In Saccharomyces Cerevisiue

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 mutagenesis, 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 caseof 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 binding 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 sequencing: 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 spotting suspensions of cells onto plates using same section of SAC6 in this next set of mutants. Thus, a 1.37- a 32-point inoculator or by streaking for single colonies. Yeast kb fragment was amplified by PCR from each genomic DNA transformation was by the lithium acetate method (SCHIESTL preparation using oligonucleotides AA039 and AA046 (Fig- and GIETZ1989). Isolation of plasmid DNA from yeast was as ure 1).These 1.37-kb fragments were then sequenced directly described previously (HOFFMANand WINSTON1987). Isolation using a double-stranded DNAcycle sequencing systemac- of genomic DNA from yeast wasby a modification of the cording to the manufacturer’s instructions, except that -p3’P- method of STRUHLet al. (1979). labeled ATP (catalogue no. NEG302H; New England Nuclear, Oligonucleotides: Oligonucleotides used for PCR and site- Boston, MA) was used instead of y-”P-labeled ATP. In each specific mutagenesis are listed below. AA037, AA039, case, the whole fragment was sequenced on at least one strand. AA046, and AA069 were used for PCR all other oligonucleo- Site-specific mutagenesis: All site-specific mutagenesis was tides listed were used in site-specific mutagenesis. The num- conducted on the Sad-BamHI fragment derived from ber to the left of the oligonucleotide sequence refers to the pAAB117 (Figure l), using a modified version of a standard 5’ nucleotide in the sequence, and that to the right refers to method (KUNKELet al. 1987). The Sad-BamHI fragment was the 3‘ nucleotide (the A of the first methionine ATG in SAC6 first cloned into theSad-BamHI sitesof pUN70 [a yeast shuttle being nucleotide No. 1). In those cases in which a mutation vector containing UR.43, CEN4, M13 ori (ELLEDGEand DAVIS was generated, the mutantnucleotide carried by the oligonu- 1988)l to generate pAAB313, which was then used to trans- cleotide is shown with an underscore, and the mutant allele form XL1-Blue (Stratagene). Single-stranded DNA prepared and residue changed by it are shown in brackets. The loca- from this strain was then used for site-specific mutagenesis, tions and orientations of the oligonucleotides in relation to either to generate a novel mutation (sac6-31; plasmid designa- the SAC6 gene are shown in Figure 1. AA037: 2146 (CTT- tion pAAB287), or to confirm that mutations identifiedabove CAGAAATACTTGAGGC) 2128; AA039: -25 (GCCCTA- were sufficient for suppression (all others). Thus, the sac6 AGGAGTACACC) -9; AA046: 1364 (GGTAAAGACTCT- 5, sac6-6, sac6-4, sac6-14, sac6-31, and sacb-10 mutations were TGCCTC) 1347; AA048: 908 (TTAATATrnTTTTA) 894 generated using the oligonucleotides AA048, AA049, Genetic Analysis of Yeast Fimbrin 93

TABLE 1 Yeast strains used in this study

Strain Genotype ~~n021 MATa sac62actl-3 tub2-201 ura3-52 uno22 MATa sac615actl-3 tub2-201 ura3-52 AAMO~~ MATa sac619actl-3 tub2-201 ura3-52 ~~~1039 MATa sac67actl-3 tub2-201 ura3-52 AA~O~O MATa/MATa sac6::LEU2/sac6::LEU2 ura3/ura3 his3/+ leu2/h2 ade2/+ AAn 108 MATa sac61 7 actl-3 ura3-52 AAn lo9 MATa sac614actl-3 tub2-201 ura3-52 ~~1112 MATa sac65actl-3 tub2-201 ura3-52 ~~~569MATa sac68actl-3 ura3-52 "572 MATa sac61actl-3 tub2-201 ura3-52 ~~~1573 MATa sac616actl-3 ura3-52 ~~~1576 MATa sac610act13 tub2-201 ura3-52 My1578 MATa sac624actl-3 tub2-201 ura3-52 ~~~1582 MATa sac623actl-3 ura3-52 ~~~1583 MATa sac611actl-3 tub2-201 ura3-52 ~~~1585 MATa sac63actl-3 tub2-201 ura3-52 ~~~1624 MATa sac64actl-3 tub2-201 ura3-52 AAYl644 MATaura3 actl-7 ~~~1692 MATa/MATa actl-l/actl-1 tub2-201/+ ura3-52/ura3his461 9/+ ~~~1697 MATa/MATa actl-l/actl-1sac610/+ tub2-201/+ ura3-52/+ his4-619/+ ~~~1699 MATa/MATa actl-l/actl-1sac62/+ tub2-201/+ ura3-52/+ his4-619/+ ~~~1701 MATa/MATa actl-l/actl-1sac67/+ tub2-201/+ ura3-52/+ his4-619/+ AAn 703 MATa/MATa actl-l/actl-1sac64/+ tub2-201/+ ura3-52/+ his4-619/+ AAn 770 MATa/MATa actl-l/actl-1 tub2-201/+ ura3-52/ura3 his4-619/+ sac630/+ DBYl993 MATa actl-2 ura3-52 DBYl995 MATa actl-3 his4-619 DBY2001 MATa actl-3 tub2-201 ura3-52 DBY2326 MATa actl-4ura3-52 &2-101 DBY5217 MATa/MATa actl-l/actl-1 tub2-201/+ ura3-52/+ his4-619/+ -05 MATa actl-l33::HIS3 nyl canl-1 his3-D200 leu2-3,112 ura3-52 tub2-201 KWY310 MATa actl-l08::HIS3 my1 canl-1 his3-D200 leu2-3,112 ura3-52 tub2-201 KWY361 MATa actl-l2O::HIS3 canl-1 ade4 his3-D200 leu2-3,112 ura3-52 tub2-201 KWY376 MATa actl-l25::HIS3canl-1 ade2 his3-D200 leu2-3,112 ura3-52 tub2-201 KWY4 10 MATa actl-l12::HIS3 ade2-101 ade4 cry1 his3-D200 leu2-3,112 ura3-52 tub2-201 M417 MATa actl-l05::HIS3canl-l his3-D200 leu2-3,112 ura3-52 tub2-201 All AAY strains were made in this laboratory.AAYl040 (hmset al. 1991) and "644 (HONTSet al. 1994) have been described previously. AAYl692 is a Ura- derivative of DBY5217, obtained by selection on 5-FOA, and presumably is ura3/ura3; however, we were unable to confirm that AAYl692 is homozygous for ura3 by tetrad analysis, as it failed tosporulate. "697, AAYl699, AAYl701, and AAYl703 are all isogenic toDBY5217 except at the SAC6 locus. AAYl770 was derived from AAYl692 by incorporation of the K610R (sac630) mutation into the genome of "692, as described in MATERIALS AND METHODS. All other AAY strains listed are sac6 segregants from sac6/+ diploids, which were spontaneous Ts+ revertants of DBY5217 (hmsand BOTSTEIN1989). The DBY strains have been described previously (~MSand BOTSTEIN1989). The KWY strains are from the study of WERTMANet al. (1992).

