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

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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
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