Assembly Properties of Altered β-Tubulin Polypeptides Containing Disrupted Autoregulatory Domains

Abstract

β-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the β-tubulin polypeptide act either directly or indirectly to control the stability of β-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian β-tubulin isotype, Mβ1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-Mβ1-specific antibody. We show that the Mβl β-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant β-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered β-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian α-tubulin isotype, Ma4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent α- and β-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent Ma4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type α-tubulin molecule. These data suggest that the universally conserved amino terminus of β-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of α-tubulin is not essential for coassembly in mammalian cells in vivo.