AA052, AA053,AA078, and AA080, respectively (Figure 1). pAABl17 (containing URA3 cEN4 SAC@ linearized with ApaI The sac61ikontaining plasmid was designated pAAB289. In in the SAC6gene (Figure l),were cotransformed into an actl- all cases, the changes made were confirmed by sequencing 3SAC6+ mutant strain (DBY2001). Ura' transformants should (SANGERet al. 1977), and the phenotypes of the mutations often contain recircularized plasmids that have incorporated were analyzed after the plasmids were used to transform vari- DNA from the mutagenized fragment, as a result of recombi- ous yeast strains. nation between the fragment and linearized plasmid, on ei- PCR mutagenesis: PCR mutagenesis was conducted as de- ther side of the cut ApaI site (hlA et al. 1987; MUHLRADet al. scribed previously(MUHLRAD et al. 1992). Thus, PCR was used 1992). Those plasmids carryinga suppressor mutation should toamplify a 1.19-kb fragment of SAC6 from wild-type give rise to Ts+ transformants. Ura+ Ts+ transformants car- pAAB117 that had been linearized with BamHI (Figure 1). rying potential sac6 suppressor mutations were therefore se- The primers used were AA069 and AA037 (Figure 1). As lected on "ura at370 (Ster several hours at 25"), and plasmids PCR under even normal conditions (i.e., no Mn2+ and no were isolated, amplified in E. coli, and then used to retrans- weighting of dNTPs) is mutagenic, these conditions were used form DBY2001. Those plasmids that again gave rise to Ts+ to minimize multiple mutations in each PCR product. The transformants were analyzed further. The plasmid carrying mutagenized 1.19-kb fragment, together withplasmid the sac630 mutation was designated pAAB291. 94 S. M. Brower, J. E. Honts and A. E. M. Adams

1 .O Kb were Ts+; 14/28 K610R and 12/28 W514C isolates were Ts+ (again, only weakly Ts+ in the case of W514C), and therefore t52 retained the mutation. As these diploids showed poor or no 69 -b sporulation, evenif first transformed with either SAC6 or t 80 ACTlcontaining plasmids, we confirmed that the observed 4- 53 4- 48 Ts+ phenotypes were due to the heterozygous sac6 mutations, 4-49 by integrating (as above) the K610R or W514C mutations into 4-46 DBY2001 (a haploid actl-3 strain). 29/30 K610R and 12/28 4-78 W514C Ura+ transformants were Ts+; however, in contrast to 39 + 4-37 what was seen with the diploids (above), growth ofthe W514C S E CI E CI Bs Ba strains was almost as strong as that of the K610R strains. Sev- b++ + eral Ura+ Ts+ transformants were then crossed to DBYl995 (act1 -3 SAC6+),generating diploids that should be identical I I-' I (except at the URA3 locus-see Table 1) to those derived Start A B stop C from AAYl692 above. "692was previously generated by FIGURE 1.-Locations on a SAC6-containingplasmid a cross between DBYl995 and DBY2001, with subsequent se- (p-117) of restriction sites and oligonucleotides used in lection on 5-FOA (see Table 1).At 37", the actl-3/actl-3 sac6- this study.A 4kb Sad-Bad1insert containing the SAC6gene K610R/+ diploid strain showed the same strong growth, and is shown; the positions of the START and STOP codons,and the actl-3/actl-3 sac6-W514C/+ diploid showed the same the locations of the three clusters of mutations (A,B, and C) weak growth, as before, providing confirmation that the het- referred to in the text, are marked (the sizes of the clusters erozygous K610R and W514C mutations were responsible for are not to scale). The Bad1site of the insert was generated the observed phenotypes. at the NcoI site downstream of SAC6 during cloning. Vector sequences (pRB720) are not shown, but are derived from RESULTS YCp50 (MA et al. 1987) by replacement of the small AatII-SphI segment of YCp50 with the small AatII-SphI region of pUCl9 Suppressor analysis has long been recognized as a (T. STEARNS,Stanford University). The vector does not con- useful tool for identifymg physically interacting pro- tain any of the restriction sites shown, except for an EcoRI site that is immediately 5' of the SacI site, and except at the teins, and the identification of an actin-binding protein junctions with the insert, as indicated. Positions of oligonucle- (Sac6p) through suppression of an actin mutation has otides (numbered and referred to in the text as AAO#) are provided a clear example of the utility of the approach indicated with arrows, the tip of the arrowhead indicating the (ADAMS et al. 1989). To determine the molecular basis location of the 3' end of the oligonucleotide; the position of of the suppression of actl-3 by the sac6 mutations, we the tip is accurate with respect to the sequence, but the rest of the arrow isnot to scale. Sequencesof the oligonucleotides first sequenced 17 different suppressoralleles of SAC6, are shown in MATERIALS AND METHODS. Restriction sites: s, and asked whether they change residues believed to be Sad; E, EcoRI; C1, CM; Bs, BstEII; H, HindIII; Ap, ApaI; Ba, important in the binding of Sac6p