis and Wilson [21]. In this procedure the incorporation of subunits is measured by the fixation of [3H]GTP into pelletable micro- tubules. In most experiments we allowed assembly to continue for 90 min at 3 2 T , to reach a steady state. Mixtures were then transferred to tubes containing [3H]GTP, from which solvent had been evaporated in a stream of nitrogen, and incubations were continued for the indicated times. Termination of the incubations was taken to be the point at which centrifugation was started. Microtubules were pelleted through 50 ”/, sucrose in bufferB, in a Beckman SW50.1 rotor operated at 45000rev./min for 2 h at 30°C. Exchange of [3H]GTP into microtubules was expressed as a percentage of maximum exchange, determined in each experiment by introducing [3H]GTP at the start of the assembly. The data were not adjusted to compensate for possible differences in average microtubule length.

For PAGE analysis samples were prepared and run in slab gels in the sodium dodecyl sulfatelurea system of Eipper [22] as previously described [2]. Slabs were stained by gentle rotary shaking in 0.05 % Coommassie blue in 25 ”/, isopropanol/lO % acetic acid and destained by rotary shaking in several changes of 10 ”/, acetic acid. Photographic negatives of wet slabs were scanned using a Quick Scan R.S.D. densitometer. For auto- radiography of 32P, slabs were dried under vacuum, and exposed to Kodak X-Omat R film.

Muteriuls

Tubulin-tyrosine ligase was prepared as described by Flavin et al. [12]. Carboxypeptidase A was obtained from Worthing- ton; acetate kinase, acetyl phosphate (Li’, K + salt) and cyclic AMP from Sigma. [‘4C]Tyrosine, [3H]colchicine, [3H]GTP and [y-32P]ATP were from Amersham.

RESULTS

Rates and E-xtents of Assembly mid the Proportions of MAPs in Assembled Microtubules

Phosphocellulose-purified, maximally modified tubulins, T-tubulin-PC and D-tubulin-PC, were allowed to assemble to steady state in the presence of MAPs. With this crude MAP fraction it was not possible to saturate tubulin for assembly; even with an MAPs : tubulin weight ratio of 5 : 1, the plateau of turbidity was continuing to increase (Fig. 2). Using four concentrations of MAPs with three different concentrations of each tubulin, no significant difference in the final extents of assembly was detected (Fig. 2). We also found no difference in the rates of assembly, as estimated (Fig. 3, inset) from the steepest portions of the turbidity curves. Fig. 3 shows the data for the highest tubulin concentration of Fig. 2, at four MAP concentrations.

Microtubules were isolated, by centrifugation, from each of the assembly mixtures of Fig. 2 and analyzed by PAGE. The mass ratio of tubulin to high-molecular-weight MAPs (mostly MAP-1 and MAP-2) was determined in each case by den- sitometric scanning. As shown in Table 1, microtubules assembled from tyrosinolated tubulin contained 30 % more MAPs. By Student’s ‘t’ test this difference was significant with p < 0.05. Assuming molecular weights of 110000 and 300000 for tubulin and MAPs, respectively, the molar ratios were 15 tubulins per MAP for T-tubulin and 21 for D-tubulin. As shown in Fig. 2, these values were obtained under conditions where the tubulins were not saturated with respect t o assembly.

0.L [Tubulinl Img/rnll

0 10 2 0 3 0 MAPs hg/rnl l

Fig. 2. Comparison of the maximum extents ofpolymerizarion of T-rubulin- PC and D-tuhulin-PC in the presence of various concentrations of MAPs. The ordinate shows the maximum change in turbidity observed after warming from 0 to 37 -C. Measurements were made at the indicated three different concentrations of both T-tubulin-PC (-), and D-tubulin-PC (-- - -). Polymerization was in butTerA containing 1 mM GTP

MAPs img/rnll

.r” 0.2 30 .--------

_____ --- ---- 2o 0 L 8 12

Time ( m i d

0 0.5 1.0 2.0 2.5 0 MAPs lrng/rnll

Fig. 3. Comparison of the rates of polymerization of T-tubulin-PC (open- bars) and D-tuhulin-PC (dotted bars), each at 1.8mglml, in the presence of various conrentration.v of MAPS. The concentrations of MAPS was the same as in Fig. 2. For purposes of comparison only, rates were estimated by drawing tangents to the steepest portions of the turbidity curves. The inset illustrates the procedure: here MAPs were added at the indicated concentration to unmodified tubulin-PC

