GENETIC-ANALYSIS OF THE FIMBRIN-ACTIN BINDING INTERACTION IN SACCHAROMYCES-CEREVISIAE

Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1). To examine the mechanism of suppression, we have sequenced 17 sac6 suppressor all eles, and found that they change nine different residues, all of which cluster in three regions of one of the two actin-binding domains of S ac6p. Two of these clusters occur in highly conserved regions (ABS1 an d ABS3) that have been strongly implicated in the binding of related p roteins to actin. The third cluster changes residues not previously im plicated in the interaction with actin. As changes in any of nine diff erent residues can suppress several different act1 alleles, it is like ly that the suppressors restore the overall affinity, rather than spec ific lost interactions, between Sac6p and actin. Using mutagenesis, we have identified two mutations of the second actin-binding domain that ran also suppress the act1 mutations of interest. This result suggest s the two actin-binding domains of Sac6p interact with the same region of the actin molecule. However, differences in strength of suppressio n of temperature-sensitivity and sporulation indicate that the two act in-binding domains are distinct, and explain why second-domain mutatio ns were not identified previously.