to actin. BamHI. The sac6 suppressor mutations map to three discrete regions of the first actin-binding domain: The muta- Incorporation of plasmid-borne sac6-30 (K610R)and sac6 tions identified are listed in Table 2, and the positions 32 (W514C) mutations into the genome: a 3.15-kb EcoRI- BamHI fragment containing an incomplete copy of the sac6 of the residues affectedby all 17 of the suppressor mutagene (beginning 38 bp downstream of the start codon; Figure tions are shown in Figures 1 and 2. A few of the muta- 1) and carrying the K610R or W514C mutation (obtained tions were isolatedmore than once, but altogether nine from PCR- or site-specific mutagenesis, respectively)was sub- different residues were affected. Two important find- cloned into the EcoRI-Bad1 site ofYIp5 (BOTSTEINet al. ings emerged from this analysis. First, the mutations 1979). Plasmid DNA was linearized with BstEII (Figure 1) to direct integration to one of the two copies of the SAC6 gene change residues that fall into three clusters (A, B, and in strain AAYl692 (uru3/uru3 actl-l/actl-1; Table 1). One of C in Figures 1 and 2) of 6, 17, and 5 residues, respec- the homologs should be unaffected by the integration, and tively. Two of the three clusters are in regionsbelieved the other should now contain one complete copy of SAC6 to be important in the actin-binding domain of this and a 5' truncated copy of SAC6, withthe URA3 gene between protein (defined by homology to the family of actin- the two. Depending on where the cross-over event occurs, the binding proteins that contains P-spectrin, a-actinin,dys- complete copy may contain the K610R or W514C suppressor mutation (ROSE1995). Ura+ transformants were therefore trophin, filamin, vertebrate fimbrin, and gelation fac- tested for growth on YEPD at 37". In the case of K610R, 23/ tor; DE ARRUDA et al. 1990; GORLIN et al. 1990), consis- 36 transformants were Ts+,and grew well at this temperature; tent with the notion that these mutations directlyaffect in the caseof W514C, 26/28 transformants were Ts+ but binding to actin (BRESNICKet al. 1990, 1991; LEVINEet showed onlypoor growth at this temperature (the remainder al. 1990, 1992; HEMMINGSet al. 1992; et al. were Ts-). The Ts+ transformants, which presumably carry KUHLMAN the suppressor mutation in the complete copy of SAC6, were 1992; FABBRIZIOet al. 1993; LEBARTet al. 1994). The retained. The URA3 gene and incomplete copy of SAC6 were third cluster may identifya previously unrecognized re- then removed from Ura' Ts+ transformants (carrying K610R gion of the actin-binding domain,or may have indirect or W514C) by selection of Ura- cells on plates containing 5- effects on the ability of Sac6p to interact with actin, FOA (BOEKEet al. 1984); such Ura- cellsusually arise by loop either through conformational changes in Sac6por via out of the extra sequences, and depending on the position of the cross-over event, may leave behind the suppressor muta- other protein(s) . tion (see ROSE 1995). Ura- strains were tested for those that The second findingwas that all 17 mutations change Genetic Analysis of Yeast Fimbrin 95

TABLE 2 sac6 Mutations

sac6 ACTI+ RecombinantsSufficiency' Residue Allele" in Ts-/Ts+? cross to Groupbchanged shown sac61 Ts+ None E140K A No sac68 Ts+ None E 140K A No sac61 7 Ts+ sac6-14, -5 E 140K A Yes sac623 Ts+ None E 140K A No sac64 Ts+ 7 sac6 F145I A Yes sac624 Ts+ None F145V A No sac61 0 Ts+ None W252C B Yes sac614 Ts+ sac617 L259W B Yes sac63 Ts+ None D264Y B No sac6-5 Ts+ sac61 7 D264Y B Yes sac66 Ts- None H268P B Yes sac67 Ts- sac64 H268P B Yes sac61 I Ts+ None L379P C No sac61 6 Ts+ None A380T C No sac615 Ts+ None A383T C Yes sac62 Ts- None A383E C Yes sac619 Ts- None A383E C Yes The sac6 suppressor mutations listed were chosen for analysis from among a collection of 24 independent isolates (ADAMS and BOTSTEIN1989). To maximize the number of different mutations analyzed, two criteria were used to select alleles for sequencing: those that yielded recombinants in crosses to at least one other suppressor (column 3, above) (ADAMS and BOTSTEIN1989); and those that were phenotypically distinct- some alleles were Ts in an ACTI+ background, and some were Ts+ in an ACTl+ background (column 2, above) (ADAMS and BOTSTEIN1989). Other alleles selected randomly were added to the group, giving a total of 17 alleles. " The sac6 allele designations shown were assigned previously (ADAMS and BOTSTEIN1989). Groups A, B, and C correspond to those shown in Figures 1 and 2. 