Table 1. Proportions of Aigii-molecular-izeig~it MAPs coassemhling with T- tubulin-PC or D-tubulin- PC Microtubules were isolated from the assembly mixtures shown in Fig. 2 by Centrifugation for 30 min at 30- 35 ‘C at 48000 x g in a Sorvall SS-34 rotor using small cellulose butyrate tubes in no. 408 adapters. The pellets were analyzcd by PAGE as described in Materials and Methods. Photographic negatives of the stained slabs were scanned, and the areas of peaks corresponding to tubulin (a + Pchain) and high-M, MAPs (mostly MAP-1 and MAP-2) were determined. Results are expressed as the mean - + standard error of the mean

Tubulin Number of Mass ratio samples analyzed tubulinlhigh-M, MAPs

T-Tubulin-PC 11 5.6 f 0.33

D-Tu bulin-PC 12 7.6 k 0.75

218

- 1 0.2 Ln c a - C

0 . _ c

._ 0.1

- 5 0 a

0

(mg/mll Iu+Pl/MAP-Z

TPC DPC

>0.5 3.4 3.8

I I I

0 2 0.L 0.6 0 8 1.0 MAP-Z[rng/rnll

Fig. 4. Comparison of maximum exients of polymerization o/' T-tubulirz- PC (M) and D-tubulin-PC lo-) in the presence of various roncentrutiuns qfpurifkd MAP-2. Tubulin was 1 mg/ml in buffer A contain- ing 1 mM GTP. After maximum absorbance was attaincd, microtubules were isolated from each mixture and analyzed as described in Tablel. The inset shows the observed proportions of tubulin (a + \I) and MAP-2 in T-tubulin-PC (TPC) and D-tubulin-PC (DPC)

Fig. 4 shows the results of allowing maximally modified tubulins to assemble to steady state in the presence of various concentrations of heat-treated, purified MAP-2. Saturation with respect to assembly was now observed at an MAP-2 concentration of 0.5 mg/ml. The two tubulins assembled to the same extent at all MAP-2 concentrations. Average mass ratios of tubulin to MAP-2, determined by PAGE analysis of microtubules pelleted from all 10 tubulin mixtures, are shown in the inset (Fig. 4). At subsaturating MAP-2 concentrations the molar ratios were 13 tubulins/MAP for T-tubulin and 16 for D-tubulin, indicating again a 20% greater MAP content in microtubules assembled from tyrosinolated tubulin. With saturating MAP-2 the molar ratios were nearly the same and approximated to the value of 9 tubulins/MAP-2 previously reported [16].

Taxol, a neutral plant product, has been shown to promote microtubule assembly [23] yielding a maximum proportion of polymer when present at about equimolar concentration with tubulin-PC [24]. Using a substoichiometric taxol concen- tration, we compared extents and rates of assembly with four different preparations of maximally modified tubulins. In contrast to the results with MAPs or MAP-2, tyrosinolated tubulin clearly assembled better with taxol: the rate was two to three times that observed with detyrosinolated tubulin, in every preparation, and the final extent of assembly was also 50% greater (Table 2). When taxol concentration was increased from 3.4 pM to 9 pM (equimolar with tubulin), the rates and extents of assembly were not significantly different (not shown). Lacking labeled taxol, we were not able to determine whether these results reflected the incorporation of different proportions of taxol in the tubulin polymers.

Incorporation of Phosphorvluted M A P-2 into Microtubules Assembled from Tyrosinolated o r Detyrosinolated Tubulin

MAP-2 is phosphorylated by a CAMP-dependent protein kinase which coassembles irz vitro [17,18]. The phosphorylation alters some parameters of microtubule steady-state dynamics [25,26]. Since this appears likely to be a physiologically

Table 2. Rates untlextents of'ussembly q f T-tubulin-PC and D-tubulin- PC iii the presence of a limiting concentration of taxol Tyrosinolated and detyrosinolated tubulin, prepared from four different batches of microtubule protein, were assernbled at a concentration of 9 pM (1 mg/ml) in bufferA containing 1 mM GTP and 3.4pM taxol, at 37'C. Extent of assembly was determined turbidimetrically from the plateau in absorbance at 350nm, and rates were estimated from tangents to the steepcst portions of the curvcs

Tubulin preparation Extent 10 ~ x Rate of assembly of assembly

1. Tyrosinolated

2. Tyrosinolated

3. Tyrosinolated

4. Tyrosinolated

Detyrosinolated

Detyrosinolated

Detyrosinolated

Detyrosinolated

0.16 0.1 1

0.09 0.03

0.13 0.12

0.09 0.06

~~

AAJS0 s- '. 16.0 5.4

6.4 2.6

4.6 2.8

5.1 2.8

important modification of MAP-2, we have also begun some studies to determine whether there is a differential incorpo- ration of phosphorylated MAP-2 into microtubules assembled from the modified tubulins.

