Mutations That Enhance the cap2 Null Mutant Phenotype in ... · standard methods (MANIATIS, FRITSH and SAMBROOK 1982). The integrating plasmids pBJO13 (cap2-AI::HZS3) and pBJl00 (cap2-Al::LEU2),

Copyright 0 1993 by the Genetics Society of America

Mutations That Enhance the cap2 Null Mutant Phenotype in Saccharomyces cerevisiae Affect the Actin Cytoskeleton,

Morphogenesis and Pattern of Growth

Tatiana S. Karpova, Marc M. Lepetit' and John A. Cooper

Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri 631 10-1093 Manuscript received April 2 1, 1993

Accepted for publication July 22, 1993

ABSTRACT Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene

encoding the /3 subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slcl- 6 6 , slcl-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complemen- tation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these resuits suggest that slcl and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

T HE genes CAPl and CAP2 of Saccharomyces cerevisiae encode the nonhomologous a and P sub-

units, respectively, of the heterodimeric actin-binding protein, capping protein (AMATRUDA et al. 1990, 1992). Capping protein is a member of a family of Ca*+-independent actin-binding proteins, found in all eukaryotic organisms examined (POLLARD and COOPER 1986; VANDEKERCKHOVE and VANCOMPER- NOLLE 1992). Capping protein binds to the barbed ends of actin filaments and accelerates nucleation of actin filament assembly (CALDWELL et al. 1989; CA- SELLA et al. 1987). In vertebrate striated muscle, capping protein is found at Z-disks (CASELLA et al. 1987), where the barbed ends of the actin-containing thin filaments are also found. During myofibrillogenesis the appearance of capping protein at Z-discs precedes the organization of actin filaments in I-bands (SCHAFER, WADDLE and COOPER 1993). These obser- vations suggest that capping protein regulates actin filament assembly in the sarcomere. In epithelial cells, capping protein is found by immunofluorescence at the junctional complex, a region where actin filaments are in close proximity to the plasma membrane (SCHAFER, MOOSEKER and COOPER 1992). Similarly, in yeast, capping protein colocalizes with the actin patches associated with the plasma membrane, although not with actin cables or the cytokinesis ring

' Present address: ENSAM-INRA-CNRS-URA 573. Biochimie et Physiol- ogie Vegetale, 34060-Montpellier, Cedex, France.

Genetics 1 3 5 693-709 (November, 1993)

(AMATRUDA and COOPER 1992). These data suggest that in vivo capping protein may mediate attachment of actin filaments to membranes.

In S. cerevisiae, loss-of-function mutations in ACTI, which encodes conventional actin (SHORTLE, HABER and BOTSTEIN 1982), and ACT2, which encodes an actin-related protein (SCHWOB and MARTIN 1992), are lethal. Mutations in the genes for some actin-binding proteins, including a dilute myosin heavy chain (JOHN- STON, PRENDERGAST and SINGER 1991) and cofilin (MOON et al. 1993) are also lethal. In contrast, CAPl and CAP2 are not essential genes. Complete disruption of CAPl or CAP2 leads to slow growth, cell size heterogeneity, loss of actin cables, and depolarization of the cortical actin patches. Loss of one protein subunit, due to gene disruption, greatly destabilizes the other subunit, thus cap lA and cap2A single mutants have similar phenotypes, which are identical to those of caplAcap2A double mutants (AMATRUDA et al. 1990, 1992).

The actin cytoskeleton is regulated by many proteins in addition to capping protein. Double mutants of cap2(capl) and sac6, the gene encoding the actin- binding protein fimbrin, are inviable. This genetic interaction is specific as it is not observed when CAP2 is disrupted in combination with other known non- essential genes for actin-binding proteins (ADAMS, COOPER and DRUBIN 1993). The mechanism for the genetic interaction between capping protein and fim-

694 T. S. Karpova, M. M. Lepetit and J. A. Cooper

brin is unknown; however, the proteins may interact indirectly via actin filaments in the cortical patches, since they are both found in patches.

To investigate the role of capping protein in regulation of the actin cytoskeleton and to search for novel components and regulators of the actin cytoskeleton, we screened for genes interacting with cap2 null mutations. We found three genetic loci, including SAC6, mutations in which lead to synthetic lethality in combination with cap2A. These enhancer mutations are semidominant and interact with each other in diheterozygotes. The mutations affect the distribution of actin and cause pseudomycelial growth in a wild-type CAP2 background.

MATERIALS AND METHODS

Strains and Plasmids: Strains used in this work are listed in Table 1. All DNA manipulations were performed by standard methods (MANIATIS, FRITSH and SAMBROOK 1982). The integrating plasmids pBJO13 (cap2-AI::HZS3) and pBJl00 (cap2-Al::LEU2), used for cap2 disruption, contain sequences flanking the CAP2 coding region. pBJ0 13 was described elsewhere (AMATRUDA et al. 1990). pBJl00 (cap2- Al::LEU2), was made by inserting the fragment of pBJO13 containing the CAP2-flanking sequences into pRS305 (SIKORSKI and HIETER 1989). The integrating plasmid pBJO53 (or AAB123) was provided by ALISON ADAMS and used for disruption of SAC6 with LEU2 (ADAMS, BOTSTEIN and DRUBIN 1991). The replicative plasmid pBJ198 (CAP2 URA3 ADE3 2 4 was constructed by inserting a 2.2-kb XbaI/ BamHI fragment, containing the entire sequence of CAP2 (AMATRUDA et al. 1990), into the polylinker of pTSV3 1A (URA3 ADE3 2 4 (kindly made available to us by MICHAEL F. TIBBETTS and JOHN R. PRINGLE, University of North Carolina). The replicating plasmid pBJO7l (CAP2 TRPl ARSHI CEN6) was constructed by inserting a 1.96-kb BamHI/PstI fragment, containing the entire sequence of CAP2 (AMATRUDA et al. 1990), into the polylinker of pRS314 (pBJ080) (SIKORSKI and HIETER 1989).

Materials, media and culture conditions: Unless otherwise noted, chemicals, materials and solvents were from Fisher Scientific (St. Louis, Missouri) or Sigma Chemicals (St. Louis, Missouri). Restriction endonucleases and other enzymes were purchased from Boehringer-Mannheim (In- dianapolis, Indiana). Yeast rich medium (YEPD) and synthetic minimal medium with different metabolites omitted for selection (DOBA), were purchased from BIOlOl (La Jolla, California). Medium containing 0.1 % 5-fluoro-orotic acid (FOA, PCR Inc., Gainesville, Florida) was prepared as described (ROSE, WINSTON and HIETER 1990). Several different high osmolarity media were used. They were YEPD with the addition of 450 mM NaCl (OSM4), 900 mM NaCl (OSM9), 1.8 M sorbitol (SORB), and 2 M ethylene glycol (EG). For assays of cell wall strength, the sorbitol-containing medium was YEPD with 1 M sorbitol. Pre-sporulation medium was YEPD with adenine (200 Kg/ml). Sporulation medium contained 1% potassium acetate and 0.1% Bacto- yeast extract. Strains were grown at 30" unless specified otherwise.

Genetic techniques: Tetrad dissection, random spore analysis, and yeast transformation were as described (ROSE, WINSTON and HIETER 1990). CAP2 disruption was performed with the gamma-loop technique (SIKORSKI and HIETER 1989); disruptions of CAP2 were verified by West- ern blot, using affinity-purified anti-Cap2p (AMATRUDA et al. 1992). For the plasmid substitution ("shuffle") assay,

strains carrying pBJ198 (CAP2 URA3) were transformed with pBJO71 (CAP2 T R P l ) or pBJ080 (TRPI), plated onto Trp- medium, which is nonselective for pBJ198, and then replica-plated on FOA. Appearance of secondary colonies on FOA signified loss of the URA3"containing pBJ198.

Mutagenesis and screen: T o induce mutations resulting in synthetic lethality with cap2A, cells grown in liquid YEPD to a density of 2 X 10' ml" were treated with 3% ethyl methanesulphonate (EMS) (10-35% viability) using a rou- tine protocol (ROSE, WINSTON and HIETER 1990) and plated on YEPD at a density of 100-500 colonies per plate. To screen for synthetic lethals, we used a colony color assay (BENDER and PRINCLE 1991). The mutagenized cap2A ade2 ade3 u r d strains contained a plasmid, pBJ198, which carried a 2~ origin of replication, ADE3, URA3 and CAP2 and which resulted in red colony color for ade2 ade3 mutants. Plasmid loss resulted in white colony color. Since cap2A strains are viable, pBJ198 could be lost, and on YEPD colonies of these strains were red with white sectors. Screens for non-sectored colonies were done at room temperature, 30" and 35"

Analysis of temperature sensitivity, osmotic sensitivity, cell wall strength and plasmid stability: Sensitivity of growth to increased temperature (Ts) and increased osmotic pressure (Os) was analyzed by plating cell suspensions onto solid media, because standard replica-plating gave inconsist- ent results. Growth on these media was monitored for 6 days.