'This column indicates which of the mutations were shown to be sufficient for suppression (see MATENALS AND METHODS).To be sure that the mutations identified were responsible for the suppression phenotype, 1) the mutation was cut out on a fragment that had been completely sequenced and used to replace the corresponding region of wild-type SAC6; this new plasmid was then shown to suppress the Ts phenotype of DBY2001; 2) themutation was shown to cause suppression by site-directed mutagenesis of wild-typeSAC6, and subsequent transformation into DBY2001; in this case, the presence of the mutation on the plasmid was verified by sequencing; or 3) the entire sac6 gene containing the mutation of interest was sequenced on at least one strand. residues in the first of the two actin-binding domains, actin-binding domains of Sac6p) (ADAMS et al. 1991), suggesting there is an importantdifference between the would cause suppression of actl-3 when placed in the two actin-binding domains of Sac6p. identical position in the second actin-binding domain. Isolation of suppressor mutations in the second actin- Isolation of suppessor mutations that change residues in binding domain: The finding that 17 of 17 sac6 muta- thesecond actin-binding domain, by PCE In the first of tions localized to the first putative actin-binding domain the two experiments, a 1.19-kb fragment of SAC6 was suggested that only the first ofthe two domains interacts amplified (from wild-type plasmid pAABl17)and muta- with actin in the region defined by the actl-3 mutation. genized by PCR. This 1.19-kb fragment included -0.1 This was surprising, as we expected that the homology kb of 3' untranslated sequence, the entiresecond actin- of the two actin-binding domains would result in them binding domain (-0.74 kb) , and the region of the first both binding to the same sites on actin. We therefore actin-binding domain that includes the third cluster of asked if it were possible to obtain suppressors of actl- mutations (-0.34 kB; see Figure 1).We chose to in- 3 in the second actin-binding domain. We used two clude the latter sequences as an internal positive con- approaches. First, we used random PCR mutagenesis to trol, so that if we failed to obtain suppressor mutations target thesecond actin-binding domain and ask in the second actin-binding domain, the negative result whether mutations that suppress the actl-3 mutation would be meaningful. Of six plasmids that yielded Ts+ can be obtained in this region of the gene. Second, transformants of strain DBY2001 (actl-3SAC@, four we asked whether a mutation corresponding toW252C showed strong suppression Le., growth in 2 days at 37" (Table 2), which causes a change in a tryptophan abso- (similar to that seen with sac6 suppressor mutations in lutely conserved between the actin-binding domains in the first actin-binding domain), and two showed weak this family of proteins (including the first and second suppression ie., growth in 4 days at 37". The six suppres- 96 S. M. Brower, J. E. Honts and A. E. M. Adams

A * * Sac6p(125) IVAGSQTGTTHTINEEERRE ETKHINSVLA GDQDIGDLLP FPTDTFQLFD Sac6p(404) ...... EEF DAEGEREARV FTLWLNSLDV DPPVIS...... LFD Hm dys (9) DCYER...... EDVQKKT FTKWVNAQFS KFGKQ...... HIENLFS FIGUREZ.-Positions of sac6 suppressor Ck sm aa (18) EDWDRDLLLD PAWEKQQRKT FTAWCNSHLR KAGTQ...... IENIEE Dd abpl20(1) ...MAAAPSG KTWIDVQKKT FTGWANNYL. KERIL...... KIEDLAT mutations in the primary sequence of yeast Ck fil (21) PATEKDLAED APWKRIQQNT FTRWCNEHL. RCVNK...... RIGNLQH fimbrin (SacGp) and their relationship to Identity ---_-______-_-______FT---N"-- ______-______residues implicated in the binding of re- ABS1 lated actin-binding proteins to actin. The actin-binding domains of Sac6p were identified by reference to the boundariesgiven Sac6p (175) ECRDGLVLSK LINDSVPDTI DTRVLN.WPK KGKELNNFQA SENANIVINS Sac6p (4361 DLKDGLILLQ AYEKVMPGAV DFKHVNKRPA SGAEISRFKA LENTNYAVDLfor the actin-binding domains in the align- Hm dys (43) DLQDGRFULD LLE.....GL TGQKL.PK.E KG.ST.RVHA LNNVNKALRV ment of DE ARRUDA et al. 1990. The seg- Ck sm aa (59) DFRDGLKLML LLE.....VI SGElU.AKPE RG.KM.RVHK ISNVNKALDF ment between the two actin-binding do- Dd abpl20(39) SLEDGVLLIN LLE.....II SSKKI.LKYN KAPKI.RMQK IENNNMAVNF Ck fil (62) DLSDGLRLIA LLE.....VL SQKRmRKYH QRPTF.RQMQ LENVSVALEF mains of Sac6p defined in this way Identity "-DG--L-- ______--N"""- (residues 387-403) has not been assigned. The boundary between the A and B subdo- B mains identified by DE ARRUDA et al. 1990 * * ** is indicated ( ] [ ) . The two Sac6p actin-bind- Sac6p(224) AKAIGCVWN VHSEDIIEGREHLILGLIHQ IIRRGLLSKI PIKLHE'ELYR ing domains were aligned relative to each Sac6p (486) GRAKGFSLVG IEGSDIVDGN KLLTLGLVWQLMRR.... NI SITM...... Hm dys (84) LQNNNVDLVN IGSTDIMGN HKLTLGLIWN II...... LHWQVKN other and to other actin cross-linking pro- Ck sm aa (101) IASKGVKLVS IGAEEIMGN VKMTLGMIWT II...... LRFAIQD teins using the PILEUP program from the Dd abpl20(82) IKSEGLKLVG IGAEDIMSQ LKLILGLIWT LI...... LRYQIQ. GCG sequence analysis software package, Ck filCk (106) WSGRASSWLS IDSKAIVDGN LKLILGLIWTLI...... LHYLISM Identity --_--_____-----I"-- "--LG--W- ______-______version 7.0. Residues altered by the suppressor mutations listed in Table 2are indi- ABS3 cated (*); residues changed by the strong, I[ single mutations listed in Table 3 are indi- Sac6p (274) LLEDDETLEQ FLRLPPEQIL LRWFNYHLKQ ANWNRRVTNFSKD.VSDGEN cated by t. In each case, residues affected Sac6p (526) .....KTLSS SGRDMSDSQI LKWAQDQVTK GGKNSTIRSFKDQALSNAHF Hm dys (123) VMKNIMAGLQ QT..NSEKIL LSWVRQST.R NYPQVNVINF .TTSWSDGLA by the mutations are underlined. Most of Ck sm aa (140) I...... SVE ET..SAKEGL LLWYQRKT.A PYKNVNIQNF .HISWKDGLG these mutations fall into three clusters A, Dd abpl2O (120) ...... MSES DN..SPKAAL LEWVRKQV.A PYKVV.VNNF .TDSWCDGRV B, or C. The positions of putative actin- Ck fil (145) PWSEDEGDDD AKKQTPKQRL LGWIQNKI.. ..PYLPITNF .NQNWQDGKA Identity -----_--_-----______L-W"""- """"-F ----____--binding sites, ABS1, ABS2, and ABS3, identified in other studies (LEVINEet al. 1990, ABS2 1992; CORRADOet al. 1994; see text) are t indicated below the sequence alignments. Sac6p 323) YTILLNQLDP ALCSKAPLQT TDLMJ3.. ..RAEQVLQNAEK .LDCRKYLTP SacGp, yeast fimbrin (ADAMS et al. 1991; Ac- Sac6p 571) LLDVLNGIAP GYVDYDLVTP GNTEEERYAN ARLAISIARX .LGALIWLVP cession No. X63867); Hm dys, human dys- Hm dys 169) LNALIHSHW DLFDWNSWC QQSATQ ...R LEHAFNIARY QLGIEKLLDP Ck sm aa ,180) FCALIHRHRP ELIDYGKLRK DDPLTN.... LNTAFDVAEK YLDIPKMLDA trophin (KOENIGet al. 1988; Accession No. Dd abpl20 159) LSALTDSLKP GVREMSTLTG D..AVQ...D IDRSMDIALE EYEIPKIMDA P11532); Ck sm aa, chick smooth muscle Ck fil 190) LGALVDSCAP GLCPDWETWD PSKPVD...N AREAMQQADD WLGVPQLLPP alpha actinin (BARONet al. 1987; Accession Identity ----""-p __--______-______"""-A-- _---______No. P05094); Dd abpl20, Dictyosteliurn dis- C coideum ABP-12O/gelation factor (NOEGEL et al. 1989; Accession No. X15430); Ck fil, **t *t Sac6p i368) SSLVAG...N PKLNmVM LF.... chicken filamin (BARRYet al. 1993; Acces- Sac6p (620) EDINEV.. .R ARLIITFIAS LMTLNK sion No. U00147). Hm dys (216) ED.VDTTYPD KKSILMYITS LFQVLP Ck sm aa (226) EDIVGTARPD EKAIMTWSS FYHAFS Dd abpl20(204) NDM..NSLPD ELSV1TW.S YFRDYA Ck filCk (237) EEI1HPDV.D EHSVMTYLYT FPKAKL Identity """"" """"" """ sor mutations were sequenced, and the changes identi- strong suppression of the Ts defect. This finding indi- fied are shown in Table 3. cates that theK610R mutation is a dominantsuppressor Of the fourplasmids that yielded strong suppression, of the actl-? defect even when present at the chromo- two had mutations in regions encoding the first actin- somal SAC6 locus, and that suppression is as strong as binding domain. Both mutations changed residues not that seen with suppressor mutations that change resi- altered by any ofthe spontaneoussuppressor mutations dues in the first actin-binding domain. This result dem- (compare with Table 2), but affected residues in the onstrates that it is possible to obtain strong, dominant third cluster of changed residues identified above (Ta- suppressor mutations of the Ts actl-? defect in the sec- ble 2; Figure 2). The other two plasmids had mutations ond actin-binding domain. in sequences encoding the second actin-binding do- On the other hand, thesuppression by K610R raised main, and one of these (K610R in mutant 10B) was a the question as to why dominant suppressors that affect single mutation (Table 3; Figure 2). Incorporation of residues in the second actin-binding domain were not the K610R (sac6-30) mutation into the genome of an identified in our first screen (ADAMS and BOTSTEIN actl-3/actl-? mutant diploid or actl-? mutant haploid 1989). One possibility was suggested by the fact that strain (see MATERIALS AND METHODS) also resulted in the actl-? mutation results in a defect in sporulation Genetic Analysis of Yeast Fimbrin 97

TABLE 3 Suppressor mutations obtained by PCR mutagenesis

ABD, Strong/weak Mutant Residue change (s) 1 or 2 suppressor“ sac6 allele’ 15A F381L 15A 1 Strong sac625 5B H384R 5B 1 Strong sac626 5D ‘ S368G, I395V, E399EI395V, S368G, 5D‘ 1‘ Weak sac627 12B‘ A414S, D484GA414S, 12B‘ 2 Strong sac628 7A F560S 17A 2 Weak sac629 1OB K610R 2 Strong sac630 In each case, the entire PCR fragment was sequenced on at least one strand to ensure the mutations shown were the only ones present. In the case of mutant 10B, the whole sac6gene (ie., including the coding sequences outside the PCR fragment) was sequenced on at least one strand to verify that K610R is the only mutation in the entire coding sequence. In the region of the K610R mutation, both strands were sequenced. “Strong” means clear growth in 2 days at 37”; “weak” means clear growth in 4 days at 37”. bThe mutations, or combinations of mutations, listed have been given the sac6 allele designations shown. ‘In mutant 5D, three mutations were identified. One of these mutations, E399E, was a nucleotide change that did not alter the amino acid at this position. A second mutation, I395V, changes a residue that occurs in a region unassigned to either of the actin-binding domains (see Figure 2). The third mutation, S368G alters a residue in the first actin-binding domain. In mutant 5D, as in mutant 12B, we do not know which of the multiple mutations cause suppression.

