Allele-specific suppression by formation of new protein-protein interactions in yeast.

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 conducted a biochemical analysis of the interaction between various combinations of wild-type and mutant actin and Sac6 proteins. Previously, we showed that actin mutations that are suppressed by sac6 mutations encode proteins with a reduced affinity for wild-type Sac6p. In the present study, we have found that mutant Sac6 proteins bind more tightly to mutant actin than does wild-type Sac6p, and thus compensate for weakened interactions caused by the mutant actin. Remarkably, we have also found that mutant Sac6 proteins bind more tightly to wild-type actin than does wild-type Sac6p. This result indicates that suppression does not occur through the restoration of the original contact site, but rather through the formation of a novel contact site. This finding argues against suppression occurring through a "lock-and-key" mechanism and suggests a mechanism involving more global increases in affinity between the two proteins. We propose that the most common kind of suppressors involving interacting proteins will likely occur through this less specific mechanism.

Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin.

Actin interacts with a large number of different proteins that modulate its assembly and mediate its functions. One such protein is the yeast actin-binding protein Sac6p, which is homologous to vertebrate fimbrin (Adams, A. E. M., D. Botstein, and D. G. Drubin. 1991. Nature (Lond.). 354:404-408.). Sac6p was originally identified both genetically (Adams, A. E. M., and D. Botstein. 1989. Genetics. 121:675-683.) by dominant, reciprocal suppression of a temperature-sensitive yeast actin mutation (act1-1), as well as biochemically (Drubin, D. G., K. G. Miller, and D. Botstein. 1988. J. Cell Biol. 107: 2551-2561.). To identify the region on actin that interacts with Sac6p, we have analyzed eight different act1 mutations that show suppression with sac6 mutant alleles, and have asked whether (a) these mutations occur in a small defined region on the crystal structure of actin; and (b) the mutant actins are defective in their interaction with Sac6p in vitro. Sequence analysis indicates that all of these mutations change residues that cluster in the small domain of the actin crystal structure, suggesting that this region is an important part of the Sac6p-binding domain. Biochemical analysis reveals defects in the ability of several of the mutant actins to bind Sac6p, and a reduction in Sac6p-induced cross-linking of mutant actin filaments. Together, these observations identify a likely site of interaction of fimbrin on actin.