Sro7p, a Saccharomyces cerevisiae Counterpart of the Tumor Suppressor l(2)gl Protein, Is Related to Myosins in Function

Corresponding author: Yasushi Matsui, Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Tokyo 113, Japan., matsui{at}biol.s.u-tokyo.ac.jp (E-mail).

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a small GTPase that maintains cell polarity. Sro7p and Sro77p, a homologue of Sro7p, possess domains homologous to the protein that are encoded by the Drosophila tumor suppressor gene lethal (2) giant larvae [l(2)gl]. sro7 sro77 mutants showed a partial defect of organization of the polarized actin cytoskeleton and a cold-sensitive growth phenotype. A human counterpart of l(2)gl could suppress the sro7 sro77 defect. Similar to the l(2)gl protein, Sro7p formed a complex with Myo1p, a type II myosin. These results indicate that Sro7p and Sro77p are the yeast counterparts of the l(2)gl protein. Our genetic analysis revealed that deletion of SRO7 and SRO77 showed reciprocal suppression with deletion of MYO1 (i.e., the sro7 sro77 defect was suppressed by myo1 and vice versa). In addition, SRO7 showed genetic interactions with MYO2, encoding an essential type V myosin: Overexpression of SRO7 suppressed a defect in MYO2 and, conversely, overexpression of MYO2 suppressed the cold-sensitive phenotype of sro7 sro77 mutants. These results indicate that Sro7 function is closely related to both Myo1p and Myo2p. We propose a model in which Sro7 function is involved in the targeting of the myosin proteins to their intrinsic pathways.

THE Drosophila tumor suppressor gene lethal (2) giant larvae [l(2)gl] was discovered in 1930 by BRIDGES. Inactivation of l(2)gl results in overgrowth of the imaginal discs and the optic lobes in the larval brain, and produces cells that lose their apical-basal cell polarity and are unable to differentiate (HADORN 1938 ; GATEFF 1978 ). Homologues of l(2)gl have been identified in humans and mice (TOMOTSUNE et al. 1993 ; STRAND et al. 1995 ; KOYAMA et al. 1996 ). The l(2)gl protein is found in both the plasma membrane and the cytosol of cells (STRAND et al. 1994A ) and interacts with the nonmuscle myosin type II (STRAND et al. 1994B ). However, its function has yet to be determined.

Rho3p is a rho-type small GTPase of the yeast Saccharomyces cerevisiae (MATSUI and TOH-E 1992A ). In rho3-deficient cells, the polarized actin cytoskeleton of budded cells is disrupted, and the bud fails to grow. In cells carrying a dominant-active RHO3 allele, cells organize the actin cytoskeleton in an abnormal position, showing a defect in maintaining the axis of cell polarity towards the bud (IMAI et al. 1996 ). These observations suggest that the role of Rho3p is to maintain cell polarity for bud growth (MATSUI and TOH-E 1992B ; IMAI et al. 1996 ). Nine SRO genes, reported as SRO1 to SRO9, suppress the growth defect of rho3 cells when overexpressed (MATSUI and TOH-E 1992B ). Several SRO genes have been characterized. Sro1p and Sro2p are identical to Bem1p and Cdc42p, respectively (MATSUI and TOH-E 1992B ). Bem1p is important and Cdc42p is essential for cell polarization in bud emergence (ADAMS et al. 1990 ; CHENEVERT et al. 1992 ). Exocytosis in growing cells is restricted at the growing bud and supplies materials for bud growth (TKACZ and LAMPEN 1972 ; FIELD and SCHEKMAN 1980 ). Sro6p is identical to Sec4p (IMAI et al. 1996 ), which controls the fusion step of secretory vesicles to plasma membrane (SALMINEN and NOVICK 1987 ). SRO9 has displayed genetic interactions with tropomyosin-encoding genes and is involved in the organization of the actin cytoskeleton [i.e., increased dosage of SRO9 can rescue the defect in tropomyosin-deficient cells and the depletion of Sro9p in tropomyosin-deficient cells results in abnormal clusters of cortical patches of actin filaments, aberrant morphology of cells, and cell lysis (KAGAMI et al. 1997 )]. In addition, similar to SRO9, TPM1, encoding a tropomyosin, can serve as a multicopy suppressor of rho3 (KAGAMI et al. 1997 ). These features of the characterized SRO genes and the genetic interactions between RHO3 and a SRO gene suggest that the other SRO gene products also participate in maintenance of cell polarity and in bud growth.

In this article, we have characterized Sro7p and Sro77p, a functionally redundant homologue of Sro7p. Both Sro7p and Sro77p possess domains homologous to the l(2)gl protein. We found that Sro7p and Sro77p are yeast counterparts of the l(2)gl protein and have genetically characterized the function of Sro7p and Sro77p. These results have demonstrated a strong relationship between Sro7 function and the myosin pathways.