As a test of the strength of the cell wall and membrane, cells were grown in liquid YEPD with or without 1 M sorbitol to late exponential phase, transferred to water for 30 min and stained with 0.01 % trypan blue in water to detect cell lysis. Lysed cells take up the blue dye. As a control, cells were also stained with 0.01% trypan blue directly in YEPD medium with or without sorbitol before transfer to water.

The frequency of pBJ 198 plasmid loss was estimated by plating serial dilutions of plasmid-bearing strains on FOA and YEPD; the ratio of single colonies grown on FOA to those growing on YEPD was determined with corrections for proper dilution. Strains were pre-grown on Ura- plates, and then multiple different colonies were analyzed by dilution and plating as above, to minimize the effects of population fluctuation.

Fluorescence microscopy: Goat anti-yeast actin anti- serum was prepared by immunizing a goat 3 times at 2-week intervals with 0.5-1 mg of yeast actin purified by DNase1 affinity chromatography (KRON et al. 1992). Antibodies were affinity-purified on a column of yeast actin, using elution with 0.1 M glycine, pH 2.8. An immunoblot documented the specificity of the antibody (data not shown). Total yeast cell lysate and 20 ng of purified yeast actin were electrophoresed, blotted and probed with 1 pg/ml afflnity- purified goat anti-yeast actin antibodies. The lysate sample showed only a single band, which co-migrated with the actin standard. Preparation of rabbit anti-yeast capping protein was as described (AMATRUDA et al. 1992).

For actin staining (DRUBIN, MILLER and BOTSTEIN 1988) affinity-purified goat anti-yeast actin antibodies at 10 rg/ml and FITC-conjugated affinity-purified rabbit anti-goat antibodies (Chemicon, El Segundo, California) were used. For capping protein staining (AMATRUDA et al. 1992) affinity- purified anti-capping protein rabbit antibodies at 1 pg/ml, secondary unlabeled affinity-purified goat anti-rabbit antibodies and tertiary rhodamine-conjugated affinity-purified donkey anti-goat antibodies (Chemicon) were used. Note that as previously (AMATRUDA et al. 1992), this procedure involves amplification with a third antibody and that nonspecific binding by the primary, secondary, and tertiary antibody was reduced by absorption against fixed null cells. Staining with rhodamine-phalloidin was as described (AMA-

Enhancers of cup2 Null Mutations

TABLE 1

Yeast strains used in thie study

695

Strain Genotype

YJC365" YJC366' YJC509 YJC508

YJC447' YJC449' YJC450' YJC452' YJC467d YJC469' YJC476 YJC479 YJC453h YJC454h YJC456h YJC457h YJC590h YJC728 YJC837 SL87' YJC727 YJC76 1 YJC762 YJC757 SL66' YJC755 YJC726 YJC792 YJC753 YJC754 SL69' YJC788 YJC730 YJC729 YJC785 YJC786 SL107' YJC766 YJC768 YJC767 YJC87 1

YJC320-9A

MATa ade2 ade3 ura3 leu2 trpl MATa ade2 ade3 ura3 leu2 lys2 MATa ade2 ade3 ura3 lysl leu1 leu2 trp2 arol metl thr 1 hisX MATa ade2 ade3 ura3 lysl lys2 leu2 trp2 arol metl thr 1 hisX arg4 MATa ade2 his3 leu2 trpl ura3 cap2-Al::HIS3 MATa ade2 ade3 ura3 leu2 trpl capZ-Al::LEV2 MATa ade2 ade3 ura3 leu2 lys2 capZ-Al::HlS3 MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 MATa ade2 ade3 ura3 leu2 trpl cap2-Al::LEU2 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 capZ-Al::HIS3 [pBJ198] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 [pBJ198] MATa ade2 his3 leu2 ura3 trpl cap2-Al::HlS3 sac6::LEU2 [pBJ198] MATa ade2 his3 leu2 ura3 trpl cap2-Al::HIS3 sac6::LEU2 [pBJ198] MATa ade2 his3 leu2 ura3 trpl cap2-Al::HIS3 sac6::LEU2 [pBJl98] MATa ade2 his3 leu2 ura3 trpl cap2-Al::HIS3 sac6::LEU2 [pBJ198] MATa ade2 ade3 leu2 ura3 lys2 cap2-Al::HIS3 sac6::LEU2 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 capP-Al::HIS3 MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HISjr MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HISP slcl-87 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 slcl-87 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 slcl-87 [pBJ198] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 slcl-87 [pBJ198] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 slcl-87 [pBJ198] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 slcl-66 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 slcl-66 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HISJ) slcl-66 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 slcl-66 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 slcl-66 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 slcl-66 [pBJI98] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 sac6-69 [pBJ198] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 sad-69 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 s a d - 6 9 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 caQ2-Al::HIS3 sac649 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 capZ-Al::HIS3 sad-69 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HlS3 sad-69 [pBJ198] MATa ode2 ade3 ura3 leu2 trpl cap2-Al::HIS3 slc2-107 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HIS3 slc2-107 [pBJ198] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HIS3 slc2-107 [pBJ198] MATa ade2 ade3 ura3 leu2 lys2 cap2-Al::HlS3 slc2-107 [pBJ198] MATa ade2 ade3 ura3 leu2 trpl cap2-Al::HlS3 slc2-107 [pBJ198]

" These two strains were provided by ALISON ADAMS (University of Arizona, Tucson). All other strains were obtained in this study.

' Meiotic segregant of YJC366XYJC320-9A. d-g YJC447, YJC449, YJC450, YJC452, respectively, transformed with plasmid pBJ198, which carries CAP2, ADE3, and URA3 and is

YJC365 transformed with pBJl00 for deletion disruption of CAP2.

described in MATERIALS AND METHODS. SAC6 disrupted by integration of pBJO53 (AABl23). A mutant of YJC472.

TRUDA et al. 1992). To examine the pattern of bud scars and the chitin distribution, cells were stained with 50 pg/ml calcofluor white M2R (PRINGLE 1991). Cells were observed with a IOOX 1.3 N.A. Plan-Neofluar objective on an Axio- vert 10 microscope (Carl Zeiss, Oberkochen, Germany) and photographed with Kodak TMax 400 or 100 film for fluorescence or TechPan film for differential interference contrast microscopy (DIC).

RESULTS

Colony color assay for mutations conferring synthetic lethality in combination with eap2A: Four

different strains (YJC467, YJC469, YJC470, YJC472) that carried cap2A, the mutations ade2, ade3 and ura3, and the plasmid pBJl98 with the wild type genes CAP2 AD3 URA3 were constructed. The basis for the colony color screen (BENDER and PRINGLE 1991) is that the viability of strains with mutations leading to synthetic lethality with cap2A depends on the presence of the plasmid with C M 2 . The presence of ADE3 confers red color to ade3 ade2 strains. Because cap2A mutants are viable, wild-type colonies of these strains on YEPD are red with white sectors arising from spontaneous plasmid loss, and when plated on FOA, these strains


TABLE 2

Frequency of colonies stable for pBJ198 (CAP2 D E 3 URA3)

Temperature

Room temperature 30" 35"

Total Strain (XlO') Stable ( X l O ' ) Stable (xl0') Stable

Total Total

YJC467 25 30 YJC469 18 14 YJC470 2.9 0 35 19 3 4 YJC472 2.6 0 28 42 3 12

Total 48.5 44 63 61 6 16

Nonsectored red colonies were induced by EMS and screened in the colony color assay.

segregate Foa+ (plasmid-less) colonies. The viability of strains with mutations leading to synthetic lethality with cap2A depends on the presence of the plasmid containing CAP2, and thus colonies of these strains are uniformly red and Foa- (unable to grow on medium containing FOA).

The development of sectors varied with incubation temperature and genotypic background. At 35" the red colony color was weak, and sectors were difficult to distinguish. Thus, the most extensive screen was performed at 30°, where color development was better. Among the four strains tested, YJC472 was the most suitable for screening because the rate of plasmid loss from this strain was the highest, and sectors were multiple and clearly visible. 1 17,000 colonies treated with EMS were screened in total, and 121 uniformly red colonies were selected (Table 2). These strains, which might carry synthetic-lethal mutations, were designated "SL."

In addition to synthetic lethals, this screen can also select (a) chromosomal or plasmid-borne mutations increasing plasmid stability, (b) plasmid integration or (c) conversion of chromosomal ade3 to ADE3. Several addition screens were performed to distinguish among these possibilities.

Strains stable for ADE3 were tested on FOA for stability of the second plasmid marker URA3. Strains from Ura- medium (conditions selective for pBJ198) were replica-plated onto FOA and screened for the ability to segregate secondary Foa+ colonies. 64 Foa' strains, which are presumably ade3 conversions or leaky synthetic lethals, were discarded in this test. Foa- strains might include synthetic lethals, as well as integrants or mutants with increased plasmid stability.