Microtubules were assembled in the presence of trace amounts [32P]MAP-2 serving as a label for the phosphorylated moiety. At both saturating and subsaturating MAP-2 con- centrations, microtubules assembled from T-tubulin-PC or D- tubulin-PC had the same amount of radioactivity. In the experiment of Table 3 microtubules were first assembled to steady state with a saturating concentration of MAP-2, and then incubated a further 60min in the presence of trace amounts of [32P]MAP-2. A large proportion of the added label was incorporated into polymer, and the amount was 30 - 40 7; higher for the detyrosinolated tubulin. Total MAP-2 was found to be preferentially enriched in microtubules assembled from tyrosinolated tubulin (Fig. 4, inset). The above results suggest that this may not be the case for some species of phosphory- lated MAP-2 which, on the contrary, seem to be preferentially incorporated into microtubules assembled from detyrosino- lated tubulin.

The [32P]MAP-2 used in these experiments, which had been purified by the heat step only without subsequent gel filtration (see Materials and Methods), contained smaller amounts of other phosphorylated proteins (Fig. 5, lane 3). However, PAGE analysis of one of the microtubule pellets from Table 3 showed (Fig. 5, lane 5) that MAP-2 was the only labeled protein that coassembled in significant amounts.

MAP-2 I s also Modtfied when Tubulin Is Enzymatically Tyrosinolated

All of the preceding experiments (except the last mentioned) were done with mixtures reconstitutcd from T-tubulin-PC or D-tubulin-PC, and MAPs or MAP-2 which had not been exposed to the tubulin-modifying conditions. We had found that detyrosinolated tubulin-3xP assembled well in a fourth cycle, but tyrosinolated tubulin-3xP assembled less extensively. In the former case (Fig. 6, lanes 5 and 6), all the high-molecular- weight MAPs were in pelleted microtubules, but in the latter

219

case substantial amounts of MAPS remained in the supernatant 2 was 1.7-times higher in the supernatant. This result is also (lanes A, 3-4). Presumably the MAPS with the tyrosinolated consistent with that of Table 3 in suggesting that phosphory- tubulin had been damaged during incubation with the lated MAP-2 may be selectively excluded from microtubules relatively crude tubulin-tyrosine ligase fraction. In addition, assembled from tyrosinolated tubulin. MAP-2 was phosphorylated while tubulin was being tyrosino- lated. ATP is required for the latter reaction, and when this was carried out with [Y-~~PIATP, autoradiography of slab gels (Fig. 6B, 1 -4) showed that MAP-2 was labeled. The labeled MAP-2 was enriched in the non-assembling supernatant. Densitometric scans of stained slab and autoradiogram showed that the ratio of [32P]MAP-2 to tubulin was 3.2-times higher in supernatant than pellet, and the specific radioactivity of MAP-

1 2 3 4 5

Table 3. Incorporation off "P]MAP-2 into microtubules afer assembly to steud.v state from unlabeled MA P-2 and T-tubulin-PC or D-tubulin-PC Microtubules were assembled for 60 min at 37 "C in 100 p1 of buffer A containing 0.1 pmol of GTP, 80 pg of unlabeled MAP-2 and 90 pg of the respective tubulin. Incubations were continued an additional 60 min after adding the indicated amounts of [32P]MAP-2. Assembled microtubules were separated from unassembled protein, and total radioactivity and protein were determined as described in Materials and Methods. Since the amount of protein in the pellet was the same in both cases. the results are expressed as total 32P in the pellet

Amount of [32P]MAP-2 added after reaching incorporated into steady state preassembled microbutules

protein T-tubulin-PC D-tubulin-PC

Amount of [32P]MAP-2

- __ -

clg counts/min

5 10000 4 500 6 500

10 20 000 6 600 11 000

Fig. 5. Components in f 32P]MAP-2 before and after its incorporation into microtubules. Lanes 1,2 and 4 are photographs of the stained PAGE slab; lanes 3 and 5 are the autoradiograms of 2 and 4, respectively. Microtubule assembly was in buffer A, containing 1 mg/ml of T-iubulin-PC, 1.04mg/ml of MAP-2, and a trace of [32P]MAP-2 (22000counts/min = 5 pg protein). Lane1 shows the unlabeled MAP-2 which had been purified by gel filtration, after the heat step. The ["P]MAP-2 which had not been gel filtered contained many trace contaminants (lane 3); however, only MAP-2 was found after microtubule assembly (lane 5)