	MATERIALS and METHODS

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

Microbiological techniques:
Yeast transformations were performed by the method of ITO et al. 1983 . Rich medium containing glucose (YPD) and synthetic minimal medium (SD) were as described (SHERMAN et al. 1986 ). Synthetic complete medium (SC) contains 0.5% casamino acid (Difco, Detroit, MI) and 100 mg/liter each of uracil, adenine sulfate, and tryptophan in SD. SC-U is SC lacking uracil.

Strains and plasmids:
The yeast strains used are listed in Table 1. All strains in this study were constructed using the wild-type strains YPH499 and YPH501 (SIKORSKI and HIETER 1989 ). The Escherichia coli strain used was strain DH5 [supE44 lacU169 (80 lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1]. Plasmid DNA was prepared as described by MANIATIS et al. 1982 . Yeast DNA was prepared as described by SHERMAN et al. 1986 .

The DNA sequence of SRO7 on the original clone was determined and was identical to the YPR032w sequence in Saccharomyces Genomic Database. SRO7 was disrupted by replacing a 2.6-kb BglII-XhoI fragment (corresponding to the amino acid residues 118–989) in the SRO7 open reading frame with a 1.1-kb fragment that contains the URA3 gene. To construct sro7::HIS3, we disrupted the URA3 marker in the sro7 allele with HIS3 using a ura3-disruption plasmid (MATSUI and TOH-E 1992B ). To construct YCpUGAL7, the 1.4-kb BamHI-XbaI fragment of YIpUGAL7 (MATSUI and TOH-E 1992B ), containing the GAL7 promoter and terminator, was inserted between the BamHI and XbaI sites of pRS316 (SIKORSKI and HIETER 1989 ). The DNA fragment containing the open reading frame of SRO7 was amplified by PCR using the primers 5'-GGGGGGGATCCAATGTTCGGTAGCAAACGTC and 5'-GGGGGGGATCCTTAAAAACCAAGGGCACCTT. The amplified fragment was digested with BamHI and inserted into the BglII site of YCpUGAL7 to be expressed under the control of the GAL7 promoter. The resulting plasmid, YCpUGAL7-SRO7, was transformed into the rho3-1 cells (strain YMR3732-2B) to examine whether the expression of SRO7 from the plasmid can suppress the temperature sensitivity of rho3-1 cells. A 1-kb fragment of the SRO77 5'-noncoding region, amplified with PCR using the primers 5'-GGGGGATCGATATGTGGCATGTTCAAAACCA and 5'-GGGGGTCTAGACCATAAAATTTTTGTATCTG, was digested with ClaI and XbaI, and was inserted between the ClaI and SpeI sites of pRS303 (SIKORSKI and HEITER 1989) to construct pRS303-SRO77-5. A 1-kb fragment of the SRO77 3' noncoding region, amplified by PCR using the primers 5'-GGGGGCTCGAGTCCATAAATAGTTTTTAT and 5'-GGGGGATCGATATTGCGTAAACTCCTTCAAG, was digested with ClaI and XhoI, and was inserted between the ClaI and XhoI sites of pRS303-SRO77-5 to construct pRS303-SRO77. The KpnI-SacI fragment of pRS303-SRO77 was inserted between the KpnI and SacI sites of pRS304 (SIKORSKI and HIETER 1989 ) to construct pSRO77. pSRO77 was digested with ClaI and used for replacement transformation. Using pSRO77, SRO77 was disrupted by replacing the complete open reading frame with the TRP1 marker (sro77::TRP1).

The SRO77 gene was amplified using the primers 5'-GGGGGATCGATATGTGGCATGTTCAAAACCA and 5'-GGGGGATCGATATTGCGTAAACTCCTTCAAG, and the amplified fragments were digested with ClaI and introduced into ClaI sites of pYO324, a high-copy-number plasmid (OHYA et al. 1991 ), for the assay for the activity to suppress the rho3 defect. Assays for the activity to suppress the rho3 defect were as described in MATSUI and TOH-E (1992b).

The coding region of LLGL was amplified from a plasmid containing LLGL cDNA (KOYAMA et al. 1996 ) by PCR using the set of primers 5'-GGGGGAATTCATGATGAAGTTTCGGTTCCGGCGGCAGGGCGC and 5'-GGGGGTCGACTCATCGCGCAGCCTGGACGC. The amplified DNA fragment was digested with EcoRI and SalI and was inserted between the EcoRI and SalI sites of pKT10, a high-copy-number plasmid carrying the TDH3 promoter (MATSUI and TOH-E 1992A ), to construct YEpULLGL. In this construct, LLGL is expressed constitutively with the TDH3 promoter.

