Complete Amino Acid Sequence of A-Tubulin from Porcine Brain (Sequence Microheterogeneity/Homology with Muscle Proteins) H

Proc. Nati. Acad. Sci. USA Vol. 78, No. 5, pp. 2757-2761, May 1981 Biochemistry

Complete amino acid sequence of a-tubulin from porcine brain (sequence microheterogeneity/homology with muscle proteins) H. PONSTINGL, E. KRAUHS, M. LITTLE, AND T. KEMPF Institute of Cell and Tumor Biology, German Cancer Research Center, D-6900 Heidelberg, Germany Communicated by Hans Neurath, January 26, 1981

ABSTRACT The amino acid sequence of a-tubulin from por- lulose with a linear gradient of 0.1-0.3 M sodium chloride. cine brain was determined by automated and manual Edman deg- Tubulin was identified by the fluorescence of its complex with radation of eight sets of overlapping peptides. It comprises 450 colchicine (5). The preparation was reduced, alkylated with io- residues plus a COOH-terminal tyrosine that is present only in doacetic acid, and assayed for protein impurities by disc gel 15% of the material. A region of 40 residues at the COOH-ter- electrophoresis in the system ofYang and Criddle (6) using 8% minus is highly acidic, mainly due to 16 glutamyl residues. This polyacrylamide gels. The gels were stained with Coomassie blue high concentration ofnegative charge suggests a region for binding and scanned in a Vernon scanner. Only tubulin of more than cations. At least six positions, most of them around position 270, 95% purity was processed further. are occupied by two amino acid residues each. Several of these For separation of a- and f-chains, the protein was chroma- exchange sites were assigned to specific peptides by analysis ofthe purified corresponding fragments. These data indicate four a-tu- tographed on hydroxyapatite in 0.1% NaDodSO4with a linear bulins in porcine brain. Although a-tubulin on the whole is un- gradient of 0.2-0.4 M sodium phosphate (7). Fractions were related to other proteins, there are regions that can be correlated assayed for purity by gel electrophoresis as above. Only a-chain to sequences of the myosin head, to actin, to tropomyosin, and to of at least 95% purity was used for sequence determination as troponins C and T. described (7, 8). To remove NaDodSO4 the protein was extensively dialyzed Tubulins occur in all eukaryotic cells as the constituents of mi- against 1 mM ammonium bicarbonate; the solution was then crotubules, which participate in cell division, intracellular concentrated by vacuum evaporation, brought to pH 5.5 with transport and secretion processes, ciliary and flagellar move- acetic acid, and treated with 9 vol of ice-cold acetone. The su- ment, morphogenesis, and cell orientation. Tubulins from pernatantwas discarded after 2 hr at -20TC, and the precipitate widely differing species and cell types appear to be remarkably was dissolved in dilute ammonium hydroxide and dialyzed similar regarding composition, molecular weight, binding of against 0.01 M ammonium bicarbonate for enzymatic digestion, cytostatic and psychopharmacological drugs, immunological which, in all cases, was done at pH 8.0 with 1-4 mg a-tubulin crossreactivity, and capacity to copolymerize. Yet even within per ml and, usually, an enzyme/substrate ratio of1:100 at 370C. one cell, there are several types of microtubules that have dif- a-Tubulin (50-100 mg) was digested with either thrombin fering stabilities and assemble into distinct organelles at various (Sigma); affinity-purified trypsin (a gift from K.