1 2 3 4 1 2 3 4 5 6

Fig. 6. MA P-2 isphosphorylatedduring tubulin tyrosinolution, undsxbseguent assembly is limited. Tubulin-3xP was tyrosinolated by the standard procedure, except that tracer amounts of [y-"PIATP were present during the incubation with ligase. The microtubules were centrifuged down and aliquots (20 pg protein for wells 1 and 3,40 pg for 2 and 4) ofpellets (lanes 1 and 2) and supernatants (lanes 3 and 4) were analyzed by SDS/PAGE. (A) The stained slab; (B) (1 -4) the corresponding autoradiogram; lanes 5 and 6 received 5-pg aliquots of pellet and supernatant, respectively, obtained when microtubules were assembled after the standard procedure for detyrosinolation

220

I I I

A

t i

0 30 60 90 Time (m id

Fig. 7. Exchange rates of subunits into microtuhules at steady state, after various mod$cations of the microtubule protein. (A) Rates observed with unmodified tubulin-3xP (M), with D-tubulin-4xP (m- 4) and T- tubulin-4xP (A-A). In the latter two cases microtubules were assem- bled after the modification procedure, disassembled, and reassembled to steady state before adding the [3H]GTP, as described in Materials and Methods. (B) Rates observed when tubulin-3xP was preincubated under phosphorylation conditions (protein at 4.8 mg/ml incubated for 20 min at 37 C in the presence of 10 mM MgS04, 10 pM CAMP. and 0.25 mM ATP) and the put through one assembly cycle as in A (M): rates observed when tubulin-3xP was incubated under the conditions for maximal tyrosinolation, but omitting tyrosine, and then put through one assembly Cycle (A-A)

Fig. 8. A representative micrograph of cross-sectioned microtubules used 10 mea.sure the number.c. c~fpro/ofilameni.rshou~~~ in Table 4. Microtubule pellets were processed for electron microscopy as described by Kim et al. [16], except that embedding was in Epon, staining was with 2 ”/, uranyl acetate in 25 methanol, and thin sections were examined in a Siemens Elmiskop 101 microscope. Magnification: field, x 220000; inset, x 580000

Fig. 7A shows an apparent effect of tyrosinolation on the kinetics of treadmilling, which actually was also caused by phosphorylation. The effect was duplicated when tubulin-3xP was incubated either with ligase + ATP but without tyrosine, or under conditions for autophosphorylation of MAP-2 (Fig. 7B). Using microtubule protein reconstituted from T- tubulin-PC or D-tubulin-PC and unmodified MAP-2, we have so far found no significant difference in treadmilling rates, but the apparent rates have varied with different batches of tubulin.

Protofilament Number and Length Distribution ?f Microtubules Formed from Tyrosinolated or Detyrosinoluted Tubulin

Unmodified tubulin-PC, D-tubulin-PC and T-tubulin-PC were separately allowed to assemble in the presence of a saturating concentration of MAP-2. The microtubule pellets were processed for electron microscopy by the tannic acid procedure, to visualize protofilaments. A typical field of microtubules seen in transverse section is shown in Fig. 8. The mean number of protofilaments per microtubule was exactly the same for all the types of tubulin (Table 4).

Length distributions of microtubules formed from the same three types of tubulin are shown in Fig. 9. Table 5 shows the mean values, along with relevant parameters of the tubulin samples. A non-parametric test (Kruskal-Wallis) was used to determine whether the mean lengths were significantly dif- ferent. Microtubules with only one end on a grid square were assumed, on the average, to have an equal length out of the square, and the observed lengths were therefore doubled. The small numbers of microtubules with neither end on a grid square (Table 5) were not included. The probability of any two samples being equal was calculated to be 0.008 for B and C , 0.045 for A and C , and 0.36 for A and B. Microtubules

Table 4. Number ofprorofIaments it1 micro tubules assembled fiom lyrosino- luted and detyrosinolated tuhulin

Number Frequency in microtubules assembled of protofilaments ~

in the presence of saturating MAP-2 and

unmodified T-tubulin-PC D-tubulin-PC tubulin-PC

~ .-

11 1 12 38 9 11 13 29 8 6 14 5 2 3 15 I

Total 78 19 20

Mean protofilamen t number 12.6 12.6 12.6

assembled from tyrosinolated tubulin were therefore signif- icantly shorter than those from detyrosinolated tubulin. The mean length for unmodified tubulin, with an intermediate proportion of C-terminal tyrosine (Table 5 ) , was also in- termediate between those of the other two samples.