The MYO1 gene was amplified using the primers 5'-CAAGGTCATGGCTTTTAAACAAAGCGT and 5'-GGGGGCTCGAGTACTGAAAATTTTACTCTGTG. The amplified fragment was digested with PstI and XhoI, and was introduced between the PstI and XhoI sites of pBluescript KS⁺ (Stratagene, La Jolla, CA) to create KSMYO1. A DNA fragment that encodes three copies of the hemagglutinin (HA) epitope, tandemly connected, and a stop codon was inserted into the XhoI site of KSMYO1 to create KSMYO1HA. In KSMYO1HA, the HA sequence is placed in frame, immediately downstream of the last codon of MYO1. The 6.5-kb PstI-SalI fragment of KSMYO1HA was inserted between the PstI and SalI site of YEplac195, a high-copy-number plasmid (GIETZ and SUGINO 1988 ), to construct a Myo1-HA-producing plasmid (YEpUMYO1HA). To construct YIpHMYO1HA, a 1.7-kb SacI-SalI DNA fragment from YEpUMYO1HA encoding the C-terminal half of Myo1-HA was inserted between the SacI and XhoI sites of pRS303. YIpHMYO1HA was digested with EcoRV and integrated at the MYO1 locus to replace wild-type MYO1 with versions of MYO1 encoding Myo1-HA and of MYO1 lacking the 5'-flanking sequence and the sequence for the N-terminal portion.

The construction of pKT10NmycSRO7 for production of Sro7-mycN was as follows. A 3.1-kb DNA fragment encoding the entire Sro7p was amplified by PCR using the primers 5'-GGGGGGGATCCAATGTTCGGTAGCAAACGTC and 5'-GGGGGGGATCCTTAAAAACCAAGGGCACCTT. The amplified fragment was digested with BamHI and inserted into the BglII site of pKT10mycN, which is a high-copy-number plasmid carrying the TDH3 promoter for production of Myc epitope–tagged protein (MATSUI et al. 1996 ), to create pKT10NmycSRO7. The construction of YIpWSRO7mycC, for production of Sro7-myc, was as follows. A 1-kb DNA fragment that was amplified by PCR using the primers 5'-GGGGGGGATCCAATGTTCGGTAGCAAACGTC and 5'-GGGGGGGTACCAAAACCAAGGGCACCTTTCA was digested with EcoRI and KpnI. The resulting EcoRI-KpnI fragment, encoding the C-terminal half of Sro7p, was inserted into the KpnI and EcoRI sites of pUC119 (VIEIRA and MISSING 1987) to create pUCSRO7C. The DNA fragment that encodes three copies of the Myc epitope, tandemly connected, and a stop codon were inserted into the KpnI site of pUCSRO7C to create pUCSRO7Cmyc. In pUCSRO7Cmyc, the Myc epitope sequence was placed in frame, immediately downstream of the last codon of SRO7. The1-kb EcoRI-SalI fragment of pUCSRO7Cmyc was inserted between the EcoRI and SalI sites of pRS304 to create YIpWSRO7mycC. YIpWSRO7mycC was digested with StuI and integrated into the SRO7 locus to construct SRO7-myc cells, where wild-type SRO7 was replaced with versions of SRO7 encoding Sro7p fused with the Myc epitope at its C terminus (Sro7-myc) and of SRO7 without the 5'-flanking sequence and the sequence for the N-terminal half. Myo1-HA suppressed the myo1 defect, and both Sro7-myc and Sro7-mycN suppressed the sro7 sro77 defect (data not shown).

The construction of YEpUMYO2HA, a high-copy-number plasmid producing an HA-tagged Myo2p, was as follows. The 3' half of MYO2 gene was amplified using the primers 5'-GATGTAATGGGAGGCGGTGC and 5'-GGGGGCTCGAGAGTGGCCGTCTTGAACGACT, and was digested with EcoRI and XhoI. To construct pMYO2HA3, the resulting DNA fragment was inserted between the EcoRI and SalI sites of the plasmid that is based on YEplac195 and carries the DNA fragment encoding three copies of the HA epitope. In pMYO2HA3, the sequence for the HA epitope was inserted just upstream of the stop codon of MYO2 in frame. The 4.0-kb EcoRI-EcoRI DNA fragment containing the 5' half of MYO2 gene from pJP10-2B (JOHNSTON et al. 1991 ) was inserted into the EcoRI site of pMYO2HA3 to construct YEpUMYO2HA. Myo2-HA suppressed the myo2-66 defect (data not shown).