The remaining 57 SL strains were crossed with strains either mutant or wild-type for CAP2 to gener- ate hybrids homozygous or heterozygous for cap2A. The cap2A strains were YJC450, YJC449, YJC452, and YJC447, and the CAP2 strains were YJC508, YJC509, YJC366, and YJC365, depending on mating type and genotype of the particular SL strain. The hybrids were screened with the colony color sectoring

assay for the ability to lose the plasmid marker ADE3. This test detected plasmid integration or dominant mutations that enhance plasmid stability as cases in which the plasmid markers are stable in both cap2Al cap2A and capZAlCAP2 hybrids. Twenty-seven mutants formed hybrids stable for plasmid markers with both strains and were discarded. All the strains selected at 35" were among this group.

Among the remaining SL strains 29 formed hybrids unstable for ADE3 with both tester strains, which indicates that the mutations are synthetic lethals and are recessive or semi-dominant. One mutant (SL87) showed plasmid marker stability in the cap2/cap2 hybrid, and instability with the cap2/CAP2 hybrid, which indicates that its mutation leads to synthetic lethality with cap2A and is dominant. The capZlcap2 hybrids of SL87 heterozygous for this mutation generated rare Foa+ secondary colonies, thus the mutation is semi-dominant, as described further below.

This group of 30 mutants could include recessive mutations that enhance plasmid stability or lead to dependence on the plasmid markers URA3 and ADE3. T o prove that we have synthetic lethals in this group, several SL strains that carried trpl were transformed with either pBJO7 1 (TRPI CAP2 CEN ARS) or pBJ080 (TRPI CEN ARS). The viability of haploid synthetic lethals should depend only on the presence of wild- type CAP2, and therefore different plasmids carrying CAP2 should be interchangeable-pBJ07 1, but not pBJ080, should be able to replace pBJ198. Alterna- tively, if an SL mutation increases pBJ198 stability, then this plasmid will remain stable either in a cap2 or CAP2 background, and neither pBJO7 1 nor pBJ080 will be able to promote loss of pBJl98.

For each SL strain tested, 36 independent transformants with either pBJO71 or pBJ080 were pregrown on Trp- medium, in conditions nonselective for pBJ198, and checked for the ability to lose URA3 on FOA. In some cases the ability to lose the other pBJ 198 marker, ADE3, was also checked in parallel to ensure that both markers behaved in a similar manner. For 9 mutants (SL87, SL66, SL69, SL107, SL81, SL96, SL91, SL77 and SL23) pBJ198 could be re- placed by pBJO71 but not by pBJ080. In 5 cases (SL103, SL70, SL101, SL71, SL25) transformants bearing pBJO7 1 behaved heterogeneously-plasmid was destabilized to different extents. The cause of this apparent slow rate of plasmid loss may be the slow growth rate of these mutants, which causes only a small number of cells to lose all the URAtcontaining plasmid copies. For 7 mutants (SL75, SL95, SL85, SL65, SL17, SL13 and SL18) pBJ198 was not destabilized by either pBJ080 or pBJO71 plasmids.

Thus from 30 SL strains chosen for future analysis, a total of 14 mutants were CAP2 dependent and so carry mutations leading to synthetic lethality in combination with cap2. For 7 mutants the results were

Enhancers of cap2 Null Mutations 697

TABLE 3

Monogenic inheritance of synthetic lethality

Parental Source of Tetrads Foa--TRPI Spore

viability, strains mutation ~ o t a ~ 2+:2- PD NPD T %a

YJC472XYJC450 Wild type 52 0 97

SL66XYJC450 SL66 15 14 2 2 10 80(80) YJC472XYJC26 SL66 12 12 0 3 9 88 Total 27 26 2 5 19

SL87XYJC450 SL87 21 20 3 0 17 72(62) YJC472XYJC727 SL87 12 10 1 1 8 86 Total 33 30 4 1 25

SL69XYJC450 SL69 18 17 2 2 13 90(87) YJC472XYJC729 SL69 13 13 3 3 7 78 YJC472XYJC730 SL69 15 14 1 1 12 87 Total 46 44 6 6 32

SL107XYJC450 SL107 15 14 2 2 10 77(86) SL107XYJC728 SL107 12 10 3 2 5 85 YJC766XYJC837 SL107 24 20 5 2 13 81 Total 51 44 10 6 28

Monogenic inheritance is shown by the high frequency of 2+:2- tetrads among the total. The absence of centromeric linkage is shown by the distribution of PD, NPD and T tetrads. Chi-square analysis in each case showed no statistically significant decrease in the number of T tetrads from that predicted for no centromeric linkage.

The percentage of Foa- among the dead spores is given in parentheses.

negative, and the remaining 9 mutants were not tested because they do not contain proper markers to intro- duce the second plasmid.

Monogenic inheritance of the synthetic-lethal phenotype: We crossed all 30 of these mutants, including those not tested or negative in the plasmid shuffle test above, with strains bearing cap2A and pBJ198 (YJC472 or YJC470, depending on the mating type of the mutant) and performed tetrad analysis. The stability of plasmid pBJ198 in the meiotic segregants was checked on FOA. Seven tetrads from two hybrids (SL66 X YJC470 and SL87 X YJC470) were also screened in the colony color assay to prove that both plasmid markers, URA3 and ADE3, co-segregate.

Hybrids fell into two groups-6 displayed 2+ : 2- segregation on FOA (monogenic inheritance of the synthetic-lethal phenotype), and 24 segregated 3+: 1- and 4+ : 0- tetrads, in addition to 2+ : 2- tetrads, which indicates polygenic inheritance. Spore viability in these hybrids heterozygous for the SL mutations was low compared to that of wild type. In hybrids displaying 2+ : 2- segregation we were able to deduce the phenotype of dead ascospores and found a slight bias towards Foa-, which indicates that mutations in the SL strains lower the viability of the meiotic segregants (Table 3).

Monogenic segregation was displayed by SL87, SL66, SL69 and SL107, which were unambiguously dependent on the CAP2 plasmid for viability above, by SL104, which was not tested in the plasmid shuffle

experiments due to poor transformation efficiency, and by SL65, which showed no clear dependence on the CAP2 plasmid. Monogenic inheritance was confirmed in additional crosses of Foa- segregants with cap2A strains (Table 3). Segregants of SL104 and SL65 were tested in the plasmid shuffle and found to carry a mutation conferring plasmid stability that was not specific for CAPP.

Thus, we conclude that SL87, SL66, SL69, SL107 and their segregants carry mutations conferring synthetic lethality with cap2A and designate them slc (for synthetic lethality with rap2). They were provisionally given the same numbers as the mutant strains (slc87, slc66, slc69, and slc107). The pedigree of segregants from these outcrosses, which were used in further tests is presented in Figure 1. None of the genes display centromeric linkage, based on the (PD+NPD) : T ratios for TRPl and Foa- (Table 3).

Recombination allelism tests: We wanted to estab- lish whether any of the mutations are allelic to each other or to sac6, null mutations in which are lethal in combination with cap2 (ADAMS, COOPER and DRUBIN 1993). To avoid effects of semi-dominant mutations and their interactions in diheterozygotes, we performed a recombinational rather than a functional test for allelism. If two slc mutations are allelic, then Foa+ segregants in meiosis should arise only by inter- allelic recombination, which occurs at a low frequency relative to intergenic recombination. In the case of allelism, O+ : 4- segregation on FOA is expected, and if the mutations are not allelic, the appearance of 2+ : Z-(NPD) and 1+ : 37T) tetrads, in addition to O+ : 4-(PD) tetrads, is expected.

For each slc mutation, two or more independent slc cap2A meiotic segregants were chosen. First, to confirm that these segregants carry single slc mutations and do not contain additional modifiers that might compromise the results of the allelism test, all these segregants were crossed to capZA, and monogenic inheritance for slc was documented in the resulting hybrids.

Next, hybrids between independent slc segregants were analyzed. Tetrad analysis data are presented in Tables 4 and 5. In cases where a small number of full tetrads was obtained, and proper statistical evaluation was not possible, we calculated the ratio of all Foa+ and Foa- spores.

We found three genetic loci for slc. The mutation of SL69 is allelic to sac6, and we designate that allele sac6-69. The other two loci were designated slcl (slcl- 66, s lc l -87) and slc2 (slc2-107).

Temperature and osmotic sensitivity of the sZc2 and sac6 mutants: Mutations in act1 lead to temperature and osmotic sensitivity (CHOWDHURY, SMITH and GUSTIN 1992; NOVICK and BOTSTEIN 1985). cap2A strains carrying a CAP2 plasmid, both haploid (YJC472) and diploid (YJC472 X YJC726), grow rea-


~ 1 ~ 1 - 8 7 (YJC757)

\ / del-66

~1~1-87 (YJC727,761,762) -x1 X -~lCl-66

(YJC753.754,

(YJC726.755)

t (SL66); SLC

792)

(YJC450)

FIGURE 1 .-Pedigree of slc strains. The thick arrows indicate induction of slc mutations by EMS, crosses are indicated by "x", and the thin arrows point to meiotic segregants, obtained in tetrad analysis. All the strains are cap2A.