-D. Jany, Stutt- times. Knowledge of the primary structure should clarify gart) (9); chymotrypsin (Merck); or protease from Staphylococ- whether there is just one tubulin for all functions or whether cus aureus (Miles) (EC 3.4.21.19), from Astacus leptodactylus there exists a family of similar proteins. It will also facilitate Esch. (EC 3.4.99.6), donated by R. Zwilling (Heidelberg) (10), mapping ofbinding sites for various ligands, production of an- from Pseudomonasfragi (EC 3.4.24) (a gift from G. Drapeau, tibodies to well-defined antigenic sites, matching of protein Montreal) (11), or from mouse submaxillary glands (EC 3.4.21) structure with that of messengers and genes, and investigation (Boehringer Mannheim). Cleavage times and exceptions from of functionally defective tubulin mutants. Comparison of the the general schedule were chymotrypsin, 3 hr; trypsin, 7 hr; structure with those of known proteins may give hints for ex- thrombin, 7 hr; submaxillary protease, 24 hr; staphylococcal periments regarding tubulin function. protease/0.2 M ammonium bicarbonate at an enzyme/sub- Tubulin in solution is assumed to exist as a heterodimer of strate ratio of 1:50, 24 hr; Astacus protease/0. 1 M ammonium two chains, a and f, each with a molecular weight4of 50,000, bicarbonate at 20'C and an enzyme/substrate ratio of 1:50, 2 and very similar amino acid compositions. Yet functional dif- hr; protease ofa Pseudomonasfragi mutant/0.01 M ammonium ferences have been reported. For example, only a-tubulin bicarbonate/2 M urea, 24 hr. For cleavage with cyanogen bro- (from blood platelets) binds cyclic AMP (1) and only /3-tubulin mide (Serva, Heidelberg) the acetone precipitate was evapo- binds exchangeable GTP (2). Here we present the sequence of rated under reduced pressure, and the residue was dissolved the a-chain from porcine brain and report on the general strat- in pure formic acid, diluted to 70%, and cleaved with a 150-fold egy used. excess of CNBr over methionyl residues for 24 hr in the dark. The product was lyophilized. The digests were fractionated on Sephadex G-50 and G-100 MATERIALS AND METHODS in 8 M urea/0. 1 M ammonium bicarbonate, and the fractions We have purified tubulin from porcine brain by a modification were desalted on Sephadex G-10. Peptides were further sep- of the methods used by Eipper (3) and by Luduena et al. (4). arated by chromatography on DEAE-cellulose, Dowex 1 x 2 The 100,000 X g brain supernatant in 0.05 M sodium pyro- and 50 x 2, cellulose thin layers, and, more recently, by re- phosphate buffer (pH 7.0) was incubated with 0.1 mM colchi- versed-phase high-pressure liquid chromatography with a Du cine for 15 min at 37°C before chromatography on DEAE-cel- Pont 850 liquid chromatograph on a Zorbax C-8 column, using 0.05 M ammonium bicarbonate brought to pH 7.5 with acetic The publication costs ofthis article were defrayed in part by page charge acid and 0-60% acetonitrile gradients at 400C. payment. This article must therefore be hereby marked "advertise- Amino acid analyses were performed on a Durrum D-500 ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. analyzer. Automated Edman degradations used the Beckman 2757 Downloaded by guest on September 23, 2021 2758 Biochemistry: Ponstingl et al. Proc. Natl. Acad. Sci. USA 78 (1981) 890 C sequencer with 0. 1 M quadrol as buffer and a single cleav- in all peptide separations. This limited the types of separation age program adapted from Brauer et al. (12). To reduce peptide methods that could be used, resulted in loss of insoluble and losses by extraction, 3 mg of Polybrene and 200 jig of glycyl- small peptides on desalting by gel filtration, and led to partial glycine were applied to the cup and subjected to three cycles blockage of a- and E-amino groups by cyanate from decompos- of degradation prior to analyzing the sample (13). Phenylthio- ing urea, producing heterogeneous fragments in low yields in hydantoin derivatives of amino acids were identified by high- any further digestion or purification. Therefore, we abandoned pressure liquid chromatography (14) and, in some cases, by ad- subdigestions ofpeptides and chose to work with a larger num- ditional thin-layer chromatography (15); both methods also