Morphological observations:
Cells, grown in liquid culture (<5 x 10⁶ cells/ml), were harvested and stained with rhodamine-phalloidin (to reveal actin filaments), 4',6-diamidino-2-phenylindole (DAPI; to reveal DNA), anti-Myc antibodies (to reveal Sro7-mycN), or anti-HA antibodies (to reveal Myo1-HA) as described (PRINGLE et al. 1989 ). For DAPI staining, cells were fixed with 70% ethanol for 5 min, washed with PBS (10 mM KH₂PO₄, 40 mM K₂HPO₄, 150 mM NaCl), and stained with 125 ng/ml of DAPI solution in PBS. For rhodamine-phalloidin staining, cells were fixed with 5% formaldehyde for 60 min, washed with PBS, stained with 1:50-diluted rhodamine-phalloidin solution (Molecular Probes, Inc., Eugene, OR) for 2 hr, and washed five times with PBS. For Sro7-mycN or Myo1-HA staining, cells were fixed with 5% formaldehyde for 120 min. The fixed cells were treated with 0.2% SDS in 100 mM potassium phosphate buffer (pH 7.5) containing 1 M sorbitol for 5 min, followed by five washes with buffer G1T [1% gelatin, 0.5 mg/ml BSA, 150 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% Tween 20, 1 mM NaN₃], submerged in methanol (-20°) for 6 min and then in acetone (-20°) for 30 sec, and incubated in G3T [3% gelatin, 0.5 mg/ml BSA, 150 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% Tween 20, 1 mM NaN₃] for 30 min. Anti-myc antibodies (9E10; 10 mg/ml), used at a 1:100 dilution, or anti-HA antibodies (16B12; BAbCo, Richmond, CA), used at a 1:200 dilution, were applied and visualized using CY3-labeled sheep anti-mouse IgG antibodies (Chemicon International, Inc., Temecula, CA) at a dilution of 1:200. These samples were mounted in p-phenylenediamine (1 mg/ml in 90% glycerol) and observed with an epifluorescence microscope (BH-2; Olympus, Tokyo, Japan).

Measurement of latrunculin-A sensitivity:
Halo assays of latrunculin-A (LA; Wako, Tokyo, Japan) were as described in AYSCOUGH et al. 1997 . Ten microliters dimethyl sulfoxide containing 0, 0.5, 1, or 2 mM LA was spotted on discs (6 mM in diameter) of a filter paper and then put on the lawn of cells to be tested, on plates of rich medium. After 2 days incubation at 25°, the sizes of the halos were measured. For measurement of LA sensitivity of cells in a liquid culture, cells were inoculated into YPD medium and incubated at 25° with shaking. LA was added to the medium at a final concentration of 0.5 µM after 4.3 hr. Cell growth was monitored by measuring the optical density at 600 nM.

Immunoprecipitation:
About 5 x 10⁷ cells were disrupted with glass beads in PBS containing 1% Triton X-100 and protease inhibitors (10 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml antipain, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A; Sigma, St. Louis, MO), and they were diluted with the same buffer to the final concentration of ~4 mg total protein/ml. Samples (250 µl) were mixed with 10 µg of anti-myc antibodies or 12.5 µg of anti-HA antibodies, and the mixture was incubated at 4° for 5 hr. A total of 60 µl of protein A-Sepharose beads (Pharmacia, Uppsala, Sweden) was added, incubated at 4° overnight, and washed five times with PBS containing 1% Triton X-100. The beads were boiled with the sample buffer for SDS-polyacrylamide gel electrophoresis for 5 min, and the resulting supernatant was subjected to immunoblot analysis.

In vitro binding assay:
Production and purification of the glutathione S-transferase (GST)-fused protein were performed as described previously (SHIRAYAMA et al. 1995 ). The DNA fragment, encoding the N-terminal half of Sro7p (amino acids 1–689), was inserted into pGEX-5X-3 vector (Pharmacia) to construct a plasmid for producing Sro7p fused with GST (GST-Sro7). The GST proteins were fixed on glutathione-agarose beads (Pharmacia). Beads carrying ~5 µg of GST-Sro7 or GST were incubated with 300 µl of the cell extract from ~1.5 x 10⁸ cells at 4° overnight. The proteins attached to the beads were then washed and eluted with glutathione as described in MATSUI et al. 1996 . The eluted proteins were analyzed by immunoblot using anti-HA antibodies.

Measurement of cell separation defect:
Cells from the log-phase culture were harvested, washed with PBS, and sonicated for 5 min (model B1260; Bransonics Corp., Danbury, CT). From more than 300 budded cells, we counted how many cells harbored more than one attached daughter cell.

	RESULTS

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

Mapping of SRO7:
The original SRO7 clone, pSRO7, was isolated from a yeast genomic library cloned in the vector YEp24 as a multicopy suppressor of rho3; i.e., rho3 cells (strain YMR505) carrying pSRO7 grew as well as wild-type cells (MATSUI and TOH-E 1992B ). pSRO7 contained a 12-kb fragment of chromosome XVI, encompassing YPR032w (located between nucleotides 634,118 and 637,219 of chromosome XVI). Deletion of an XhoI fragment (located between nucleotides 635,798 and 637,091 of chromosome XVI) from the plasmid disrupted YPR032w and eliminated its ability to suppress the rho3 defect. The temperature sensitivity of rho3 Ts^- mutant cells (strain YMR3732-2B) was suppressed by expression of YPR032w under the control of the GAL7 promoter (data not shown). We therefore conclude that SRO7 is YPR032w, encoding a protein of 1033 amino acids (Figure 1A). A homology search against the yeast genomic DNA sequence and GenBank revealed that yeast possesses a homologous gene (YBL106c on chromosome II), encoding a protein of 1010 amino acids (55% amino acid identity with Sro7p, Figure 1A), and that both SRO7 and YBL106c encode proteins with domains homologous to the l(2)gl protein (Figure 1B). Introduction of YBL106c on pYO324, a high-copy-number plasmid, enabled rho3 cells (strain YMR505) to grow slightly better than the rho3 cells with pYO324 alone (data not shown), indicating that YBL106c can serve as a weak multicopy suppressor of rho3. We designated YBL106c as SRO77.