(NOVICKet al. 1989; see also Table 4), in addition to strain carrying the K610R mutation showed a complete causing a Ts growth defect. The original screen de- failure to sporulate (Table 4). These results show that manded suppression of both defects (A. E. M. ADAMS, the sporulation defect of actl-3/actl-3, which is sup- unpublished data), so we asked whether the failure to pressed by sac6 mutations affecting residues in the first obtain seconddomain mutations such as K610R in that actin-binding domain, is not suppressed by the K610R screen could be explained by a failure of this mutation mutation in the second actin-binding domain, and is to suppress the sporulation defectof actl-3/ actl-3cells. even exacerbated by this mutation (Table 4). This result actl-3/actl-3 sacdx/+ strains (isogenic except for the explains why K610R (and probably others like it), was allele present at the SAC6 locus and in whether they not identified in the previous suppressor analysis (AD- were Ura+ or Ura-; see Table 4), carrying K610R (sec- AMS and BOTSTEIN1989). ond-domain mutation), F1451,W252C, H268P, or The finding that K610R not only fails to suppress, A383E (all firstdomain mutations) were incubated in but even exacerbates, the sporulation defect of actl-3/ sporulation medium at23”. These strains, as well as the actl-3 diploids is interesting. The increased sporulation parent strains DBY5217 and AAYl692 (both actl-3/actl- defect is not dueto additive effects, as the K610R muta- 3 SAC@/SAC6+), were examined microscopically after tion does not have a significant sporulation defect of 5 days. As shown in Table 4, AAYl692 and DBY5217 its own-plasmid pa291 carrying mutant sac630 is yielded <1% sporulation. Strains carrying sac6 muta- able to complement the sporulation defect of a sa&/ tions affecting residues in the first actin-binding do- sac6 null strain (AAYl040) approximately as well as can main allgave sporulation rates of15-280/0, butthe plasmid pAAB117 carrying wild-type SAC6 (28 and 33%

TABLE 4 Ability of various sad mutations to suppress the actl-3/actl-3 sporulation defect

sac6 mutant ABD“ Percent Strain Relevant genotype residue altered sporulated

~~~692actl-3/actl-3 +/+ none - 0.3 DBY5217 actl-3/actl-3 +/+ none - 0.8 ~~~1699actl-3/actl-3 sac6-2/+ A383E 1 23 AAYl701 actl-3/actl-3 sac6-7/+ H268P 1 28 AAYl703 act1-3/adl-3 sac64/+ F145I 1 22 AAYl697 actl-3/actl-3 sac6-10/+ W252C 1 15 AAYl770 actl-3/actl-3 sac630/+ K610R 2 0 Strains listed are isogenic except for the allele at the SAC6 locus and in whether they are Ura+ or Ura-. AAY 1692 and AAYl770 are Ura- whereas all others are Ura+ (see Table 1). In each case ~500cells were counted. In the case of “770, no tetrads were found in numerous additional fields examined. “ABD, actin binding domain. 98 S. M. Brower, J. E. Honts and A. E. M. Adams sporulation, respectively, compared with 0.25%sporula- beyond that defined by just. the actl-3 mutation, we tion with vector alone). This result therefore indicates asked what other actl alleles are suppressed by muta- that it is the specific combination of the actl-? and sac6- tions affecting either of the two actin-binding domains. 30 mutations that is so detrimental during sporulation. In particular, we asked whether it was possible to iden- One possible explanation for the finding thatthe tify any actl mutations that are suppressed by one, but K610R mutation suppresses the actl-? defect under veg- not the other, of the W252C and W514C mutations. etative conditions, but exacerbates the defect under Plasmids pAAB287 (carrying W514C) and pAAB289 sporulation conditions, is that the intracellular environ- (carrying W252C) were therefore used to transform 10 ment (e.g., ionic conditions or proteins that interact temperature-sensitive actl alleles that change residues with actin-Sac6p) during vegetativegrowth optimizes scattered over the surface of actin (WERTMANet al. the interaction between the two mutant proteins, 1992). Transformants were tested for growth at 37", whereas the intracellular environment during sporula- and the results are shown in Table 5. The spectrum of tion interferes with the interaction. mutations suppressed by the two mutations is identical Isolation of a suppasor mutation by site-spea$c mutagene- and, except in the case of actl-3 (see above),the sis: In the second of the two experimentsoutlined strength of suppression is about the same. This result above, we generated a mutation in the second actin- therefore supports the idea that the first and second binding domain identical to one in the first by site- actin-binding domains both bind to the same region specific mutagenesis. The sac6-10 mutation (W252C; of actin and that this site of interaction comprises a Table 2) causes a change in a tryptophan residue of significant part of subdomains 1 and 2 of actin (see the first actin-binding domain that is absolutely con- Table 5). However, as W514C is phenotypically distinct served in all actin-binding domains (including the two from W252C in suppression of both the Ts growth and in Sac6p) in this family ofproteins (Figure 3B of ADAMS sporulation defects of actl/actl diploids, the two actin- et al. 1991). We therefore generated a tryptophan to binding domains of Sac6pare notabsolutely equivalent. cysteine change at the corresponding residue in the second actin-binding domain (W514C; see MATERIALS DISCUSSION AND METHODS), so that we could directly compare the phenotypes of the two mutations. In this study, we have analyzed 17 independent sac6 Centromere-containing plasmids carrying the W514C mutations that were isolated as spontaneous suppres- or W252C mutations were used to transform DBY2001 sors of actl mutations, as well as a collection of sac6 (actl-3 SAC6+), and transformants were tested for mutations obtained by in uitro mutagenesis. The rela- growth at 25 and 37" on YEPD. Both mutations resulted tionship of the altered residues to those implicated in in similar growth at 25". However, the W514C mutation the binding of related proteins to actin, as well as the showed weakerand morevariable suppression than the relationship between the two actin-binding domains, is W252C mutation at 37". When the W514C mutation discussed below. In addition, we consider the implica- was integrated into the genome of DBY2001, it showed tions of our findings for themechanism of suppression, suppression almost as strong as that by W252C. How- both in this case and in general. ever, when W514C was incorporated into the genome Comparison of residues