The tubulin protein family includes polypeptides of at least three subclasses, alpha-, beta- and gamma-tubulin. The classical form of tubulin consists of the alpha- and beta-tubulin subunits that combine non-covalently to form the tubulin alpha/beta-heterodimer unit. This 100 kDa protein, referred to as tubulin, is the functional unit that self-assembles into microtubule biopolymers (Ludueña et al., 1977). Tubulin can be made to polymerize reversibly in vitro. Polymerization can be promoted by magnesium, GTP, warming and accessory proteins, while calcium, GDP and cooling inhibit assembly (Burns, 1991a). The process of going from individual alpha- and beta-subunits to the heterodimers and finally to the microtubule is shown schematically in Figure 4. From sequence data, alpha- and beta-subunits each have a molecular weight of 49-50 kDa and contain about 450 amino acid residues depending on species (Ponstingl et al., 1981; Valenzuela et al., 1981; Krauhs et al., 1981). Furthermore, they are highly homologous with respect to amino acid sequence (Burns and Surridge, 1994).
The gamma-tubulins are the most recently characterized subclass of the tubulin protein family (Oakley and Oakley, 1989). Gamma-tubulins are believed to be assembled into high molecular weight entities, and are essential for microtubule functions (Oakley et al., 1990; Stearns et al., 1991; Horio et al., 1991). The gamma-tubulin subclass is primarily associated with microtubule nucleation and can be found at microtubule organizing centres (MTOCs), which act as the point of initiation of self assembly in cells (for example, the spindle poles, the centrosomes or the chromosome-end nucleation site of microtubule assemblies) (Oakley et al., 1990; Zheng et al., 1991; Joshi et al., 1992; Joshi, 1993).
Just recently discovered are two new members of the tubulin family, tentatively named delta- and epsilon-tubulin (Burns, 1995). Both proteins resemble gamma-tubulin, in that the amino-terminal domain binds to alpha/beta-heterodimers, but have low sequence identity to gamma-tubulin. These potentially new tubulin classes bind GTP but have no GTPase activity. Burns and colleagues also found that the carboxyl-terminal domain binds a non-tubulin protein.
Multiple tubulin genes are expressed by most organisms, with different isoforms being expressed in specific cell types or at specific stages of cellular development (MacRae and Langdon, 1989; Ludueña et al., 1992). Within a single tissue only two to three genes may be expressed at one time. Protein isotypes can also arise from different post-translational modifications (such as phosphorylation, tyrosination, acetylation or glutamylation) (Bulinski and Gundersen, 1991). Phosphorylation has been described in both alpha- and beta-tubulins; alpha-tubulin being phosphorylated at the carboxyl-terminal (Wandosell et al., 1987) and beta-tubulin phosphorylated on Ser444 (Ludueña et al., 1988). Tyrosination occurs at the carboxyl-terminal tyrosine on alpha-tubulin. The residue is removed by a carboxypeptidase and may be replaced by an RNA-independent tubulin-tyrosine ligase (reviewed by Barra et al., 1988). Thus, intracellular tubulin is a mixture of molecules with and without tyrosine at the -carboxyl terminus. The acetylation modification occurs on -tubulin at Lys40 (LeDizet and Piperno, 1987). Gamma-glutamylation is an unusual modification in which as many as six glutamic acids may be added to Glu445 of alpha-tubulin and Glu435 of beta-tubulin (Rudiger et al., 1992; Redeker et al., 1992; Edde et al., 1990).
Despite this diversity in post-translational modification, these tubulin variants still generally polymerize into structurally indistinguishable microtubules (Mandelkow et al., 1995). Moreover, most tubulin isotypes can be functionally interchangeable, different isotypes can each participate in forming typical microtubule organelles. For example, the two different -tubulins in Saccharomyces cerevisiae are entirely interchangeable (Schatz et al., 1986).
