Genetics, Vol 139, 267-286, Copyright © 1995

INVESTIGATIONS

Structural Analysis of Mutations in the Drosophila {beta}2-Tubulin Isoform Reveals Regions in the {beta}-Tubulin Molecule Required for General and for Tissue-Specific Microtubule Functions

J. D. Fackenthal, J. A. Hutchens, F. R. Turner and E. C. Raff
Present address: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.

We have determined the lesions in a number of mutant alleles of {beta}Tub85D, the gene that encodes the testis-specific {beta}2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the {beta}2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all {beta}-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t(6) contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate {beta}-tubulins. Correspondingly, B2t(6) disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that {beta}3, a developmentally regulated Drosophila {beta}-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type {beta}2-tubulin. We show here by complementation analysis that {beta}3 and the B2t(6) product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the {beta}2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the {beta}2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the {beta}2 variant lacking the carboxy terminus and the B2t(6) variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate {beta}-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type {beta}-tubulins. 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 microtubule morphology as a function of tubulin assembly kinetics.

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