Sequence of a Highly Divergent h ' Tubulin Gene Reveals Regional Heterogeneity in the / 3 Tubulin Polypeptide

The nucleotide sequence of a chicken genomic DNA segment containing the chicken/34 tubulin gene has been determined. The predicted amino acid sequence of/34 is surprisingly divergent from that of the chicken/32 gene that encodes the dominant neural tubulin. /34 differs from /32 at 36 residue positions and encodes a polypeptide that is four amino acids longer, yielding a divergence of 8.9% between the two/3 tubulin isotypes. While many of the amino acid substitutions are conservative, several involve significant alteration in the physiochemical properties of the residue. Furthermore, the amino acid substitution positions are not randomly located within the primary sequence but are distinctly clustered: major divergence occurs in the carboxy-terminal region beyond residue 430 and within the second protein coding exon segments of the genes. In addition, large regions of absolute sequence conservation are also present. Certain sequences within the heterogeneous regions are conserved in other species, indicating that these regions are under positive evolutionary selection pressure and are therefore probably essential for some aspect of/3-tubulin function. These findings strongly suggest that regional amino acid sequence heterogeneity may play an important role in the establishment of functionally differentiated/3 tubulin polypeptides. Microtubules, composed principally of dimeric subunits of 1 o~ and 1/3 tubulin polypeptide, are fdamentous polymers that are employed in a number of diverse functions in eucaryotic cells. For example, microtubules are the major components of mitotic and meiotic spindles and of cilia and flagella. Furthermore, they participate in various types of intracellular transport and, in conjunction with intermediate filaments and actin filaments, establish the structure and functional properties of the eucaryotic cytoskeleton. Yet in spite of this functional diversity, the ultrastructure of microtubules is, with a few notable exceptions (6, 10), identical in all cells. Moreover, the tubulin polypeptides themselves have been conserved throughout evolution to such an extent that tubulins from all sources investigated have been shown to co-polymerize efficiently in vitro (see reference 18 for review). Despite these apparently conserved features, several lines of evidence suggest that the microtubule systems of different cells or within single cells are distinguishable on the basis of physical, biochemical, and pharmacological properties. For example, Benke and Forer (2) originally reported evidence for four classes of microtubules in individual cells which differed in their drug sensitivities or thermal stability. Similarly, Brinkley and Cartwright (5) have demonstrated that the interpolar and kinetichore microtubules of mammalian spindles differ in their response to drugand cold-induced depolymerization. Additional compelling evidence has come from the work of Thompson et al. (31) and Cumming et al. (9) who have independently demonstrated, using monoclonal antibodies to a tubulin determinants, that a tubulin related epitopes are apparently segregated into distinguishable subsets of microtubules within single cells or subsets of cells. Thus, it seems clear that although microtubules appear morphologically identical, they represent a heterogeneous class of structures. One obvious potential level of control of microtubule properties is at the level of the tubulin dimers which constitute the major structural subunit. This hypothesis would require that multiple, functionally distinct tubulin subunits be present within cells. Immunological and biochemical support for this hypothesis has emerged from a number of investigators who have reported heterogeneous pools of c~ and/3 tubulins in a number of tissue and cell types (e.g., references 11, 12, 22, 24, 25, 30) and within single cells (13). More definitively, protein sequence analysis of porcine brain tubulins has demonstrated the presence of two /3 tubulins (19) and four a tubulins (27) that differ subtly in primary sequence. More recently, sequence analysis of two different human/3 tubulin genes has demonstrated the presence of at least two ~ tubulin isotypes that differ significantly in sequence at the carboxy terminus of the polypeptide (15). We previously demonstrated that the chicken contains four THE JOURNAL OF CELL BIOLOGY VOLUME 99 NOVEMBER 1984 1754-176