ber of overlapping primary fragments. A summary of the frag- served for the assignment of amides. Manual Edman degra- ments generated for sequence analysis is given in Fig. 2. dation plus dansylation was performed as described (16). A striking feature of the sequence is the COOH-terminal region, which we already have discussed in detail (17). The last RESULTS AND DISCUSSION 66 residues are entirely devoid ofasparagine, glutamine, thre- onine, cysteine, proline, and isoleucine, and the last 40 posi- The sequence of the 450 amino acid residues of porcine brain tions have 47% acidic side chains, 16 glutamic and three as- a-tubulin (Mr =50,000, depending on the variant), is given in partic, rendering this segment one of the most acidic known. Fig. 1. It is consistent with the amino acid composition and was Its high content of glutamyl residues suggests that it may be established from the eight sets of peptides generated by cyan- responsible for binding cations, for instance Ca2 , or for the ogen bromide, trypsin, chymotrypsin, staphylococcal protease, basic microtubule-associated proteins, which play antagonistic the less-frequently used thrombin and mouse submaxillaris pro- roles in microtubule assembly in vitro (for review, see ref. 18). tease, and by two enzymes that may not have been used before This part is predicted to have a helical structure, quite different in sequence studies, one recently isolated from a mutant of from the rest of the chain. Pseudomonasfragi, which specifically cleaves at the NH2-ter- In agreement with x-ray data, no indications were found for minal side ofaspartyl groups, and the other a protease from the a sterical organization in domains-e.g., there are no major se- digestive tract of the crayfish Astacus leptodactylus Esch., quence repeats and the 12 cysteines are spaced unevenly, four cleaving preferentially at the NH2-terminal side ofalanine, gly- cysteinyl together with two methionyl residues forming a prom- cine, threonine, and serine. inent "sulfur" cluster at residues 295-316. Some other amino Tubulin peptides strongly aggregate in Solution, as does the acids also show a highly asymmetric distribution: although po- parent molecule; hence it was necessary to include 8 M urea sitions 55-135 and 288-378 are devoid of serine with the ex- 25 MET-ARG-GLU-CYS-ILE-SER-ILE-HIS-VAL-GLY-GLN-ALA-GLY-VAL-GLN-ILE-GLY-ASN-ALA-CYS-TRP-GLU-LEU-TYR-CYS- 50 LEU-GLU-HIS-GLY-ILE-GLN-PRO-ASP-GLY-GLN-MET-PRO-SER-ASP-LYS-THR-ILE-GLY-GLY-GLY-ASP-ASP-SER-PHE-ASN- 75 THR-PHE-PHE-SER-GLU-THR-GLY-ALA-GLY-LYS-HIS-VAL-PRO-AXG-ALA-VAL-PHE-VAL-ASP-LEU-GLU-PRO-THR-VAL-ILE- 100 ASP-GLU-VAL-ARG-THR-GLY-THR-TYR-ARG-GLN-LEU-PHE-HIS-PRO-GLU-GLN-LEU-ILE-THR-GLY-LYS-GLU-ASP-ALA-ALA- 125 ASN-ASN-TYR-ALA-ARG-GLY-HIS-TYR-THR-ILE-GLY-LYS-GLU-ILE-ILE-ASP-LEU-VAL-LEU-ASP-ARG-ILE-ARG-LYS-LEU- 150 ALA-ASP-GLN-CYS-THR-GLY-LEU-GLN-GLY-PHE-SER-VAL-PHE-HIS-SER-PHE-GLY-GLY-GLY-THR-GLY-SER-GLY-PHE-THR- 175 SER-LEU-LEU-MET-GLU-ARG-LEU-SER-VAL-ASP-TYR-GLY-LYS-LYS-SER-LYS-LEU-GLU-PHE-SER-ILE-TYR-PRO-ALA-PRO- 200 GLN-VAL-SER-THR-ALA-VAL-VAL-GLU-PRO-TYR-ASN-SER-ILE-LEU-THR-THR-HIS-THR-THR-LEU-GLU-HIS-SER-ASP-CYS- 225 ALA-PHE-MET-VAL-ASP-ASN-GLU-ALA-ILE-TYR-ASP-ILE-CYS-ARG-ARG-ASN-LEU-ASP-ILE-GLU-ARG-PRO-THR-TYR-THR- 250 ASN-LEU-ASN-ARG-LEU-ILE-GLY-GLN-ILE-VAL-SER-SER-ILE-THR-ALA-SER-LEU-ARG-PHE-ASP-GLY-ALA-LEU-ASN-VAL- ILE HIS TH'Y-L- 275 ASP-LEU-THR-GLU-PHE-GLN-THR-ASN-LEU-VAL-PRO-TYR-PRO-ARG-ALA- IEILEPHE-PRO-LEU-ALA-~~ARGHR-TYRPHE ALA.PRO-VAL-ASX GLY ~~~~~~~~~300 ILE-SER-ALA-GLU-LYS-ALA-TYR-HIS-GLU-GLN-LEU-SER-VAL-ALA-GLU-ILE-THR-ASN-ALA-CYS-PHE-GLU-PRO-ALA-ASN- 325 GLN-MET-VAL-LYS-CYS-ASP-PRO-ARG-HIS-GLY-LYS-TYR-MET-ALA-CYS-CYS-LEU-LEU-TYR-ARG-GLY-ASP-VAL-VAL-PRO- 350 LYS-ASP-VAL-ASN-ALA-ALA-ILE-ALA-THR-ILE-LYS-THR-LYS-ARG- ILE-GLN-PHE-VAL-ASP-TRP-CYS-PRO-THR-GLY- SER 375 PHE-LYS-VAL-GLY-ILE-ASN-TYR-GLU-PRO-PRO-THR-VAL-VAL-PRO-GLY-GLY-ASP-LEU-ALA-LYS-VAL-GLN-ARG-ALA-VAL- 