Various research groups have examined the sequence similarities among the known alpha-, beta- and gamma-tubulins. In general the three tubulin subunits share a 35-45% identity with each other (Little and Seehaus, 1988; Oakley and Oakley, 1989). This represents a high degree of homology among the different classes. Additionally, there is significant sequence conservation within a tubulin class (Figure 5). If the highly heterogeneous carboxyl-terminal segment of 20-25 amino acids is ignored, the remainder, including the amino-terminus of the alpha-tubulin (amino acids 1-436) and beta-tubulin (amino acids 1-430) are highly conserved (Little and Seehaus, 1988; Burns, 1991b) with 72% sequence conservation among alpha-tubulins, 78% among beta-tubulins (Burns and Surridge, 1994) and at least 66% identity among known gamma-tubulin sequences (Oakley and Oakley, 1989). Work by Oakley and Oakley (1989) indicated that the gamma-tubulins have a greater homology to beta-tubulins (59%) than alpha-tubulins (51%). This homology increases for all three cases when the non-identical residues are matched for conservative amino acid substitutions.
It is only the carboxyl-termini of the tubulin subunits which tend to be highly divergent. Including the carboxyl-termini (i.e. the complete sequence) there is between 30-40% sequence conservation within each of the alpha-, beta- and gamma-tubulins. Again, this sequence conservation increases if conservative substitutions are accounted for (Burns and Surridge, 1994; Oakley, 1994).
The homology existing among the tubulin classes strongly suggests that the folded structures for all three classes are similar, with the highly conserved regions being indicative of nucleotide binding sites. Regions in the sequences which are not conserved may be expected to fold into the same tertiary structure, as suggested by the hypothesis of similarity for all tubulin classes. This is supported by structural studies which detect only minimal gross structural differences between alpha- and beta-tubulin in electron density maps of the lattice (Mandelkow and Mandelkow, 1994). Sequence differences would be due to specific properties such as variations in GTPase activity and ligand binding. It is also implicit that there must be differences between alpha- and beta-tubulin at the intersubunit contact surfaces, since the two subunits are not interchangeable.
Dissimilar sequences which fold into similar structures have been observed for other proteins, such as the two domains of actin (Kabsch et al., 1990), the alpha-subunit of tryptophan synthase (Stackhouse et al., 1988) and the parallel eight-stranded alpha/beta-barrel class represented by triose phosphate isomerase (the so-called TIM barrel) which is duplicated in a variety of other enzymes of highly divergent sequences (Scheerlinck et al., 1992; Lesk et al., 1989).
Previous research by other groups on other GTP-binding proteins has yielded structural information; the three-dimensional structures of transducin-alpha and Ras protein, both of which are GTP proteins, have been determined. The structure of transducin alpha-subunit complexed to GTPalphaS has been determined to 2.2 Å resolution (Noel et al., 1993). The alpha-subunit of the native transducin heterotrimer is approximately 325 residues in length, which is somewhat smaller than any of the tubulin monomers. The structure obtained for the transducin-alpha-GTPalphaS complex showed a GTPase domain which forms a cleft structure common to all other GTPases. This cleft is present in Ras protein (Pai et al., 1990) and in EF-Tu (Berchtold, 1993). It is important to note that these proteins are not homologous in sequence, yet structural similarities are maintained in at least the nucleotide binding domain. From these studies it may be possible to model a partial structure for tubulin; that is, the tubulin heterodimer structure may contain the same GTP binding cleft that other GTPases have. This suggestion is supported by studies which demonstrate that fold homologies can occur which yield similar structures even though sequence homology is minimal (Harrison and Durbin, 1985; Chou and Carlacci, 1991).
Two previous reviews have alluded to the major similarities and differences between actin and tubulin (Mitchison, 1992; Cleveland, 1982). Actin, a protein containing approximately 385 residues (which is slightly smaller than tubulin) is arranged into four domains which surround a deep cleft containing ATP or ADP, together with a tightly bound divalent cation (Kabsch et al., 1990). Both tubulin and actin self-associate into structural polymers via a process called helical polymerization. In the polymer lattices all subunits occupy fairly equivalent positions with respect to their neighbours. Both polymers are polar, where faster growth extension occurs at one end, while the opposite end is either slower in elongation or does not grow at all. There are also some sequence homologies between actin and tubulin, chiefly in five regions found in both proteins. The structure of actin has been elucidated (Kabsch et al., 1990). Therefore, since both proteins have some similarities in these general properties and most particularly in the sequence, perhaps homology also extends to some aspects of the tertiary structure.