View larger version (125K): In this window In a new window Download PPT slide   Figure 1. Alignments of the amino acid sequences of Sro7p, Sro77p, and the l(2)gl proteins. (A) Alignments of the amino acid sequences of Sro7p and Sro77p. The identical residues are boxed in black. Asterisks denote the domains homologous to the l(2)gl protein that are shown in B. (B) The homologous domains of Sro7p, Sro77p, the Drosophila l(2)gl protein, and the LLGL protein, a human l(2)gl protein. Numbers of the amino acid residues are indicated in parentheses. Shadowed residues indicate conserved residues with either the Sro7p or Sro77p domains; identical residues are boxed in black.

Phenotypes of sro7 sro77 cells:
Deletion of either SRO7 or SRO77 did not affect cell growth at 15°, 20°, 25°, 30°, and 37°, but sro7 sro77 double mutants did not grow at temperatures <20° (Figure 2A). This synthetic defect of sro7 with sro77, along with the structural similarity between Sro7p and Sro77p, indicates that Sro7p and Sro77p are functionally redundant.

View larger version (42K): In this window In a new window Download PPT slide   Figure 2. Phenotypes of sro7 sro77 cells. (A) Cold sensitivity of sro7 sro77 cells. Cells indicated (WT, strain YPH499; sro7, strain YMK025-1; sro77, strain YMK026; and sro7 sro77, strain YMK027) were streaked on a YPD plate and incubated at 20° for 4 days. (B) Actin filaments of sro7 sro77 cells. (a) Wild-type cells (strain YPH499) stained with rhodamine-phalloidin. (b) sro7 sro77 cells (strain YMK027) grown at 30° were shifted to 20°, harvested 18 hr after the shift, fixed, and stained with rhodamine-phalloidin. Bar in B represents 5 µm. (C) LA sensitivity of sro7 sro77 cells. The arrow indicates the time when LA was added to the medium at a final concentration of 0.5 µM. Open circles, wild-type cells (strain YPH499); filled circles, sro7 cells (strain YMK025-1); squares, sro77 cells (strain YMK026); triangles, sro7 sro77 cells (strain YMK027).

Eighteen hours after the shift to 20°, the growth of sro7 sro77 cells was arrested randomly, and the cells became slightly rounded. In wild-type cells, cortical actin patches are localized mostly to the bud, and actin cables are observed clearly (Figure 2B, panel a). In sro7 sro77 cells, however, actin patches were dispersed, and only short actin cables were observed (Figure 2B, panel b). These results indicate that organization of the polarized actin cytoskeleton is partly disrupted in the sro7 sro77 mutants.

LA is a drug that sequesters G-actin and then inhibits the formation of F-actin in cells. Several mutants carrying defects in actin cytoskeleton show significantly increased sensitivity to LA (AYSCOUGH et al. 1997 ). The partial disruption of polarized actin cytoskeleton in sro7 sro77 cells may suggest that Sro7p and Sro77p play a positive role in actin organization. To address this hypothesis, we examined the sensitivity of sro7 sro77 cells to LA using halo assays. Cell growth was inhibited in a circle ~22 mm in diameter around the spot where 10 µl of 0.5 mM LA was placed in a lawn of wild-type, sro7, or sro77 cells. In contrast, the circle was ~27 mm in diameter around the spot of 10 µl of 0.5 mM LA in a lawn of sro7 sro77 cells, suggesting that sro7 sro77 cells are hypersensitive to LA. When LA was added to the culture medium at final concentration of 0.5 mM, the growth of sro7 sro77 cells was inhibited (Figure 2C). In contrast, 0.5 mM LA had no effect on the growth of wild-type, sro7, and sro77 cells (Figure 2C). These indicate that simultaneous disruption of SRO7 and SRO77 results in hypersensitivity to LA.

Functional complementation by a human counterpart of l(2)gl:
We introduced a plasmid expressing LLGL, a human counterpart of l(2)gl (KOYAMA et al. 1996 ), into sro7 sro77 cells to examine whether the l(2)gl protein is functionally similar to Sro7p. sro7 sro77 cells carrying the LLGL-expressing plasmid grew as well as wild-type cells at 20°, but sro7 sro77 cells carrying a control plasmid did not (Figure 3). This result demonstrates that LLGL can complement sro7 sro77.

View larger version (91K): In this window In a new window Download PPT slide   Figure 3. Complementation by LLGL. sro7 sro77 cells (strain YMK027) with either control plasmid pKT10 (Cont.) or with YEpULLGL (LLGL) were streaked on an SC-U plate and incubated at 20° for 5 days.