400 CYS-MET-LEU-SER-ASN-THR-THR-ALA-ILE-ALA-GLU-ALA-TRP-ALA-ARG-LEU-ASP-HIS-LYS-PHE-ASP-LEU-MET-TYR-ALA- 425 LYS-ARG-ALA-PHE-VAL-HIS-TRP-TYR-VAL-GLY-GLU-GLY-MET-GLU-GLU-GLY-GLU-PHE-SER-GLU-ALA-ARG-GLU-ASP-MET- 450 ALA-ALA-LEU-GLU-LYS-ASP-TYR-GLU-GLU-VAL-GLY-VAL-ASP-SER-VAL-GLU-GLY-GLU-GLY-GLU-GLU-GLU-GLY-GLU-GLU-(TYR) FIG. 1. Amino acid sequence of a-tubulin from porcine brain. Positions 265, 266, 271-273, and 340 are heterogeneous. The COOH-terminal tyrosine is present in only 15% of the material. Downloaded by guest on September 23, 2021 Biochemistry: Ponstingl et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2759

RESIDUE NUMBER 100 200 300 400 0 W OR ROMP mmff-_. 0 0 T a rl- rg ra 0m 0m m m B E iffyOMMMM/ FMS //F/ //g N// V L- I u Vi/// m~ em 6

S IILz D EI::] V/v ml I I El EM 0 gm 0 M E:::] Q 0 CH V/Z//- M-/l EJ 0 % rg mm, 0IE --I A E TO- F//7//7///7//- D_ ,,,,,,, I n Th. F V~~~ ~ ~ ~~~~~If ~ ~ ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~VZX^, ] l u MI E/7/Y1//1, INTACT CHAIN

FIG. 2. Summary of fragments generated for the sequence analysis of a-tubulin. The hatched section of each bar indicates the portion of the sequence determined. Peptides were generated by trypsin (T); cyanogen bromide (B); protease from Staphylococcus aureus V8 (V), from mouse sub- maxillary glands (S), chymotrypsin (CH), protease from Astacus (A), thrombin (TO), and a protease from a mutant ofPseudomonas fragi (F).

ception of a single residue in a variant, 10 serine residues are mixture of two amino acid residues in a given position. How- rather regularly spaced between positions 136 and 198. Posi- ever, position 265 appears to have three-isoleucine, glycine, tions 43-45 and 142-148 carry clusters of glycines, suggesting and alanine-in four different linkage groups. Hence at least that these areas may be flexible regions, while positions 163-231 four different a-chains may be present in our preparation. and 247-309 are devoid ofthis frequent amino acid in tubulin, Most of these exchanges can be explained by a single base whose abundance may be responsible for the low amount of substitution in the codon except that the isoleucine to glycine, secondary structure. and isoleucine to alanine at 265 and isoleucine to histidine at Although tubulin has been reported to be present in or 266 each require two base changes. closely associated with membranes (19, 20), there are no regions Although this heterogeneity might be due to alleles, it may of the sequence that are predominantly hydrophobic. Also tu- also reflect the presence of different tubulins in different cell bulin, its isolated a-chain, and most of the peptides obtained types ofthe brain-e.g., nerve and glia cells. An organ-specific from digests were fairly soluble in aqueous solution at pH =7.5. /3-tubulin has already been described in Drosophila (22). Al- Thus, the tendency for tubulin and its fragments to aggregate ternatively, more than one a-tubulin may be required even and the interaction of tubulin with membranes may be due to within one cell. ionic forces. Secondary Structure Prediction. We have tried to predict One possible way ofregulating tubulin assembly is posttrans- the secondary structure of a-tubulin according to Chou and lational modification of side chains. So far, however, we have Fasman (23). a-Tubulin appears to be rather irregularly folded: not detected any modified amino acids. An additional COOH- only 26% of the chain is predicted to be helical and 33% is pre- terminal tyrosine is present in 15% of our material (17) and, dicted to have a ,3-sheet conformation, which is similar to results recently, a ligase has been isolated from porcine brain (21), ofearlier circular dichroism studies with native a- and ,3-tubulin which specifically adds this residue to the COOH-terminal from pig (22% a, 