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In Saccharomyces Cerevisiue Copyright 0 1995 by the Genetics Society of America Genetic Analysis of the Fimbrin-Actin Binding Interaction in Saccharomyces cerevisiue Sharon M. Brower, Jerry E. Honts and Alison E. M. Adams Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721 Manuscript received December 8, 1994 Accepted for publication February 9, 1995 ABSTRACT Yeast fimbrin is encoded by the SAC6 gene, mutationsof which suppress temperature-sensitivemuta- tions in the actin gene (ACTl).To examine the mechanism of suppression,we have sequenced 17 sac6 suppressor alleles, and found that they change nine different residues, all of which cluster in three regions of one of the two actin-binding domainsof Sac6p. Two of these clusters occur in highly conserved regions (ABS1 and ABS3) that have been strongly implicated in the binding of related proteinsto actin. The third cluster changes residues notpreviously implicated in the interaction with actin. As changes in any of nine different residues can suppress several differentactl alleles, it is likely that the suppressors restore the overall affinity,rather than specific lost interactions, between Sac6p and actin. Using mutagen- esis, we have identified two mutations of the second actin-binding domain that can also suppress the actl mutations of interest. This result suggests the two actin-binding domainsof Sac6p interact with the same region of the actin molecule. However, differences in strength of suppression of temperature- sensitivity and sporulation indicate that the two actin-binding domains are distinct, and explain why seconddomain mutations were not identified previously. HE actin cytoskeleton is a complex structure com- analyses have suggested that, at least in vitro, these resi- T prised of a large number of interacting proteins, dues may not beessential for the interaction(BRESNICK whose distribution and function has been well charac- et al. 1990, 1991; LEVINEet al. 1990, 1992; HEMMINCSet terized througha combination of microscopic, bio- al. 1992; KUHLMANet al. 1992; FABBRIZIOet ul. 1993; chemical, and immunological tools. More recently, ad- CORRADOet al. 1994; LEBARTet al. 1994). It therefore ditional insights have been gained from theapplication seems likely that there are several regions that contrib- of genetics. For example, mutations have provided a ute to the overall actin-binding activity, and that any powerful tool for analyzing the in vivo roles of individual one interaction may be dispensable. Moreover, the rela- proteins and domains in cytoskeletal function, as well tive importance of the different regions may vary from as a means toidentify interacting components through one protein to another (e.g., FABBRIZIOet al. 1993; COR- genetic screens and selections. In addition, mutations RADO et al. 1994; LEBARTet al. 1994). In the case of have been used to provide information as to likely sites fimbrin, almost nothing is known about the details of of interactions on the surfaces of proteins. the interaction with actin. For example, it is not known Yeast and vertebrate fimbrins are members of a family whether both domains bind to actin, and if they do, of actin-filament cross-linking proteins that share exten- whether both bind to the same sites on the actin mole- sive homology in their27-kD actin-binding domains (DE cule, or which residues are directly involved in the inter- ARRUDA et al. 1990). Most members of this family of action. proteins i.e., a-actinin, P-spectrin, filamin, ABP120, and Yeast fimbrin is encoded by the SAC6 gene, and was dystrophin have just a single actin-binding domain and identified previously by affinity chromatography on F- cross-link adjacent actin filaments as multimers (DE AR- actin columns, as well asthrough dominantsuppression RUDA et al. 1990; GORLINet al. 1990). The fimbrins, of a temperature-sensitive actin mutation (DRUBINet however, have two homologous actin-binding domains al. 1988; ADAMS and BOTSTEIN1989; ADAMS et al. 1989, per molecule, and presumably cross-link actin filaments 1991). Interestingly, several of the sac6suppressors have a Ts phenotypein an genetic background, indi- as monomers (BRETSCHER1981; GLENNEYet al. 1981; DE ACTlf cating that suppression is reciprocal, and likely due to ARRUDA et al. 1990). Biochemical studies of the various members of this family of proteins have identified sev- compensatingchanges in physically interacting pro- eral groups of residues within the 27-kD actin-binding teins (ADAMS and BOTSTEIN1989). Recently, we ob- domains that interactdirectly with actin, but mutational tained evidence that several actl mutations that show suppression with these sac6alleles are defective in bind- ing to wild-type Sac6p (HONTSet al. 1994). If the sac6 Corresponding author: Alison