STRUCTURAL-ANALYSIS OF MUTATIONS IN THE DROSOPHILA BETA-2-TUBULIN ISOFORM REVEALS REGIONS IN THE BETA-TUBULIN MOLECULE REQUIRED FOR GENERALAND FOR TISSUE-SPECIFIC MICROTUBULE FUNCTIONS

We have determined the lesions in a number of mutant alleles of beta T ub85D, the gene that encodes the testis-specific beta 2-tubulin isofor m in Drosophila melanogaster. Mutations responsible for different clas ses of functional phenotypes are distributed throughout the beta 2-tub ulin molecule. There is a telling correlation between the degree of ph ylogenetic conservation of the altered residues and the number of diff erent microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved i n all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) con tains an amino acid substitution within an internal cluster of variabl e amino acids that has been identified as an isotype-defining domain i n vertebrate beta-tubulins. Correspondingly, B2t(6) disrupts only a su bset of microtubule functions, resulting in misspecification of the mo rphology of the doublet microtubules of the sperm tail axoneme. We pre viously demonstrated that beta 3, a developmentally regulated Drosophi la beta-tubulin isoform, confers the same restricted morphological phe notype in a dominant way when it is coexpressed in the testis with wil d-type beta 2-tubulin. We show here by complementation analysis that b eta 3 and the B2t(6) product disrupt a common aspect of microtubule as sembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of corr ect axoneme morphology but not for general microtubule functions. As w e have previously reported, the beta 2-tubulin carboxy terminal isotyp e-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carbo xy terminus and the B2t(6) variant complement each other for mild-to-m oderate meiotic defects but do not complement for proper axonemal morp hology. Our results are consistent with the hypothesis drawn from comp arisons of vertebrate beta-tubulins that the two isotype-defining doma ins interact in a three-dimensional structure in wild-type beta-tubuli ns. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtu bule morphology as a function of tubulin assembly kinetics.