30% /3) (24) and calfbrain (26% a, 47% ,3) (25). glutamate. All major helix potentials reside in the COOH-terminal half There is no evidence for a carbohydrate moiety, nor for y- of the chain around residues 275-291 and the three helices at carboxyglutamic acid. Also, not having any radioactive label in the COOH-terminus already reported in an earlier paper (17), our material, we did not detect any phosphorylated residues. residues 383-403, 413-435, and 440-450. Major /8-sheet re- Microheterogeneity. The establishment ofthe sequence was gions are expected at positions 49-94 (five strands), 169-195 impeded by microheterogeneity in several positions. Although (three strands), and 223-239 and 340-378 (four strands). A series the electrophoretic homogeneity of the starting material made of overlapping turns are predicted at positions 31-49 and the presence ofimpurities unlikely, it was nevertheless possible 139-149. that the preparation contained similar peptides derived from In these regions, there is only one position with well-docu- different regions of the protein or that incomplete degradation had resulted in the presence of more than one residue in a po- 270 sition. The first possibility could be excluded by extensive over- ILE-HIS-PHE-PRO-LEU-ALA-THR-TYR-ALA lapping and the second by separating variant peptides by high- pressure liquid chromatography and analyzing the homogene- GLY-HIS-PHE-PRO-LEU-ALA-THR-TYR ous fractions. A total of at least six positions carry amino acid exchanges (Fig. 3) and most of them are concentrated in a "hot GLY-ILE-PHE-PRO-LEU-ALA-ARG-PHE-ASX spot" around position 270. Analyses of homogeneous fractions of these variant allowed identification peptides unambiguous X -PHE-ASX ofthe residues at several exchange sites. Other peptides in the ALA-HIS-PHE-PRO-LEU-ALA- same area and around residue 160, however, were found to yield FIG. 3. Assignment of sequence variants around position 270. Sep- heterogeneous degradation products at one position. Discus- arate peptide fractions were degraded and yielded homogeneous sion of them is omitted from this paper. As a rule, we found a sequences. Downloaded by guest on September 23, 2021 2760 Biochemistry: Ponstingl et al. Proc. Natl. Acad. Sci - USA 78 (1981) mented microheterogeneity-the threonine to serine exchange edge of a structure alone does not explain function, it forms a at position 340-presumably at the beginning of a strand of f3- basis on which to tackle functional problems, and comparison sheet that is not likely to be greatly influenced by this substi- of protein structures may suggest further experiments. tution. The other amino acid substitutions are located in areas a-Tubulin on the whole is unrelated to any other known pro- with less clearcut structural potentials; thus, their effect on the tein, but some parts of the sequence appear to be variations of respective structures would be difficult to predict. known motifs. Because several regions of a-tubulin resemble The known lability of the tubulin molecule, as measured by areas ofvarious proteins, we can not assume a genetic relation- its capacity to polymerize and to bind colchicine, may be ex- ship. More likely, the similarities indicate that a given func- plained by its low level of secondary structures, as predicted tion-for example, binding and hydrolysis ofa nucleotide-can by this model. be performed by a limited number ofsimilar structures. Below Tubulin Peptides from Other Sources. Some sequence in- we give a few examples (Fig. 4). formation for tubulin peptides from other sources has been re- Four regions of a-tubulin are similar to actin sequences (28) ported: Comparison with previous data on the NH2-terminal and, with one exception, they are in the same order in both 25 residues ofchicken brain a-tubulin (26) shows six differences. proteins. Between 32% and 70% ofthe residues in these regions Residues 10, 13, and 17 have been identified as threonine in are identical, comparable with the similarity of a- and 13-tu- chicken brain a-tubulin and are glycine in the porcine protein. bulin. The first of these segments includes a thrombic cleavage Cysteine residues are present in porcine a-tubulin at positions site in a-tubulin and in actin. 