Physical interaction of Sro7p and Myo1p:
The l(2)gl protein interacts with the nonmuscle myosin type II (STRAND et al. 1994B ), and yeast possesses a type II myosin that is encoded by MYO1 (WATTS et al. 1987 ). We therefore examined the physical interaction of Myo1p and Sro7p. Sro7-myc in extract from the cells, producing Myc epitope-tagged Sro7p (Sro7-myc) and HA-tagged Myo1p (Myo1-HA), was immunoprecipitated with anti-Myc antibodies. In the immunoprecipitation of Sro7-myc, we also detected Myo1-HA (Figure 4A, lane 1). In addition, Myo1-HA was precipitated from the extract of cells producing Myo1-HA by using the beads fixing GST-fused Sro7p (Figure 4B, lane 2), whereas Myo1-HA was not precipitated by the beads with GST (Figure 4B, lane 3). These results suggest that Sro7p forms a complex with Myo1p in cells.

View larger version (31K): In this window In a new window Download PPT slide   Figure 4. Interaction of Sro7p with Myo1p. (A) Coimmunoprecipitation of Myo1p with Sro7p. Extracts from SRO7-myc (lanes 1 and 2, strain YMK031) or SRO7 cells (lanes 3 and 4, strain YMK030), integrated with YIpHMYO1HA for producing Myo1-HA, were immunoprecipitated with anti-HA antibodies (lanes 2 and 4) or anti-Myc antibodies (lanes 1 and 3). The immunoprecipitates were analyzed by immunoblotting with anti-HA antibodies. (B) Coprecipitation of Myo1p with Sro7p. Extract from wild-type cells (lane 1, strain YPH499) or cells integrated with YIpHMYO1HA (lanes 2–4, strain YMK030) was incubated with GST beads (lane 3) or GST-Sro7 beads (lanes 1 and 2). After washing, elutes from beads were analyzed by immunoblotting using anti-HA antibodies. (Lane 4) Total cell lysate.

Subcellular localization of Sro7p and Myo1p:
We determined the localization of Myo1p and Sro7p in wild-type cells by using Myo1-HA and Sro7p with a Myc-epitope at the N terminus (Sro7-mycN). Myo1-HA was localized exclusively to the bud neck (Figure 5, left). Sro7-mycN was detected over most of the cell surface (Figure 5, right). We detected Sro7-mycN in both the supernatant and pellet, which were prepared by disruption of cells with PBS and centrifugation at 10,000 g for 10 min of the cell extract (data not shown). This result suggests that Sro7p may also localize in the cytosol fraction.

View larger version (90K): In this window In a new window Download PPT slide   Figure 5. Localization of Myo1p and Sro7p. Cells (strain YPH499) carrying YEpUMYO1HA for Myo1-HA (left) and cells (strain YPH499) carrying pKT10NmycSRO7 for Sro7-mycN (right) were stained with anti-HA antibodies and anti-Myc antibodies, respectively. Upper panel, immunofluorescence; lower panel, phase contrast.

Genetic interactions between SRO7/SRO77 and MYO1:
To assess the functional relationship between Sro7p and Myo1p, we tested for genetic interactions between them. Disruption of MYO1 resulted in slow growth, a defect in cell separation [a portion of the myo1 cells do not separate from each other, as reported in WATTS et al. 1987 and RODRIGUEZ and PATERSON 1990 ], and reduced efficiency of colony formation from germinated spores. To analyze the effect of the deletion of SRO7 and SRO77 on the reduced efficiency of colony formation from germinated myo1 spores, we crossed myo1 cells with sro7 sro77 cells, sporulated the resulting diploid cells, and performed tetrad analysis. Only 6 of 22 (27%) myo1 spores formed a visible colony. In contrast, 15 of 25 (60%) sro7 myo1 spores, 13 of 31 (42%) sro77 myo1 spores, and 21 of 34 (62%) sro7 sro77 myo1 spores formed a visible colony, indicating that loss of Sro7p and Sro77p suppresses the myo1 defect in colony formation. Concerning the myo1 defect in cell separation, among the budded cells in a culture of myo1 cells, 42% of myo1 cells had more than one daughter cell attached, indicating a cell separation defect. In contrast to this, only 19% of sro7 myo1 cells, 32% of sro77 myo1 cells, and 11% of sro7 sro77 myo1 cells among the budded cells in the cultures showed the cell separation defect, indicating that the deletion of SRO7 and SRO77 suppresses the myo1 defect in cell separation. Furthermore, sro7 sro77 myo1 colonies were almost the same size as colonies of wild-type cells and were larger than colonies of myo1 cells (Figure 6), indicating that the slow growth phenotype of myo1 cells is suppressed by sro7 sro77. These results, therefore, indicate that loss of Sro7p, and Sro77p suppresses the myo1 defects.

View larger version (107K): In this window In a new window Download PPT slide   Figure 6. Reciprocal suppression between sro7 sro77 and myo1. Cells indicated were streaked on a YPD plate and incubated at 20° for 5 days.

sro7 sro77 myo1 cells grew as well as wild-type cells at 20°, at which sro7 sro77 cells could not (Figure 6), indicating that disruption of MYO1 suppresses the cold-sensitive phenotype of sro7 sro77 cells. This reciprocal suppression involving sro7 sro77 and myo1 suggests that the function of Sro7p and Sro77p (Sro7 function) is closely related to that of Myo1p.