E. M. Adams, Department of Molecular and Cellular Biology, Life Sciences South, University of Arizona, Tuc- mutations suppress these actl defects by restoring defec- son, AZ 85721. E-mail: adams%[email protected] tive interactions between mutant actins and SacGp, the Genetics 140 91 - 101 (May, 1995) 92 S. M. Brower, J. E. Hontsand E. A. M. Adams sac6 mutations would be expected to affect the region [sac6-5=D264Y]; AA049: 921 (CTCAGGAGGCAGCTT) 907 ofSac6p to which actin binds and to increase the [sac66=H268P]; AA052: 551 (TTAGTAAmCACGT) 537 strength of the interaction with the mutant actin. In [ sac64=F145I] ; AA053: 894 (ACTTAAC(&4CCTCT) 880 [sac6-14= L259Wl; AA069: 957 (GCAAmGAGGTT- this study, we set out to analyze the mechanism by which GCC)974; AA078: 1660 (CCCATCAATTGACACACTAA- the sac6 mutations suppress the actl mutations. In par- CCC) 1638 [sac6-31=W514C]; AA080: 879 (CCTAATAAT- ticular, we examined whether the sac6 mutations change TTGACAAATCAAACC) 856 [sac610=W252C]. residues likely, by homology with other actin-binding Molecular cloningof sac6 mutationsby “gap repair”: Eight proteins, to be involved in the interaction with actin. of the sac6 mutant alleles were copied from the genome onto plasmids by “gap repair” (ORR-WEAVERet al. 1983). Thus, We found that 17 sac6 mutations analyzed change resi- pAABl17 (Figure l),awild-type SAC6containing plasmid that dues that cluster in three regions of one of the two carries the URA3 selectable marker and a centromere, was Sac6p actin-binding domains. Two of these regions are cut with CluI and Hind111 to remove a 2.5-kb fragment con- believed to be important in the actin-binding domains taining the wild-type SAC6gene (from -100 bp upstream of of related proteins, suggesting that these mutations the translation start to-340 bp downstream of the translation stop-see Figure 1).This cut plasmid was then purified and cause suppression of the actl defect through a direct used to transform ura3 actl sac6 strains carrying various sac6 effect on the binding of Sac6p to actin. However, as mutant alleles (AAYl021,AAYl022, “023, AAYl039, changes in any one of nine different Sac6p residues AAYl108,AAYl109, AAYl112, and AAYl624; see Table l), can suppress a variety of actl alleles, it is unlikely that and Ura+transformants were selected. Transformants should include cells containing plasmids in which the ClaI-Hind111 each suppressor restores a single lost interaction. gap has been filled by gap repair, using the genomic, mutant Rather, it seems more plausible that the sac6 mutations sac6sequences as template (ORR-WEAVERet al. 1983). Plasmid suppress by increasing the overall affinity betweenactin DNA was then isolated from yeast and used to transform E. and Sac6p. coli strain HB101. Plasmids were isolated from these amp‘ As all 17 mutations fell into just oneof the two actin- transformants, screened by restriction analysis for those of the correct size, and then used to transform DBY2001, a tem- binding domains, it seemed likely that the two actin- perature-sensitive ura3 actl-3 SAC6+ strain (Table 1). Ura+ binding domains are not equivalent. We tested this hy- transformants were tested for temperature sensitivity on YEPD pothesis by studying mutants of the second actin-bind- to check they carried a dominant sac6 suppressor mutation, ing domain and found that equivalent mutations in and the mutant sac6 genes of those plasmids that gave rise to the two domains have overlapping, but not identical, Ts+transformants were then sequenced by the dideoxy method (SANGERet al. 1977). An additional allele, sac6-6, phenotypes. This result indicates that the two domains which was previously isolated on plasmid pRB1275 (ADAMS are not absolutely equivalent, but likely interact with and BOTSTEIN1989) was also sequenced. the same region of actin. Identification of sac6 mutations by PCR and cycle sequen- cing: Eight additional sac6 mutations were sequenced directly from the genome, using PCR and a double-stranded DNA cy- MATERIALSAND METHODS cle-sequencing system (catalogue no. 8196SA,GIBCO BRL, Yeast strains, media and genetic techniques: Yeast strains Gaithersburg, MD) . Thus, genomic DNA was first isolated from used in this study are listed in Table 1. Media for yeast growth each of the strains ”569, AAYl572, “573, “576, and sporulation, and methods for mating, sporulation and ~~~1578, ~~~1582,and ~~~1585 ~~~1583,(Table 1). AS we tetrad analysis were as described by SHERMANet al. (1974). had found that all of the mutations isolated by gap repair We obtained 5-fluoro-orotic acid (5-FOA) from PCR Incorpo- (above) changed residues in the more N-terminal of the two rated via the Genetics Society of America. Growth on plates actin-binding domains (see RESULTS),we firstanalyzed the was scored by
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