4, 20, and 25 whereas, in the chicken protein, the residues at A particularly interesting relationship exists between a-tu- these positions have been tentatively identified as serine. Res- bulin positions 192-238 and a fragment from the globular head idues 22-36, 303-313, 389-398, and 414-425 correspond to ofmyosin (29). This segment ofthe myosin heavy chain includes unlocated fragments and residue 426-450 corresponds to the two cysteines, whose alkylation modifies the ATPase activity of COOH-terminal cyanogen bromide peptide isolated from calf myosin (31). The head ofmyosin can form acrossbridge between brain and sequenced by Lu and Elzinga (27). However, two the thick and thin filaments by attaching to an actin molecule. other peptides designated a by these authors have no counter- The reaction between actin and myosin is cyclical, and each part in our a-sequence but resemble porcine /3-tubulin (un- cycle includes the hydrolysis ofone molecule ofATP. Residues published observations). 1-46 of this fragment appear to be similar to positions 192-238 Homology to Other Proteins. Several conflicting hypotheses in a-tubulin. In particular, the cysteine SH(1), which can be have been advanced to explain microtubule function in intra- alkylated in the absence of bound nucleotides with the result cellular transport. It has been suggested that microtubules are that the Ca2+-ATPase activity is stimulated, resembles the cys- passive skeletons or pointers for directional movement, provide teine-213, and the SH(2), which can be alkylated in the presence scaffolds to which force-generating molecules are attached, or of ADP, occupies a position comparable with cysteine-200 in even actively function as motors ofmovement. Although knowl- a-tubulin. Myosin alkylated at both sulfhydryl groups is devoid a 57- 68 G A G KH V P R AV F V ACTIN 21- 32 F A G D D A P R A V F P a 95-127 G K E D A 'A N Y A R G H Y[T I G K E I I D L V L D RIR K L A D ACTIN 289-321 R K D L Y A N N VM S G G T T M Y P G T A D R M Q K E I T A L A P a 239-254 T HS L F D IG A L N V D L T E ACTIN 173-187 H A I ML L L A G R - D L T D a 299-312 A Q iM V K C DlP R H GHKY ACTIN 278-291 Y N S I M KC D I D I R K D

a 191-215 T H T T L E H S - D[ A F M V D Nf A I Y D I C R R MYOSIN 1- 23 E H E L V L H Q L RU- - N G V LWE GW- RLI CRL K HEAD a 216-239 N L D I E R P T T N L-N[ L I G N I V S SHT MYOSIN 24- 47 G F P - S[R I LIJ A D F K Q Y K V L N A S A II P HEAD a 430-448 K Y|E E VIG VD S VGEG E E E|G

TNT 1- 16 s - |E E V - - E HV E E E AIE E EIA

FIG. 4. Homology of a-tubulin with actin (28), a fragment ofthe myosin head (29), and troponin T (30) from rabbit skeletal muscle. A, Ala; B, Asx; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr; Z, Glx. Downloaded by guest on September 23, 2021 Biochemistry: Ponstingl et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2761 ofATPase activity. Although direct participation of this region 6. Yang, S. & Criddle, R. S. (1970) Biochemistry 9, 3063-3072. in actin or nucleotide binding has not been proven, the evidence 7. Little, M. (1979) FEBS Lett. 108, 283-286. suggests that they are at or near the catalytic site for myosin 8. Ponstingl, H., Krauhs, E., Little, M., Kempf, T. & Hofer-War- binek, R. (1980) in Methods in Peptide and Protein Sequence ATPase. No secondary structure could be predicted for the re- Analysis, ed. Birr, C. (Elsevier/North-Holland, Amsterdam), gion between SH(2) and SH(1), which is also the case for the pp. 225-234. corresponding tubulin sequence. Sulfhydryls are essential for 9. Jany, K. D., Keil, W., Meyer, H. & Kiltz, H. H. (1976) Biochim. tubulin polymerization, and blockage ofas few as one or two SH Biophys. Acta 453, 62-66. groups inhibits the assembly of microtubules by an as yet un- 10. Sonneborn, H. H., Zwilling, R. & Pfleiderer, G. (1969) Hoppe- determined mechanism (32, 33). Seyler's Z. Physiol. Chem. 350, 1097-1102. 