~1~2-107 (SL107) X SLC (YJC450)

1 sad-69 (YJC729,730) - XI SLC (YJC837) X ~1~2-107 (YJC766,767,768)

sac669 (YJC785,786,788) ~1~2-107 (YJC871)

TABLE 4

Recombination allelism test between SZC mutants

TABLE 5

Recombination allelism test between slc and sac6

in full tetrads regation Segregation Total seg-

crossed Strains

PD NPD T Foa+ Foa- Conclusion

Segregation in Total seg

Strains crossed PD NPD T Foa+ Foa- Conclusion

fir11 tetrads gregation

slc87Xslc69 SL87XYJC730 2 2 8 20 42 SL87XYJC729 2 2 5 12 36 Total 4 4 13 32 78 Digenic

slc87Xslc66 SL87XYJC755 15 0 0 0 60 Homozygote < 1.7 cM

slc87Xslc87 SL87XYJC727 8 0 0 0 44 Homozygote< 2.2 cM

slc66Xslc107 YJC726XSL107 0 2 4 14 32 Digenic

slc66Xslc69 YJC726XSL69

slc66Xslc66 SL66XYJC726

slc69Xslc107 SL107XYJC730 SL107XYJC729 Total

slc69Xslc69 SL69XYJC730 SL69XYJC729 Total

1 1 3 6 20 Digenic

5 0 0 0 26 Homozygote < 3.8 cM

2 0 0 13 30 1 0 0 13 22 3 0 0 26 52 Digenic

8 0 0 0 3 2 9 0 0 0 3 6

17 0 0 0 68 Homozygote < 1.5 cM

All parental strains carry cap2A and plasmid pBJ 198 (CAP2 ADE3 URA3), without which they are inviable. PD was 4 Foa-:O Foa', NPD was 2 Foa-:2 Foa+, and T was 3 Foa-:l Foa+. Conclusions were confirmed by chi-squared analysis of the distribution of total segregants, using the null hypothesis that the Foa+:Foa- ratio was 1:3. For homozygotes, the maximum possible distance between the loci is indicated in cM; 1 cM corresponds to 2.92 kbp (MORTIMER et al. 1989).

sonably well at elevated temperatures, up to 41 O on solid YEPD. In liquid YEPD during exponential phase, the doubling time of YJC472 X YJC726 is 1.7

sac6XSAC6 YJC454XYJC728 35 0 1 71 73 Monogenic

sac6Xsac6 YJC456XYJC590 3 0 0 0 27 Homozygote< 3.7 cM

sac6Xslcl07 YJC457XYJC766 1 0 0 16 27 YJC729XSL107 1 0 0 13 22 YJC73OXSL107 2 0 0 13 30 Total 4 0 0 39 79 Digenic

sac6Xslc69 YJC453XYJC730 3 0 0 0 59 YJC453XYJC729 5 0 0 0 60 Total 8 0 0 0 119 Homozygote < 1.7 cM

sac6Xstc66 YJC454XYJC726 3 6 6 18 42 YJC454XYJC755 9 14 39 67 181 Total 12 20 45 85 223 Digenic

sac6Xslc87 YJC454XYJC727 4 0 5 5 31 YJC454XYJC761 6 4 35 43 137 Total 10 4 40 48 168 Digenic

All parental strains carry cap2A and plasmid pBJ 198 (CAP2 ADE3 URA3), without which they are inviable. PD was 4 Foa-:O Foa+, NPD was 2 Foa-:2 Foa+, and T was 3 Foa-:l Foa'. Conclusions were confirmed by chi-squared analysis of the distribution of total segregants, using the null hypothesis that the Foa+:Foa- ratio was 1:3. Other criteria are discussed in the text. For homozygotes, the maximum possible distance between the loci is indicated in cM; 1 cM corresponds to 2.92 kbp (MORTIMER et al. 1989).

hr at 30°, 1.8 hr at 37", and 2.8 hr at 41". These strains are also osmoresistant-they grow well on OSM4 (450 mM NaCI), OSM9 (900 mM NaCl), SORB (1.8 M sorbitol) and relatively well on EG (2 M ethylene

Enhancers of cup2 Null Mutations 699

glycol). In the absence of plasmid, the only difference of the cap2A strains compared to wild type is impaired growth on YEPD at 41 " and on OSM4 at 39".

The original SL mutants and diploids homozygous for a given mutation, which carry the CAP2 plasmid, grow more slowly than wild type strains at 30". In liquid YEPD in exponential phase, the doubling time of a slcI-87/slcl-87 homozygote (SL87 X YJC727) is 3.75 hr, the doubling time of a sac6-69/sac6-69 homozygote (SL69 X YJC729) is 3.4 hr, and the doubling time of a slc2-107/slc2-107 homozygote (YJC918 X YJC869) is 4.3 hr. The mutants are also temperature sensitive. On plates SL66, SL87, SL69 and diploids homozygous for slcl and sac6 grow slowly at 37"-39", and do not grow at 41". SL107 and a slc2- 107/slc2-107 homozygote (YJC918 X YJC869) grow slowly at 37" and do not grow at 39-41 ". Similar results were obtained with cultures grown in liquid YEPD. The slc mutants are also osmotic sensitive. The haploid SL69 and homozygous diploid sac6-69/sac6- 69 (SL69 X YJC729) do not grow on OSM4 and OSM9. SL87, SL66, and SL107 and diploids homozygous for slcl or slc2 grow slowly on OSM4 and OSM9 at 30", and do not grow on OSM9 at 39". Those strains are also sensitive to 2 M ethylene glycol and grow more slowly in the presence of 1.8 M sorbitol. To determine whether these effects were caused by the slc mutations, we analyzed the growth of segregants from tetrad analysis of the slc homozygotes at elevated temperature and in the presence of NaCI.

In one slcI-66/SLCI hybrid (YJC472 X YJC726-12 tetrads), and one slcl-87/SLCI hybrid (YJC472 X YJC727-10 tetrads), the SLC segregants grew well at 39 " , but slc segregants displayed different degrees of sensitivity to 39". Thus, alleles of slcl are weak Ts, and this phenotype is modified by genetic background. On OSM9, osmotic sensitivity segregated predominantly 2+ : 2- but independent of the Foa- phenotype. To confirm the independence of this Os mutation, we performed tetrad analysis of the SLCI SLC cap2A/cap2A hybrid YJC472 X YJC728. YJC472, the parent of all the SL strains, is OsR and YJC728 is an SLC Os9 segregant of SL87 X YJC472. As expected, we found predominantly 2+ : 2- segregation for Os9 (9 of 10 tetrads, with one 3+ : 1- tetrad), which confirms that Os9 in SL87 is due to a mutation not found in YJC472 and not s l c l .

For three slc2-107/SLC2 hybrids (SL107 x YJC450-8 tetrads; SL107 X YJC728-8 tetrads, YJC766 X YJC837-11 tetrads), cosegregation of Foa- and Ts (37") was observed. At 30" on OSM4 growth of the slc2-107 segregants was slower than that of wild type. At 30" on OSM9 growth of the slc2-107 segregants was also slow. The segregants of SL107 X YJC450 and YJC766 X YJC837, but not SL107 x YJC728, were analyzed on OSM9 because YJC728 carries an additional mutation conferring sensitivity

to OSM9, as noted above. Thus, slc2-107 is Ts at 37" and slightly Os on 450 mM NaCI.

For two sac6-69/SAC6 hybrids (YJC472 X YJC729- 10 tetrads; YJC472 X YJC730-15 tetrads) and one sac6AISAC6 hybrid (YJC453 X YJC728-10 tetrads), osmotic sensitivity to 450 mM NaCl (Os4) and temperature sensitivity to 37-39" (Ts) cosegregated with sac6. Thus, both sac6A and sac6-69 are Os to 450 mM NaCl and Ts.

The order of severity among the mutations is sac6 > slc2 > cap2 > slcl = wild type for osmotic sensitivity and slc2 > sac6 > slcl > cap2 = wild type for temperature sensitivity. The fact that the order of severity is different for temperature and osmotic sensitivity agrees with previous work in which temperature and osmotic sensitivity of act1 mutants are sepa- rable by suppression analysis (CHOWDHURY, SMITH and GUSTIN 1992).

Effects of slc mutations on cell wall strength To test whether the slc mutations affect the strength of the cell wall, we determined whether cells grown in high osmolarity media would lyse when placed in water. We grew cells in high osmolarity media (1 M sorbitol/YEPD), then transferred them to water. The fraction of lysed cells was measured before and after transfer to water (Table 6). For wild-type and cap2 strains, 1% were lysed before transfer to water and 2-4% were lysed after transfer to water. For sac6-69 and sacbA, 8-9% of cells were lysed before transfer to water, but 43-45% were lysed after transfer to water, indicating that the cell walls were more fragile than those of wild-type. For s lc l , two different strains showed a lower percentage of lysis than the sac6 strain, both before and after transfer to water; however, both strains did show a substantial increase in the amount of lysis after transfer to water. The slc2 strain also showed an increased fraction of lysed cells after transfer to water vs. before transfer to water; however, the fraction of lysis before transfer to water was higher than that for any other strain. Therefore, by this test, all the mutants have increased fragility of cell walls.