Morphology of sro7 sro77 myo1 cells:
Judging from the size of colonies, sro7 sro77 myo1 cells can grow as well as wild-type cells. However, DAPI staining of these cells revealed that the morphology of sro7 sro77 myo1 cells was very abnormal (Figure 7C) when compared with that of either sro7 sro77 cells or myo1 cells (Figure 7, a and b). Either mother or daughter nuclei were frequently observed with a filamentous shape between the mother and daughter cells (indicated by arrowheads in Figure 7C). The abnormal morphology of sro7 sro77 myo1 cells may suggest a defect in nuclear migration and/or in chromosome segregation. A defect in nuclear migration of myo1 cells has been reported (WATTS et al. 1987 ), but another group has reported that myo1 cells did not show a defect in nuclear migration (RODRIGUEZ and PATERSON 1990 ). At least in the genetic background of cells used in this study, we did not observe in myo1 SRO7 SRO77 cells an apparent defect in nuclear migration or the defect similar to that observed in sro7 sro77 myo1 cells (Figure 7B). Therefore, the defect observed in sro7 sro77 myo1 cells is specific for the cells lacking both Sro7 function and Myo1p.

View larger version (129K): In this window In a new window Download PPT slide   Figure 7. DAPI staining of sro7 sro77 myo1 cells. Cells (a, sro7 sro77 cells, strain YMK027; b, myo1 cells, strain YMK024; c, sro7 sro77 myo1 cells, strain YMK029), cultured at 20°, were fixed and stained with DAPI to reveal DNA. Arrowhead in c indicate the cells showing abnormality.

Interactions between SRO7 and MYO2:
Deducing from the phenotypes of myo1 cells, Myo1p does not contribute significantly to bud growth. However, SRO7 can serve as a multicopy suppressor of the rho3 defect, suggesting that Sro7p acts positively in bud growth in addition to its function related to Myo1p. Therefore, it might be possible that Sro7p interacts with the pathways of other motility factors that contribute to bud growth. Thus, we tested for genetic interactions between SRO7 and MYO2. MYO2 encodes the type V myosin that is essential for polarized secretion (JOHNSTON et al. 1991 ). Cells carrying myo2-66, a temperature-sensitive allele of MYO2, grew very slowly at 30°. myo2-66 cells with a high-copy-number plasmid carrying SRO7 grew still slowly compared to wild-type cells; however, they grew better than the myo2-66 cells with a control plasmid at 30° (Figure 8A), indicating that SRO7 can serve as a multicopy suppressor of myo2. Moreover, sro7 sro77 cells with a high-copy-number plasmid carrying MYO2 could grow at 20° as well as wild-type cells (Figure 8B), indicating that MYO2 can serve as a multicopy suppressor of sro7 sro77.

View larger version (41K): In this window In a new window Download PPT slide   Figure 8. Genetic interactions between SRO7/SRO77 and MYO2. (A) myo2-66 cells (strain YMK021) with SRO7 on YEp24, a multicopy plasmid carrying the URA3 marker, (pSRO7, left) or with YEp24 as a control (Cont., right) were streaked on a SC-U plate and incubated at 30° for 4 days. (B) sro7 sro77 cells (strain YMK027) with YEpUMYO2HA for overproduction of Myo2p (left) or with YEplac195 as a control (right) were streaked on a SC-U plate and incubated at 20° for 5 days.

Motivated by the genetic interactions between SRO7 and MYO2, we tested for protein-protein interactions between Sro7p and Myo2p. We carried out similar experiments to those of Myo1p, shown in Figure 4, using an HA epitope-tagged Myo2p (Myo2-HA). Beginning with extracts from cells producing both Sro7-myc and Myo2-HA, Sro7-myc was immunoprecipitated with anti-Myc antibodies. However, we did not detect any Myo2-HA in the Sro7-myc immunoprecipitate (data not shown). Moreover, there was no interaction between Myo2-HA and GST-Sro7 (data not shown). Using immunoblot analysis, we could detect Myo2-HA in whole-cell lysates with an intensity similar to that of Myo1-HA. Therefore, if Sro7p interacts with Myo2p, it most likely does so at a much lower affinity than when it interacts with Myo1p.

	DISCUSSION

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

SRO7 and SRO77 are the yeast counterparts of l(2)gl:
We have found several similarities between Sro7p/Sro77p of S. cerevisiae and the l(2)gl protein of D. melanogaster. First, both Sro7p and Sro77p possess domains homologous to the l(2)gl protein (Figure 1). Second, similar to the fact that inactivation of l(2)gl produces cells that have lost their apical-basal cell polarity (GATEFF 1978 ), the simultaneous deletion of SRO7 and SRO77 perturbs the organization of the polarized actin cytoskeleton, i.e., it results in a cell polarity defect (Figure 2B). Third, the l(2)gl protein in Drosophila cells and Sro7p in yeast cells are found in the plasma membrane (STRAND et al. 1994A ; Figure 5, right). Fourth, coimmunoprecipitation analysis and an in vitro binding assay suggest that Sro7p and Myo1p form a complex in cells, which is similar to the fact that the l(2)gl protein interacts with the nonmuscle myosin type II (STRAND et al. 1994B ). In addition to these similarities, the human l(2)gl can complement Sro7 function for cell growth at low temperatures (Figure 3). These results indicate that SRO7 and SRO77 are the yeast counterparts of l(2)gl.