altered by sac6 suppressor of an actl-3/actl-3strain (AAYl692),the resulting strain mutations with residues implicated in the binding of showed very weak to no suppression of both thetemper- related proteins to actin: Previous analysis of the con- ature-sensitivegrowth and sporulation defects com- served actin-binding domain has led to the identifica- pared with an isogenic strain carrying the W252C mutation of three regions that likely interact with actin (Fig- tion (see MATERIALS AND METHODS). This result ure 2). Two of these regions were first identified in indicates that the W514C and W252C mutations can dystrophin, by proton NMR spectroscopy of synthetic both suppress the actl-? defect in haploids, but that peptides that correspondto defined regions of the pro- only W252C is a fully dominant suppressor. This result tein: ABS (actin-binding site) 1 at residues 17-26 and explains why mutations like W514C were not identified ABS2 at residues 128-156 (LEVINEet al. 1990, 1992; in the original selection for dominant suppressors (AD- Figure 2). Within ABS1, four residues (KTFT at posi- AM~and BOTSTEIN1989). tions 19-22) were shown to be particularly important The spectra of actl alleles suppressed by mutations for the interaction, and these residues are absolutely changing residues in the first and second actin-binding conserved between P-spectrin, a-actinin, dystrophin, domainsare identical. The analysisabove demon- and gelation factor (LEVINEet aE. 1992; Figure 2). HOW- strated that mutations that change residues in either of ever, mutagenesis of sequences encoding the corre- the two actin-binding domains of Sac6p can suppress sponding residues of a-actinin (HEMMINGet al. 1992) the actl-3mutation. This result suggested that both the and dystrophin (CORRADOet al. 1994) results in reduc- first and second actin-binding domains of Sac6p inter- tion, but not elimination, of the actin-binding activity act with the region of actin identified by the actl-3muta- of fusion proteins, indicating that these are not theonly tion. To test whether this region of interaction extends regions involved inthe interaction. In thepresent study, Genetic Analysis of Yeast Fimbrin 99

TABLE 5 Suppression of various actl alleles by W514C and W252C Suppressed by actl Altered Actin Strain allele residues subdomainW252C W514C KWY361 actl-120 99,100 1 DBY2001 actl-3 32 1 KWY305 actl-133 24, 25 1 DBYl993 actl-2 58 2 AAYl644 actl-7 61 2 KWY376 actl-125 50, 51 2 KWY417 actl-105 311, 312 3 DBY2326 actl-4 258 4 KWY310 actl-108 256, 259 4 KWY410 actl-112 213-215 4

Ten temperature-sensitive ura3 actl strains were transformed with plasmids carrying sac6 mutant genes containing either the W252C or W514C mutation. "+" indicates the transformed strain grew significantly better at 37" than did the untransformed parent; "-" indicates lack of suppression of the Ts defect in comnarison to the untransformed Darent. The actl alleles of the KWY strains are all marked with the HIS3 gene' (Table 1). we show that two residues (E140 and F145) of Sac6p (Table 3)], occur in a region of the 27-kD actin-binding that are changed by suppressor mutations occur in or domain not previously implicated in the binding to ac- adjacent to this region. One of the mutant alleles F145 tin. It is possible that we have identified yet another changes a phenylalanine that corresponds to F21of region that interacts directly with actin. Alternatively, it dystrophin (Figure 2). This result suggests that this resi- may be that the sac6 mutations affect the interaction due is important in the binding of fimbrin to actin more indirectly, perhaps througha more global change and makes it likely that this conserved phenylalanine is in the conformation of SacGp, or via some other pro- involved in the interactionof most, if not all, members tein. Interestingly, suppressor analysis of the tempera- of this family of actin-binding proteins to actin. ture-sensitive A383E (sac62) mutation, which changes A third region of the broadly defined actin-binding a residue in this region, results in an extragenic suppres- domain, ABS3 (CORRADOet al. 1994; Figure 2), is a sor mutation in a gene other than ACTl (ADAMS and highly conserved 27-residue peptide thatwas first shown BOTSTEIN1989). In contrast, suppressor analysis of the to be involved in the binding of Dictyostelium gelation temperature-sensitive H268P (sac66 and sac6-7) muta- factor to actin (BRESNICKet al. 1990, 1991). More re- tion (which changes a residue in ABS3; see above and cently, the homologous region has been shown to be Figure 2), results in extragenic suppressor mutations in important in the binding of Bcr-Ab1 (MCWHIRTERand ACTl only. Further analysis should reveal whether the WANG1993), filamin (LEBARTet al. 1994), and a-actinin suppressor of sac62 identifies anothergene whose (KUHLMANet al. 1992) to actin. In the case of a-actinin product binds to Sac6p. (KUHLMANet al. 1992) and filamin (LEBARTet al. 1994), Mechanism of suppression: The results described in thehydrophobic residues in this 27-merhave been this study haveimplications for understanding the mo- shown to be particularly important for the interaction. lecular basis of suppression. Because individual muta- Interestingly, one of the sac6 suppressor mutations we tions in SAC6 can suppress a wide variety ofactin muta- identified, W252C, changesan absolutely conserved tions in subdomains 1 and 2, it is unlikely that each tryptophan thatis found within the hydrophobic region suppressor restores a specific lost interaction. More of this highly conserved 27 amino acid sequence; in plausible is the possibility that thesac6suppressor muta- addition, three other sac6 suppressors also change resi- tions increase the overall af€inity between Sac6p and dues in this region (Figure 2). These findings make it actin, thereby overcoming weaker interactions caused extremely likely that this segment is also important in by any one of a number of actl alleles. In this view, the the binding of fimbrin to actin, and again suggest that mutant sac6 alleles might also increase the interaction this region of the actin-binding domain is important with wild-type actin, and those sac6 mutant alleles that for the interaction of most, if not all, members of this show defects in an ACTl+ genetic background (ie., family of actin-binding proteins with actin. H268P and A383E; Table 2) may give rise to interac- The remaining sac6 mutations change residues at the tions with wild-type actin that are too strong. It might end of the first