We were interested to determine whether the lysis observed before transfer to water was enhanced or inhibited by the added sorbitol, especially in the case of slc2, where the lysed fraction was quite high before transfer to water. The experiment was repeated with YEPD without added sorbitol (Table 6). Comparing the fraction of lysis before transfer to water in YEPD vs. YEPD plus sorbitol, one sees that sorbitol lowered the amount of cell lysis for all the strains, except for wild type and slc2. Therefore, sorbitol appears to protect the cap2, sac6, and slcl mutants from lysis. Perhaps the cell walls of slc2 cells are so weak that the protective effect of sorbitol is not sufficient for these cells. As expected, for each strain, the fraction of lysis after transfer to water was higher than that before transfer to water, and the increased amount of lysis


TABLE 6

Test of cell wall fragility

Sorbitol + YEPD YEPD

Before water After water Before water After water

Strain No. Lysed/total % No. Lysed/total % No. Lysed/total ?6 No. Lysed/total %

wt YJC472 2512308 1.1 14816813 2.2 1011373 0.7 3911630 2.4 cap2A YJC452 1812656 0.7 24715833 4.2 10211923 5.3 16312601 6.3 sac6-69 YJC729 14311735 8.2 83911852 45.3 26911828 14.7 45711804 25.3 sac6A YJC456 25512728 9.3 107112467 43.4 29211624 18.0 42311852 22.8 ~ 1 ~ 1 - 8 7 YJC762 8712223 3.9 37812315 16.3 119/1183 10.1 20911837 11.4 ~ 1 ~ 1 - 6 6 YJC792 1811654 1.1 112/2323 4.8 6911487 4.6 12112336 5.2 ~1~2-107 YJC768 43811855 23.6 73711933 38.1 1721856 20.1 46211485 31.1

Cells were grown to late exponential phase in sorbitol + YEPD or YEPD alone, then transferred to water for 30 min. Lysis was determined by trypan blue staining, both before and after transfer to water.

(after transfer to water vs. before transfer to water) is less than that seen with sorbitol.

Based on all these data, the approximate order of severity for this phenotype is slc2 > sac6 > slcl > cup2 > wild type.

Cytological effects of slc mutations: Mutations in genes encoding components of the actin cytoskeleton often affect cell morphology and the distribution of the actin cytoskeleton. Previously, we observed that disruptions of cap2 lead to the loss of actin cables, the depolarization of actin patches, and accumulation of abnormally large cells (AMATRUDA et al . 1992). In the strains used in this study, we observed the same effects of cap2 disruption, but in addition a small number of cells (3/1736, 0.17%) with elongated buds were also seen (Figure 2b).

We examined cell morphology and the distribution of both actin and capping protein of slc mutants with a Cap+ background. For each mutation, slcl-87, slc2- 107, and sac6-69, we tested three different mutant strains-the original SL mutant, which carries CAP2 on a plasmid, and two segregants, which have a wild- type copy of CAP2 in the chromosome. T o obtain these segregants, we crossed slcl-87 cap2A [CAPB], slc2-107 cap2A [CAP2], and sac6-69 cap2A [CAP21 strains (YJC757, YJC766, and YJC788, respectively) with an SLC CAP2 strain (YJC366). For each hybrid, we analyzed all four segregants from one PD tetrad (2 Foa- (slc) cap2A : 2 Foa+ (SLC) CAP2) and one NPD tetrad (2 Foa- (slc) CAP2 : 2 Foa+ (SLC) cap2A). Therefore, the results with the slc CAP2 segregants were compared with those from the other segregants as controls. We always found the same results in all three strains for a given mutation, regardless of whether the strain carried CAP2 in the chromosome or on a multicopy plasmid.

The cell morphology of the slc mutants differs from that of wild-type strains (Figure 2). Elongated mothers and/or buds were seen in 5% (14/266) of slcl cells and 30% (1 14/384) of slc2 cells. Some other cells were mildly enlarged and round. slcl and slc2 cells grew in

clumps that resisted sonication. Approximately 70% (269/384) of slc2 cells and 25% (67/266) of slcl cells grew in clumps, in contrast to wild type, where 3% (15/513) of cells grew in rare clumps. The slcl and slc2 cells in clumps grew in pseudomycelial chains, which were longer and more frequent in the slc2 strains. The chains had very little branching. To confirm the absence of branching, we stained cells with calcofluor to localize the chitin-containing bud scars and observed that most cells in chains had no bud scars except for the ones where they touched their neighbors. Colonies of slc2 on a plate have a rough surface as a consequence of the pseudomycelial growth, slcl strains, which show less pseudomycelial growth, have smooth colonies. These slcl and slc2 strains with pseudomycelial growth are haploids, which is unexpected because pseudomycelial growth is associated with a polar, as opposed to an axial, budding pattern (GIMENO et al. 1992). However, the wild-type background of these strains has a polar, not an axial, budding pattern as a haploid. Therefore, the pseudomycelial growth may be the consequence of more than one mutation.

Abnormally large round cells are formed by the sac6-69 strain. The frequency of sac6-69 cells with elongated buds is less than that for slcl and slc2, near that for cap2A. Neither sac6-69 nor cap2A strains show pseudomycelial growth. Overall, the features of sac6-69 strains resemble those of sac6A strains (AD- AMS, BOTSTEIN and DRUBIN 1991).

The slc mutations also affect the actin cytoskeleton (Figures 3, 4, and 5). In these experiments, we used both anti-actin antibodies (Figures 3 and 5 ) and rhodamine phalloidin (Figure 4) to localize actin by fluorescence microscopy. Antibodies have an advantage in visualization and photography of cables, probably because the cells are flattened. Antibodies are necessary to detect bars. Phalloidin, on the other hand, provides better signal-to-noise in these strains. Note that in these figures, we selected fields of single cells, not growing in clumps or chains, to provide the best

Enhancers of cap2 Null Mutations 70 I

immunofluorescence images of the actin distribution. Cells in clumps and chains had similar effects to those seen in single cells. In slcl cells, cortical actin patches are distributed over the mother and bud, instead of being restricted to the bud tip. Fewer cells contain actin cables, and cables that are present show reduced fluorescence intensity. In slc2 cells, patches are also delocalized, and cables are lost. The sac6-69 mutation, similar to the sac6A null mutation and the SAC6-2 dominant point mutation (ADAMS, BOTSTEIN and DRURIN 1989, 1991), leads to pronounced delocali- zation of patches and loss of cables in the majority of cells. Actin bars are observed in about 1 cell out of 1000 for the slc2 and sac6 strains (Figure 5). In slc2 cells, 8 bars were observed in approximately 6000 cells, and in sac6 cells, 6 bars were observed in a p proximately 6000 cells. This frequency is low, but readily noticeable in all of several strains examined and clearly increased over that of wild-type, in which bars were never seen. Therefore, bars are probably a direct effect of the mutation but with incomplete penetrance.

Immunofluorescence staining of s k mutants for capping protein showed that capping protein is found predominantly in the cortical actin patches, as in wild- type cells (Figure 6) . In the slcl and slc2 mutants, the diffuse component of cell staining described previously in wild-type cells (AMATRUDA and COOPER 1992) was increased in intensity, more so in slc2-107. This

h.

FIGURE 2.-Differential interference contrast (DIC) images of SLC and SIC strains. (a) Wild-tvpe SLC CAP2. (b) SLC cap2A. Cells are het- erogeneous in size. with some enlarged cells. Some cells possess elongated buds and thick necks. (c) sur6 CAP2. Many cells are enlarged. (d) slcl CAP2. Cells growing in short pseudomvcelial chains are shown. Cells with elongated buds are found more frequently than in the cap2A strain. (e) sic2 CAP2. The ma.jority of cells are arranged in pseudomvcelial chains, and many cells form elongated buds. Bar = I O pm.

observation may reflect partial mislocalization of c a p ping protein.

Cells with extremely elongated mothers and/or buds are observed among the population for slcl and slc2 strains, and far less frequently in such strains. The distribution of both actin and capping protein in these unusual cells is similar to those of the more normal- shaped cells of the population (Figure 7). Actin cables can be seen, although with reduced number and intensity relative to wild-type cells. Cortical actin and capping protein patches partially polarize to the tips of the buds but are predominantly depolarized through the mother and bud. Therefore, the abnormal shape is not associated with extreme or unusual abnormalities of the actin cytoskeleton.

Interactions between slc mutations in haploids: To determine the phenotype of double mutant h a p loids, we performed tetrad analysis of diploids diheterozygous for pairs of sic mutations and homozygous for cap2A but carrying a CAP2 plasmid. For all mutants except sac6A.-:LEU2, the s h mutations were not marked. Therefore, to monitor the segregation of each gene. we performed a statistical analysis of segregation of ability to lose the CAP2 plasmid on FOA. If the double mutants are viable and the genes are not linked, the predicted segregation is 1 PD (4 Foa-): 1 N PD (2 Foa+:2 Foa-):4 T ( 1 Foa+:Q Foa-). If double mutants are inviable, the predicted segregation is 1 PD (4 Foa-):] NPD (2 Foa+:O Foa-):4 T(l Foa+: 2 Foa-).