11. Drapeau, G. R. (1980)J. Biol. Chem. 255, 839-840. The highly acidic COOH-terminal part of a-tubulin resem- 12. Brauer, A. W., Margolies, M. N. & Haber, E. (1975) Biochem- bles the NH2-terminal sequence oftroponin T (see Fig. 4) (30). istry 14, 3029-3035. This protein, as a component of the troponin complex, partic- 13. Tarr, G. E., Beecher, J. F., Bell, M. & McKean, D. J. (1978) ipates in the Ca2+ regulation ofactin-myosin contacts. One may Anal. Biochem. 84, 622-627. speculate that these similar structures ofa-tubulin and troponin 14. Zimmerman, C. L., Appella, E. & Pisano, J. J. (1977) Anal. T perform analogous physiological functions that, in view ofthe Biochem. 77, 569-573. 15. Edman, P. & Henschen, A. (1975), in Protein Sequence Deter- clusters of glutamyl residues, could involve cation binding. mination, ed. Needleman, S. B. (Springer, New York), pp. In addition to the sequence similarity to troponin T, some 232-279. similarities have been observed to the structures of a-tropomy- 16. Ponstingl, H., Nieto, A. & Beato, M. (1978) Biochemistry 17, osin and troponin C. Also a tripeptide, His-Gly-Lys, that has 3908-3912. been isolated from cat spinal cord and reported to impair firing 17. Ponstingl, H., Little, M., Krauhs, E. & Kempf, T. (1979) Nature of neurones in the dorsal horn (34) is present at positions (London) 282, 423-424. 18. Kirschner, M. W. (1978) Int. Rev. Cytol. 54, 1-71. 309-311 of a-tubulin. 19. Zenner, H. P. & Pfeuffer, T. (1976) Eur. J. Biochem. 71, As these proteins are quite unrelated, it is difficult to explain 177-184. the similarities on the basis ofevolutionary relationship. We feel 20. Kelly, P. T. & Cotman, C. W. (1978)J. Cell Biol. 79, 173-183. that a more useful approach would be to evaluate the sequence 21. Murofushi, H. (1980)1. Biochem. 87, 979-984. similarities strictly on the basis of structure-function criteria. 22. Kemphues, K. J., Raff, R. A., Kaufman, T. C. & Raff, E. C. Thus, one would expect that two unrelated proteins or regions (1979) Proc. Natl. Acad. Sci. USA 76, 3991-3995. 23. Chou, P. Y. & Fasman, G. D. (1978) Annu. Rev. Biochem. 47, of proteins that perform analogous functions should also have 251-276. similar amino acid sequences regardless of the evolutionary 24. Ventilla, M., Cantor, C. R. & Shelanski, M. (1972) Biochemistry relationship. 11, 1554-1561. Note Added in Proof. After we had communicated this article, the nu- 25. Lee, J. C., Corfman, D., Frigon, R. P. & Timasheff, S. N. (1978) cleotide sequence ofcDNA from chicken brain tubulin messengers was Arch. Biochem. Biophys. 185, 4-14. acid 26. Luduena, R. F. & Woodward, D. 0. (1973) Proc. Natl. Acad. published by Valenzuela et al. (35). From these data, an amino Sci. USA 70, 3594-3598. sequence for chicken brain a-tubulin was deduced, corresponding to 27. Lu, R. C. & Elzinga, M. (1978) Biochim. Biophys. Acta 537, residues 41-451 ofour sequence and differing only in residues 175 (ar- 320-328. ginine), 295 (tyrosine), and 358 (glutamate) from one of our variants. 28. Collins, J. H. & Elzinga, M. (1975) J. Biol. Chem. 250, We wish to thank Mr. Jurgen Kretschmer, Mrs. Ch. Orlando, and 5915-5920. Miss Herta Scherer for their skillful technical assistance; Dr. G. Os- 29. Elzinga, M. & Collins, J. H. (1977) Proc. Natl. Acad. Sci. USA terburg for programming the method ofsecondary structure prediction, 74, 4281-4284. discussions. This work was 30. Pearlstone, J. 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