actin-binding domain (Figure 2). These seem surprising if suppressors that change absolutely mutations [L379P, A380T, A383T and A383E (Table conserved residues (F145 and W252; Figure 2) result 2), as well as F381L and H384R from PCR mutagenesis in a stronger interactionwith actin. However, for actin- 100 S. M. Brower, J. E. Honts andE. A. M.Adams filament bundling, and thus the fimbrin-actin interac- fecting residues in either domain restore the overall tion, to be reversible, the optimal wild-type interaction interaction to some required level. might not necessarily be the strongest. Although we favor the notion that thetwo actin-bind- Precedent for this mechanism of suppression comes ing domains of Sac6p do bind to thesame site on actin, from an analysis of second-site mutations of lambda we suggest there are differences between the two do- repressor that suppress defects in the bindingof repres- mains that result in nonidentical interactions with that sor to operator sequences; such mutations suppress by site on actin, as the W514C and W252C mutations show globally increasing the affinity ofrepressor for operator different levels of suppression of both temperature-sen- sequences (NELSONand SAUER1985). We suggest that sitivity and sporulation and the K610R mutation sug- suppression may frequently occur by this type of mecha- gests that different residues may be involved in the in- nism, especially as the target size for such suppressors teraction of the second actin-binding domain with is much larger than that for more specific suppressors. actin-no mutations corresponding to K610R were ob- Comparison of the two actin-binding domains of tained among the 17 spontaneous suppressors in the Sac6p: Chicken fimbrin is a monomeric protein first actin-binding domain. However, a moreexhaustive (BRETSCHER1981; GLENNEYet al. 1981) and presumably mutational analysis ofthe second actin-binding domain cross-links actin filaments via the two homologous actin- would be required to clarify this point. Differences be- binding domains (DE ~UDAet al. 1990). Possible ways tween some of the actin-binding domains in this family in which this cross-linking might occur are the two ac- of proteins have been noted before (e.g., LEBARTet al. tin-binding domains might both bind to the same sur- 1993; SENTERet al. 1993). face of actin, and thereby cross-link adjacent actin fila- We thank ROYPARKER, BRUCE PATTERSON, DANNY BROWER, SHARON ments symmetrically or the two actin-binding domains STROBEL,KURT TOENJES,and DANADAVIS for critical comments on might bind to different regions of actin, and thuscross- the manuscript, and members of our lab for helpful discussions during the course of this work. This work was supported by National link actin filaments asymmetrically. Inthe former Institute of General Medical Sciencesgrant (GM-45288) and the PEW model, we would expect that thesame spectrum of actl Scholars Program (to A.E.M.A.), and a postdoctoral National Re- mutations would be suppressed by mutations affecting search Service Award (CA-09213) to J.E.H. either actin-binding domain, whereas in thelatter model, we would expect that different classes of actl LITERATURE CITED mutations (that affect different surfaces of actin) would be suppressed by sac6 mutations affecting one, but not ADAMS, A. E. M., and D. BOTSTEIN, 1989 Dominant suppressors of yeast actin mutations that are reciprocally suppressed. Genetics the other, actin-binding domain. Previously, we found 121: 675-683. that actin mutations that show suppression with sac6 ADAMS, A. E. M., D. BOTSTEINand D. G. DRUBIN,1989 A yeast actin- mutations all change residues in subdomain 1 or 2 of binding protein is encoded by SAC6, a gene found by suppression of an actin mutation. Science 243 231-233. actin (HONTSet al. 1994). As the sac6 mutations exam- ADAMS, A. E. M,, D. BOTSTEINand D. G. DRUBIN,1991 Requirement ined in that study all affect residues in the first actin- of yeast fimbrin for actin organization and morphogenesis in vivo. Nature 354: 404-408. binding domain of Sac6p (see above), these results sug- BARON,M. D., M. D. DAVISON, P.JONES and D. R. CRITCHLEY, 1987 gest that subdomains 1 and 2 of actin interact with the The sequence of chick a-actinin reveals homologies to spectrin first actin-binding domain of Sac6p. and calmodulin. J. Biol. Chem. 262: 17623-17629. BARRY, C. P.,J. NE,V. LEMMONand A.P. YOUNG, 1993 Molecular In the present study, we found that mutations in the characterization of a multipromoter gene encoding a chicken second putative actin-binding domain of Sac6p alsosup- filamin protein. J. Biol. Chem. 268: 25577-25586. press actl alleles in subdomain 1 or 2, but not those in BOEKE,J. D., F. LACROUTEand G. R. FINK,1984 A positive selection for mutants lacking orotidine6’-phosphate decarboxylase activ- subdomain 3 or 4 (Table 5). This result suggests that ity in yeast: 5-fluoro-orotic acid resistance.Mol. Gen. Genet. 197: both Sac6p domains bind subdomains 1 and 2 of actin. 345-346. BOTSTEIN,D. S. C. FAICO, S. E. STEWART,M. BRENNAN,S. SCHERER The homology between the two actin-binding domains et al., 1979 Sterile host yeasts (SHY): a eukaryotic system of is, of course, consistent with this conclusion, although biological containment for recombinant DNA experiments. in the case of scruin, an unrelated actin-cross-linking Gene 8: 17-24. BRESNICK,A.R., V. WARRENand J. CONDEEIJS, 1990 Identification protein, there is precedent for two homologous actin- of a short sequence essential for actin binding by Dictyostelzum binding domains binding to different surfaces on actin ABP-120. J. Biol. Chem. 265 9236-9240. (OWENand DEROSIER 1993;SCHMID et al. 1994). If both BRESNICK,A. R., P. A. JANMEYand J. CONDEEUS, 1991 Evidencethat a 27-residue sequence is the actin binding site of ABP-120. J. actin-binding domains ofSacGp do bind to the same Biol. Chem. 266 12989-12993. region of actin, it is interesting that suppression can BRETSCHER, A,, 1981 Fimbrinis a cytoskeletal protein that crosslinks occur through a change in just one domain at a time, F-actin in vitro. Proc. Natl. Acad. Sci. USA 78: 6849-6853. CORRADO,R, P. L. MILLS and J. S. CHAMBERLAIN,1994 Deletion ie., that interactions with both Sac6p actin-binding do- analysis of the dystrophin-actin binding domain. FEBS Lett. 344: mains do not have to be altered simultaneously. The 255-260. most likelyexplanation is that theactl mutations reduce DE ARRUDA, M. V., S. WATSON,C.S. LIN,J. LEA~and P. 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