702 T. S. Karpova. M. M. Lepetit and J. A. Cooper

FIGURE 3.-Anti-actin immunofluorescence of SLC and SIC strains. Note that panels c, f, h and j were printed in the same way, to allow for compar- ison of staining intensity. However, all the other panels-a, b, d, e. g and i-were printed with different exposures to allow optimal visuali7ation of the morphology in each case. (a-d) SLC CAP2. Budding cells with cables in the mother cells and cortical patches in the buds are seen. (e and r) s lc l -87 CAP2 The population includes cells with faint or absent cables and patches predominantly localized to buds (e) as well as cells without cables and with patches both in the mother and the bud (9. (g and h) slc2-107 CAP2. Cells generally lack cables and have patches in both mother and bud. (i and j) sac6-69 CAP2. Cells generally lack cables and have patches in both mother and bud. .%me cells are quite enlarged ti). Bar = 10 Pm .

Tetrad dissociation of slc diheterozygotes generated viable double mutant haploids for slcl sac6 and slcl slc2, and the phenotype of the double mutants was similar to that of the single mutants in tests of temperature-sensitive growth, osmotic-sensitive growth, and growth without the CAP2 plasmid.

On the other hand, sac6 slc2 double mutants were inferred to be inviable. In two sac6-69 slc2-107 diheterotygous hybrids (SLlO7 X YJC729 and SLlO7 X YJC730) and one sac6A slc2-107 diheterozygous hybrid (YJC766 X YJC457). spore viability was poor, and tetrads generally contained only two or three viable spores. All of four four-spored tetrads were PD. No NPD or T four-spored tetrads were found. In the

sac6A X slc2-107 diheterotygous hybrid (YJC766 X YJC457) the sac6 disruption was marked by LEU2 integration. If double mutant segregants are inviable, the expected ratio of Leu+ to Leu- among all segregants is 1:2. The observed ratio was 1 1:36 (X?:p = 2.10 < 3.84). Therefore, we conclude that the sac6 slc2 haploid was inviable.

The distribution of different types of tetrads further supports this conclusion. If double mutants are inviable, then four-, two- and three-spored tetrads are PD (4 Foa-:O Foa+); NPD (2 Foa+:O Foa-) and T ( 1 Foa+:2 Foa-). respectively. I f in such tetrads additional random death of a single mutant or wild-type asco- spore occurs, then three- and two-spored tetrads in-


FIGURE 4.-Localization of- filamentous actin by fluoresccnct. microscopy of cells stained with rI~otia~rline-ph;~lloidirl. Photography was optimized to demonstrate cables. (a-d) M'ild type. Cables are present in mothers, and patches are restricted to buds. (e-g) sac6-69. Cables are absent, and cortical patches are prominently delocalized through the mother and bud. The characteristic large. round appearance of these cells is shown here, also. (h, i). slcl -87. Cables are absent, and patches are partially delocalized. + I ) slc2-107. Cables are absent, and patches are delocalized. Bar = 10 pm.

clude 3 Foa-:O Foa+ for PD and 1 Foa+: 1 Foa- and 2 Foa-:O Foa+ for T. Thus the expected tetrad ratio for unlinked genes is 1 PD (4 Foa-:O Foa+, 3 Foa-:O Foa+): 1 NPD (2 Foa+:O Foa-):4 T ( 1 Foa+:2 Foa-, 1 Foa+:l Foa-, 2 Foa-:O Foa+). In both sac6-69 slc2-107 diheterozygous hybrids combined the observed segregation was 7 (PD + NPD):22 T, which is consistent with the predicted 9.6 (PD + NPD):19.4 (T) = 1.04 < 3.84). Similarly, in the sac6A slc2-107 diheterozygous hybrid, the segregation was 3 (PD + NPD):14 T(X$':4 = 1.88 < 3.84).

Alternatively, if double mutants are viable and poor

spore viability is caused by random spore death, then the expected ratio of three-spored tetrads will be 7 NPD + T (1 Foa+:2 Foa-):l NPD (2 Foa+:l Foa-):4 PD + T (3 Foa-:O Foa+), assuming nonlinkage. In two sac6-69 slc2-107 diheterozygous hybrids, the difference between experimental and predicted data was statistically significant (13:O:l in total; X?:5 = 7.3 > 3.84), which indicates that death of double mutants is not random.

Dominance tests of slc mutations: T o determine whether the slc mutations are dominant, we studied their effects in heterozygotes. We tested three phe-

704 T. S . Karpova, M. M. Lepetit and J. A. Cooper

FIGURE 5.-Anti-actin immunofluorescence of suc6-69 (a) and slc2-107 (b) cells. The rare cells with actin bars were selected for this figure. Bar = 10 pm.

FIGURE B.-Anti-capping protein immunofluorescence of SLC and slc cells. (a) Wild-type SLC CAP2. Capping protein is localized in the cortical patches. A minor component has a diffuse cytoplasmic distribution. (b) SLC cupZA. A small amount of diffuse fluorescence is also seen in these cells, which lack capping protein. All the panels in this figure were printed in the same manner; therefore, part but not all of the diffuse component seen in the other panels is due to nonspecific binding of the antibodies. In sac6 C A P 2 (c). slcl C A P 2 (d), and slc2 CAP2 (e) cells, as in wild type, the capping protein is localized to patches. The staining of these cells differs from that of wild type in that the patches are not confined to the bud and the diffuse component is increased, particularly in some cells. Bar = IO pm.

notypes, based on our findings above in haploids- temperature sensitivity, osmotic sensitivity and the frequency of loss of the CAP2 plasmid. Overall, we found that these phenotypes were complemented, but only partially, in heterozygous diploids. For each slc mutation, two or three cap2A/cap2A slc/SLC diploids were constructed by crossing independent meiotic segregants with a cap2A SLC strain. The diploids were Cap+ because they contained the CAP2 plasmid pBJ 198. For each heterozygous diploid, we compared its phenotypes to those of its respective homozygous diploid and haploid mutant strains as well as wild-type SIC+ Cap+ haploid and diploid strains (YJC472, YJC728, and YJC472 X YJC728).

Temperature sensitivity: The wild-type strains are viable at 41 " on rich media. For s lc l , the mutant hap-

loids slcl-87 (SL87, YJC727) and slcl-66 (SL66, YJC726, YJC754) and the homozygous diploids slcl- 87/slcl-87 (SL87 X YJC727) and slcl-66/slcl-66 (SL66 X YJC726) displayed poor growth at 39" on rich media, but the heterozygous diploids slcl- 87/SLCI (YJC472 X YJC727) and slcl-66/SLCI (SAL66 X YJC728, YJC472 X YJC754) displayed a level of growth intermediate between those of wild type and mutant homozygotes. At 41 ", the heterozygous diploids are inviable, in contrast to the wild-type strain, as mentioned above. Therefore, slc-66 and slcl- 87 are semi-dominant for Ts at 39" and dominant at 41".

For slc2, the mutant slc2-107 haploids (SL107, YJC767, YJC766, YJC871) and homozygous diploid slc2-107/slc2-107 (YJC766 X YJC87 1) grew poorly at


37" bu It nc )t at all a .t 39". The heterozygous slc2-107/ SLC2 diploids (SL107 X YJC728, YJC837 X YJC767, YJC837 X YJC766) grew at 39", although worse than wild-type diploids, but not at 41 ". Therefore, slc2 is also semi-dominant for Ts at 39" and dominant at 41 ".

For sac6, the mutant sac6-69 haploid (SL69, YJC729, YJC730) and homozygous diploids sac6-691 sac6-69 (SL69 X YJC729, SL69 X YJC730) grew poorly at 39". The sac6-69/SAC6 heterozygous diploids (SL69 X YJC728, YJC729 X YJC472, YJC730 X YJC472) grew at 39", although worse than wild-type diploids, but not at 41 ". Therefore, sac6 is also semi- dominant for Ts at 39" and dominant at 41 ".

Osmotic sensitivity: The strains described in the pre- ceding section were also tested for osmotic sensitivity. The wild-type strains are viable at 30" on high osmolarity media, either OSM4 (450 mM NaCI) or OSM9 (900 mM NaCI). For s lc l , the mutant haploid segregants and their homozygous and heterozygous hybrids grew well on OSM4. Growth on OSM9 was not analyzed because the analysis of phenotypes in haploids described above showed that some of the diploids carry a mutation, different from s lc l , which causes osmotic sensitivity on OSM9 but that slcl haploid strains did grow on OSM9. For slc2, the mutant haploids and homozygous diploids grew slowly on OSM4 and very slowly on OSM9. The slc2/SLC2 heterozygous diploids grew well on both OSM4 and OSM9. Therefore, slc2 is recessive for Os. For sac6, the mutant haploids and homozygous diploids were not viable on OSM4, but the heterozygous diploids were. Therefore, sac6 is recessive for Os to 450 mM NaCI. However, the heterozygous diploids were not viable on OSM9, but the wild-type was. Therefore, sac6 is dominant for Os to 900 mM NaCI.