DISCUSSION

Several parameters of assembly in vitro have been found to be modified by the presence of C-terminal tyrosine in the a chain of tubulin. The most striking difference was the greater rate and extent of polymerization of tyrosinolated tubulin when assembly was induced by taxol. Until more is known about the binding site for taxol, little can be inferred about the

22 1

Average Length = 6 . M p n r Average Length = 4.72 prn .

n

MICROTUBULE LENGTH Ipm)

Fig. 9. Computer-generated histograms showing microtubule length rlistrihutioris. Microtubules were assembled to steady state, in the presence of a saturating concentration of MAP-2, from: (A) unmodified tubulin-PC; (B) D-tubulin-PC; (C) T-tubulin-PC. Aliquots were applied to carbon over formvar-coated 200-mesh copper grids, and negatively stained as previously described [4]. Lengths were measured by using a Summagraphics 1. D. digitizer

Table 5. Mean 1engih.s of microtubules assembled,fi.om T-tubulin- PC or D-tubulin- PC The tubulin samples used were those shown in Fig. 9

Tubulin Colchicine r: Chains with Number of microtubules measured Mean Variance sample binding

capacity preexisting ability to both ends one end neimer end - -- length ___.- ___

TY r accept further on grid on grid on grid TY r square square squarc

nmoljl10 pg :; pm pmz - - ~~ protein

A. Tubulin-PC 0.55 21 45 90 48 4 5.80 27.1

B. D-Tubulin-PC 0.52 6 42 83 54 6 6.54 29.7

C. T-Tubulin-PC 0.48 42 4 131 71 4 4.72 12.2

molecular basis for this effect. As a neutral compound which does not compete with MAP-2 [24], taxol is not an obvious candidate to bind to the acidic C termini of tubulin.

MAPs, the brain proteins which may promote assembly physiologically, are likely candidates to bind to the tubulin C termini [lo]. We could not detect any effect of tyrosinolation on either the rate or extent of assembly induced by crude MAPs or purified MAP-2. The composition of the resultant micro- tubules differed, however; those formed from tyrosinolated tubulin contained a 20 - 30 "/, higher proportion of MAPs, or

All these effects were observed only when the taxol or MAPs were present at subsaturating concentrations that allowed less than maximal assembly.

The evidence is more tentative that phosphorylated MAP-2 is selectively excluded from microtubules assembled from tyrosinolated tubulin, and conversely that the relative en- richment of MAP-2 in these microtubules, noted above, may be in the non-phosphorylated moiety. Ideally, these experiments should have utilized MAP-2 having different known phosphate contents. We used tracer amounts of ["P]MAP-2 and assumed that the bulk MAP-2 consisted of a mixture of both species. Available evidence indicates that this is the case and that MAP-2, which can contain a maximum of about 12 mol phosphate/mol, has 6 - 9 mol/mol as it is isolated [27] (and R. Vallee, personal communication).

The average number of protofilaments were the same in microtubules assembled, with saturating MAP-2, from either type of tubulin. Under these conditions the MAP-2 content was

MAP-2.

also the same. Microtubules assembled from tyrosinolated tubulin were found to be significantly shorter, suggesting that tyrosinolated tubulin may be more effective in nucleation and/or less so in elongation. Experiments are in progress to compare the elongation of seeds (sheared microtubules) by subunits, with tyrosinolated tubulin confined either to the seeds or the subunits.

The results reported here do not define the functions of tubulin tyrosinolation in vivo. But such marginal effects as the moderate preferences for incorporation of MAPs, manifested only when the latter are at suboptimal concentration for assembly, might have important consequences in a delicately balanced cell milieu.

We thank Terry Jones for performing the electron microscopy, and Marvin Shapiro, of the Division of Computer Research at the National Institutes of Health, for the statistical analysis of microtubule lengths.

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Kumar, Chemical Immunology Section, National Institutes of Arthritis, Digestive Diseases and Kidney, National Institutes of Health, Building 10, Room 9N204, 9000 Rockville Pike, Bethesda, Maryland, USA 20205

M. Flavin, Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Building 3, Room B1-22, 9000 Rockville Pike, Bethesda, Maryland, USA 20205