SITE-DIRECTED MUTAGENESIS OF PUTATIVE GTP-BINDING SITES OF YEAST BETA-TUBULIN - EVIDENCE THAT ALPHA-TUBULINS, BETA-TUBULINS, AND GAMMA-TUBULINS ARE ATYPICAL GTPASES

The exchangeable GTP-binding site on beta-tubulin has been extensively studied, but the primary sequence elements which form the binding sit e on beta--tubulin remain unknown. We have used site-directed mutagene sis of the single beta-tubulin gene of Saccharomyces cerevisiae to tes t a model for the GTP-binding site on beta-tubulin, which was based on sequence comparisons with members of the GTPase superfamily [Sternlic ht, H., Yaffe, M. B., & Fan, G. W. (1987) FEBS Lett. 214, 226-235]. We analyzed the effects of D295N, N298K, and N298Q mutations in a propos ed base-binding motif, (295)DAKN(298), On tubulin-GTP binding and on n ucleotide-binding specificity. We also examined the effects of a D203S mutation in a putative phosphate-binding region, (203)DNEA(206), On n ucleotide binding affinity, on the assembly-dependent tubulin GTPase a ctivity in vitro, and on the dynamic properties of individual ''mutant '' microtubules in vitro. The effects of the mutations on cell phenoty pe and on microtubule polymerization in cells were also measured. The results do not support the proposal that the (203)DNEA(206) and (295)N KAD(298) motifs are cognate to motifs found in GTPase superfamily memb ers. Instead, the data argue that the primary sequence elements of bet a-tubulins that interact with bound nucleotide, and presumably also th ose of the alpha- and gamma-tubulin family members, are different from those of ''typical'' GTPase superfamily members, such as p21(ras). Th e GTPase superfamily should thus be broadened to include not just the typical GTPases that show strong conservation of primary sequence cons ensus motifs (GxxxxGK, T, DxxG, DxKN) but also ''atypical'' GTPases, e xemplified by the tubulins and other recently identified GTPases, that do not show the consensus motifs of typical GTPases and which also sh ow no obvious primary sequence relationships between themselves. The t ubulins and other atypical GTPases thus appear to represent convergent solutions to the GTP-binding and hydrolysis problem.