Frequency of CAP2 plasmid loss: Haploids and homo-

FIGURE 7.--Anti-actin immunofluorescence of extremely elongated slc cells. These representative examples show that the actin distribution of the most abnormally-shaped cells was not different from that of more normal-shaped cells in the population. In this figure. negatives were printed in two ways-the upper row of panels were printed to examine actin cables and the lower row to examine actin cortical patches. (a and b) s lc l CAP,?. Faint cables are seen in the mother, and patches are confined to the bud. (c and d) sk,? CAP,?. Cables are absent, and patches are partially polarized to the tip of the bud. (e and r) sac6 CAPP. Cables are absent, and patches are delocalized. Bar = 10 pm.

zygous diploids for slcl , slc2, and sac6 are not viable in a cap2A background, since this criterion was the basis for their isolation. On the other hand, each of the three heterozygous diploids, s l c l /SLCl , slc2/ SLC2, and sacGISAC6, are viable in a cap2A background, as shown by the ability of the cap2Alcap2A strains to lose the CAP2 plasmid and remain viable at 30" on rich media. We determined the rate of plasmid loss-the frequency of viable plasmid-less colonies-for the three heterozygous diploids and a wild-type SLC/ SLC strains by plating on FOA following pregrowth on Ura- media (Table 7). At 30" , each of the three heterozygotes generated Foa- ( i . e . , plasmid-less) colonies at a frequency less than that of wild-type but greater than zero-the frequency observed for mutant haploids and homozygotes. By this test, all three mutations are semi-dominant. At 37 ", the slc2/SLC2 and sac6lSACb heterozygous diploids also generated Foa- colonies at a frequency less than that of wild-type but greater than zero. However, at 37" the slcl/SLCl heterozygous diploid generated zero Foa- colonies. By this test, slc2 and sac6 are semi-dominant, and slcl is dominant. Based on the frequencies of plasmid loss, the mutations can be ranked in the order slcl-66 > sac6-69 > slc2-107. Note that this order is quite different from the rank orders seen for the phenotypes of these mutants in a Cap+ background above. In those cases, the phenotype of slcl is less severe than those of sac6 or slc2. Therefore, one can interpret this disparity to mean that the phenotype of slcl is influenced more by the presence of CAP2 than the phenotypes of the other mutations, suggesting that the proteins interact via different mechanisms.

Having generated these Cap-strains that were mono- heterozygous for the slc mutations, we were also able to test how the temperature and osmotic sensitivity of these strains compares to those of the respective


TABLE 7

Frequency of loss of a CAP2 plasmid from slc/SLC monoheterozygotes that are cafi2A/cap2A

Frequency of Foa+ colonies

30" 37"

Cross Relevant genotype Foa+/total Percent of control Foa+/total Percent of control

YJC472XYJC728

UJC472XUJC785 YJC472XYJC786 SLC69XYJC728

YJC837XYJC766 YJC837XYJC767

YJC472XYJC753 YJC472XYJC754 SL66XYJC728

SLCXSLC

SLCXsac6-69 SLCXsac6-69 SLCXsac6-69

sLcxs1c2-107 sLcxs1c2-107

SLCXslcl-66 SLCXslcl-66 SLCXslcl-66

7911 72

23/98 8611240 12/45

24/94 9/54

60/6460 43/10200 10/600

100

51 15 58

56 36

2.0 0.9 3.6

1191748

4319800 16/12400

114500

731940 71540

0/6460

0/6000 011 0200

100

2.8 0.8 0.1

49 8

co. 1 C0.06 co. 1

~ ~ ~~ ~ ~~ ~~

Each strain contained the CAP2 LIRA3 plasmid pBJ198. For each strain listed, two to five colonies were taken from a Ura- plate, diluted serially 10-fold and plated on FOA and YEPD at either 30" or 37" to form individual colonies. The number of colonies was counted and multiplied by the dilution coefficient and is listed in the left column. The fraction of Foa+/Total colonies was normalized to that of the wild- type control, YJC472 X YJC728, and is listed on the right as percent. The data from the two to five colonies for each strain was added together because the results were similar in each case. A sac6/sac6 homozygous diploid (SL69 X YJC730) and a slcl-66/slcI-66 homozygous diploid (SL66 X YJC726) generated no Foa- colonies at either temperature, as expected.

~~ ~~ ~~ ~~ ~~~~~~

Cap+ strains, as an additional test of interaction between the CAP2 and SLC genes. We observed that the temperature sensitivity and osmotic sensitivity of each of the three heterozygous diploids that were Cap- was enhanced relative to those that were Cap+. In fact, in each case, the Cap- heterozygous diploids have phenotypes similar to those of Cap+ homozygous diploids. Cap- slcllSLC1, slc2/SLC2, and sac6lSAC6 strains grew poorly at 39" on rich media, in contrast to their respective Cap+ strains described above. On synthetic media, the Cap- slcl/SLCl, slc2/SLC2, and sac6/SAC6 strains were viable at 30 O but not 37 " , while the Cap+ strains in each case were viable at both 30 " and 37 " . For slc2/SLC2, the Cap- strain is inviable on OSM9, while the Cap+ strain is viable, as noted above. For sac61SAC6, the Cap- strain is inviable on OSM4, and the Cap+ strain is viable. For each case, we documented that retransformation of the Cap- heterozygous diploid with the CAP2 plasmid pBJ198 restored the level of temperature and osmotic sensitivities to those of the appropriate Cap+ strain. These results demonstrate that the slc mutations are also semidominant with respect to the synthetic lethality phenotype.

Genetic interactions between slc mutations in diploids: In haploids sac6 and slc2 interact, leading to inviability of the double mutant, described above. To test whether the slc mutations interact in diploids, we compared the phenotypes of slc monoheterozygotes and diheterozygotes in pairwise combinations. As in the previous experiments, all the diploids were homozygous for ca$2A, but carried the CAP2 plasmid, pBJ198. For each pairwise combination of slc mutations, we prepared hybrids from 2 or 3 meiotic segregants. These hybrids were pregrown on Ura- medium and then tested for the ability to lose the CAP2 URA3

plasmid on FOA at 30" and 37" (Table 8). At 30" the diheterozygous slcllSLC1 sac6lSACb

hybrids formed ten times fewer colonies on FOA than did the more severely affected monoheterozygote, slcl/SLCl (Table 7). At 37", neither the diheterozygotes nor the slcllSLC1 monoheterozygote formed any Foa- colonies. The diheterozygous slc2/SLC2 sac61SAC6 hybrids formed somewhat fewer and ten times fewer colonies on FOA at 30 " and 37 " , respectively, than did the more severely affected monoheterozygote, sac6ISAC6 (Table 7). The diheterozygote slc2-107/SLC2 slcl-66ISLCl had a level of plasmid stability intermediate between those of the individual monoheterozygotes.

Formally, these results indicate that slcl interacts with sac6, and that slc2 interacts with sac6, but do not show interaction between slcl and slc2. The interpre- tation of these experiments is somewhat limited by the fact that the monoheterozygote for each mutant is abnormal relative to wild type. The phenotype expected for a diheterozygote may be that of the more severely affected monoheterozygote or some additive combination of the phenotypes of the two monoheterozygotes. In the latter respect, the results with the slcllSLC1 slc2/SLC2 diheterozygote could be inter- preted as suppression of the slcllSLC1 phenotype by the slc2 mutation.

Because the slcl-66/SLCl sac6lSAC6 and slc2-107/ SLC2 sacblSAC6 diheterozygotes do form a few colonies on FOA, we were interested to determine whether these colonies are in fact still heterozygous for both slc genes. Another alternative would be that these colonies are the result of mitotic homozygotization to wild type at one genetic locus ( i e . , gene conversion), leading to formation of a viable strain mono-

707 Enhancers of cap2 Null Mutations

TABLE 8

Frequency of loss of a CAP2 plasmid from slc/SLC diheterozygotes that are cap2A/cap2A

Frequency of Foa+ colonies

30" 37"

Cross Relevant genotype Foa+/total Percent of control Foa+/total Percent of control

YJC472XYJC728 SLCXSLC 791172 100 1 191748 100

SL66XYJC729 slcl-66Xsac6-69 811 3000 0.13 011 3000 C0.05

SL66XYJC730 slcl-66Xsac6-69 1011 2000 0.17 011 2000 C0.05 SL87XYJC730 slc-87Xsac6-69 3/6000 0.1 1 0/6000 co.11

SL107XYJC726 ~1~2-107X~lcI-66 101130 17 211 3000 0.10 SL107XYJC730 slc2-107Xsac6-69 71140 1 1 4114000 0.18 SL107XYJC729 slc.2-107Xsac6-69 2311000 5 211 0000 0.13

YJC453XYJC726 sac6AXslcl-66 711 300 1.2 0/13000 C0.05 YJC453XYJC754 sac6AXslcI-66 10/6000 0.4 0/6000 eo . 1 1

YJC453XYJC753 sac6AXslcl-66 211 3000 0.04 011 3000 C0.05

The experiment was performed and the data are presented as in Table 7.

heterozygous at the other locus. Therefore, we were concerned that the apparent different rates of plasmid loss may reflect different rates of mitotic homozygotization instead of viability of diheterozygotes. To determine the frequency of mitotic homozygotization, we isolated colonies growing on FOA at 30" from several diheterozygotes. For the slcl-66 sac6-69 diheterozygote, 81% of colonies (78196) grow at 30" but not at 37 ". Nineteen percent of colonies (1 8/96) grow at both 30" and 37", which suggests that they are the result of mitotic homozygotization. In sac6A slcl-66 hybrids, where sac6 is disrupted by LEU2, the frequency of mitotic homozygotization for SAC6 can be determined directly. SAC6+ homozygotes (Leu-) constitute 9.4% (9196) of colonies growing on FOA at 30". If the frequency of S L C P homozygotization in these hybrids approximately the same as that for SAC6, then the total frequency of mitotic homozygotes should be 18.8%. Thus mitotic recombinants constitute approximately 20% of the colonies, but the majority of the colonies are indeed probably diheterozygous for slc and sac6.

DISCUSSION

In S. cermisiae, actin-binding proteins regulate the actin cytoskeleton, which is essential for viability. Null mutations in the genes CAP1 and CAP2 are viable (AMATRUDA et al. 1990, 1992), but the additional disruption of SAC6, which encodes fimbrin, leads to inviability (ADAMS, COOPER and DRUBIN 1993). Therefore, to identify new genes for components of the actin cytoskeleton and to characterize essential genetic interactions, we screened for mutations that enhance the cap2A null mutant phenotype with a colony color assay (BENDER and PRINGLE 1989). Four synthetic-lethal mutations were inherited monogeni- cally. Based on recombinational allelism tests, they belong to three genetic loci. One locus is allelic to

sac6. The other two loci are not allelic to sac6 or to each other. The mutations are designated slcl-66, slcl-87, and slc2-107, for synthetic lethality with cap2. slc2-107 displays synthetic lethality with sac6, and sac6A is synthetic lethal with abpIA, but slc2-107 should not be allelic to abpl because abpIAcap2A strains are viable (ADAMS, COOPER and DRUBIN 1993). The majority of mutants showed irregular segregation for the synthetic lethal phenotype, which suggests that some mutations lead to a slight impairment of phenotype, and that the interaction of several such mutations is necessary for detection in the screen.

Genetic characteristics of the slc mutations: The synthetic lethal phenotype is semi-dominant. s lc l /SLCI, slc2/SLC2 and sac6/SAC6 heterozygotes in a capPA/ cap2A background display reduced viability relative to wild type. In addition, the phenotypes of slc diheterozygotes were more severe than those of monoheterozygotes for two pairs of mutations. The slcI-66/ SLCl sac6/SAC6 and slc2-107/SLC2 sac6/SAC6 diheterozygotes were able to lose a CAP2 plasmid and become Cap- at a frequency far lower than that of the corresponding monoheterozygotes. On the other hand, the rate of loss of the CAP2 plasmid in the slc2- 1071SLC2 slcI-66/SLCI diheterozygote was intermediate between those in the monoheterozygotes. Therefore, slcl interacts with sac6, and slc2 interacts with sac6, but slcl and slc2 do not interact, in a formal sense.

For genes encoding enzymes, a semi-dominant phenotype is often characteristic of a gain-of-function mutation. Null mutations also can be semi-dominant if protein concentration is important, as has been seen for genes encoding regulatory or structural proteins (HADWIGER et al. 1989; STEARNS and BOTSTEIN 1988). Therefore, the slc mutations can be either gain-of- function or loss-of-function mutations. The latter pos- sibility is more probable. First, mutations that simply


inactivate a gene product are more frequent than those which change its properties. Second, a sac6A null mutation is also semi-dominant. Thus, the semi- dominance of each mutation and the interactions between the mutations in the diheterozygotes are most probably due to loss-of-function mutations.

Phenotypes of slc mutations: The slc mutations have phenotypic effects of their own in a Cap+ background. These phenotypes include temperature sensitivity, osmotic sensitivity, cell wall fragility, abnormal morphogenesis, and abnormal distribution of actin. Together, these phenotypes are consistent with the hypothesis that SLCl and SLC2 encode components of the actin cytoskeleton. The phenotypes of sac6-69, the allele isolated here, are consistent with this being a null mutation.

slc2-107 confers temperature sensitivity and slight osmotic sensitivity. sac6-69, like sac6A, confers temperature and osmotic sensitivity. Strains carrying slcl- 66 and slcl-87 are not osmotic sensitive and are somewhat temperature sensitive, but this phenotype is weak and is modified extensively by genotypic background. Several known genes encoding components of the actin cytoskeleton are important for stress viability. High osmotic pressure induces rearrangement of the actin cytoskeleton (CHOWDHURY, SMITH and GUSTIN 1992). Temperature sensitive mutants of actl are also osmotic sensitive, as are heterozygotes for an actl null mutation (NOVICK and BOTSTEIN 1985). Specific sup- pressors of the osmotic sensitivity, as opposed to the temperature sensitivity, can be isolated. At least one of these genes probably encodes an actin-binding protein (CHOWDHURY, SMITH and GUSTIN 1992). Null mutants of sac6 (fimbrin) (ADAMS, BOTSTEIN and Drubin 1991) and tpml (tropomyosin) (LIU and BRETSCHER 1992) are temperature sensitive, and those of my01 are temperature sensitive and osmotic sensitive (WATTS, SCHIELDS and ORR 1987).

The morphology of the slc strains is abnormal. The sac6-69 cell population contains large, round cells, as seen previously with other sac6 alleles and other actin cytoskeleton mutations (ADAMS, BOTSTEIN and DRUBIN 1991; ADAMS, COOPER and DRUBIN 1993; AMATRUDA et a l . 1992; LIU and BRETSCHER 1992). Although the slcl and slc2 mutant strains include some round large cells, most cells are elongated, both as mothers and buds, somewhat similar to the effects of overexpression of capping protein (AMATRUDA et al . 1992). An increased fraction of cells in slcl and slc2 strains are dead, in particular the more abnormally shaped cells, and again more often in slc2. In addition, cell wall fragility is increased in slcl, slc2, and sac6 strains, based on a test for lysis upon a rapid decrease in osmotic pressure. Abnormalities of morphogenesis and cell wall structure are often seen in actin cytoskeleton mutants.

The actin distribution is also altered in the slc

mutants. Both slcl and slc2 show depolarization of actin cortical patches. Both mutations lead to defects in actin cables, with slc2 more severely affected than s l c l . Bars of actin are seen in slc2 and sac6 cells, but not slcl cells. These features are commonly seen in mutations of genes encoding components of the actin cytoskeleton (ADAMS, BOTSTEIN and DRUBIN 1991; ADAMS, COOPER and DRUBIN 1993; AMATRUDA et al . 1992; DRUBIN 1990; LIU and BRETSCHER 1992).

The slc mutants also show pseudomycelial growth. A substantial fraction of cells growing in rich media, either in liquid or in agar, formed short linear chains. This pattern of growth has been noted as an effect of starvation on certain a/a diploid strains of S. cerevisiae (GIMENO et a l . 1992), but this phenomenon is generally not seen on rich media, in haploids, or in strains with backgrounds like those used in our study. Pseu- domycelial growth requires several features, including a polar budding pattern, bud site selection away from the founder by the terminal cell, and a persistent connection between mother and daughter cells. The wild-type background of these haploid strains does have a bipolar budding pattern, so the slc mutations may provide the persistent connection and terminal bud site selection. Haploid strains with null mutations of my01 also show pseudomycelial growth without starvation (WATTS, SCHIELDS and ORR 1987). Al- though the actin cytoskeleton is generally not consid- ered to be involved in bud site selection and growth pattern (DRUBIN 1991), these results, together with the observation that overexpression of Abplp alters the haploid budding pattern (DRUBIN, MILLER and BOTSTEIN 1988), suggest that the relationships may be more complicated.

We are grateful to ALISON ADAMS for advice on designing the screen and to her and ALAN BENDER for providing important reagents, some of which were prepared by MICHAEL TIBBETTS and JOHN PRINGLE. We are grateful to JIM AMATRUDA, DOROTHY SCHAFER and JIM WADDLE for reading the manuscript. This work was supported by grants from National Institutes of Health, GM38542 and GM47337, and from the Lucille P. Markey Chari- table Trust. J.A.C. was a Lucille P. Markey Biomedical Scholar and is currently an Established Investigator of the American Heart Association.

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Documents

Mutations That Enhance the cap2 Null Mutant Phenotype in ... · standard methods (MANIATIS, FRITSH and SAMBROOK 1982). The integrating plasmids pBJO13 (cap2-AI::HZS3) and pBJl00 (cap2-Al::LEU2),