Interactions of Myo1p and Sro7p:
Myo1p was localized exclusively to the bud neck (Figure 5, left; WATTS et al. 1985 ; BROWN 1997 ; LIPPINCOTT and LI 1998 ). This is consistent with the localization of the nonmuscle myosin type II in cells of other species, where the nonmuscle myosin type II is localized at the site of cell separation, i.e., in the cleavage furrow (SATTERWHITE and POLLARD 1992 ). In contrast, Sro7p was detected over most of the cell surface. By using the immunoprecipitation method, we found the complex formation of Sro7p with Myo1p in cells (Figure 4A). However, it is unlikely that the interaction of Sro7p with Myo1p plays a critical role in the subcellular localization of Stro7p and Myo1p because Myo1-HA in sro7 sro77 cells was still localized at the bud neck and the localization of Sro7-mycN in myo1 cells was indistinguishable from that in wild-type cells (data not shown).

Although Myo1p colocalizes with actin ring at the bud neck during anaphase and the ring seems contractile during cytokinesis (LIPPINCOTT and LI 1998 ), it is still unclear how the actin ring and Myo1p act in cell separation. However, myo1 cells have aberrant septa, indicating that Myo1p is required for the construction of the cell wall between mother cell and daughter cell and the aberrant septa may cause the cell separation defect (RODRIGUEZ and PATERSON 1990 ). The myo1 defect, including the cell separation defect, is suppressed by the disruption of SRO7 and SRO77. This indicates that yeast cells can separate independently of Myo1p when Sro7 function is missing and that the presence of Sro7 function represses Myo1p-independent cell separation. In the absence of Sro7 function, perhaps other motile factors can substitute for Myo1 function to induce the Myo1p-independent cell separation. Among the myosins in yeast, Myo2p is observed at the mother bud neck just before cell separation (LILLIE and BROWN 1994 ; BROWN 1997 ). Therefore, it is conceivable that Myo2p is responsible for Myo1p-independent cell separation, which causes a defect in nuclear migration and/or chromosome segregation (Figure 7C). It is possible that Sro7p/Sro77p and Myo1p are required to prevent cells from the aberrant cell separation.

sro7 sro77 myo1 cells grew as well as wild-type cells at low temperatures, indicating that the cold sensitivity of sro7 sro77 cells is Myo1p dependent (Figure 6). Considering that Myo1p can form a complex with Sro7p, this result suggests that Myo1p may inhibit cell growth when it fails to form a complex involving Sro7p or Sro77p. Consistently, overexpression of Myo1p reduced the growth rate of both sro7 sro77 cells and wild-type cells (data not shown). The cold sensitivity of sro7 sro77 cells was suppressed by increased dosage of Myo2p (Figure 8B), leading to the possibility that the Myo2 pathway is perturbed in the absence of Sro7 function. Therefore, it is highly likely that Sro7 function restricts Myo1p to specialize in the process of cell separation and not to affect cellular processes involving Myo2p.

Sro7p plays a positive role in organization of actin cytoskeleton:
Rho3p is required to maintain cell polarity for bud growth (MATSUI and TOH-E 1992B ; IMAI et al. 1996 ). Because SRO7 can serve as a multicopy suppressor of rho3, Sro7p may also participate in the maintenance of cell polarity and in bud growth. sro7 sro77 mutants consistently show partial disruption of the organization of the polarized actin cytoskeleton (Figure 2B, panel b). In addition, sro7 sro77 cells are hypersensitive to LA (Figure 2C). Yeast cells with a defect in the factor that plays a positive role in organization of actin cytoskeleton, such as Srv2, Cap2, Tpm1, Sac6, or Sla2, are hypersensitive to this drug (AYSCOUGH et al. 1997 ). The phenotypes of sro7 sro77 cells, partial disruption of the polarized actin cytoskeleton and hypersensitivity to LA, suggest that Sro7p and Sro77p play a positive role in organization of the polarized actin cytoskeleton.

Myo1p has an inhibitory effect on organizing the actin cytoskeleton because we detected only faint actin cables in cells carrying MYO1 on a high-copy-number plasmid (data not shown). It might be possible that the positive role of Sro7p in actin organization is to repress the inhibitory activity of Myo1p on it by complex formation. Whereas the growth of sro7 sro77 cells was rescued by myo1, however, the polarized actin cytoskeleton was still disrupted in sro7 sro77 myo1 cells and in sro7 sro77 cells (data not shown). Concerning the LA sensitivity, sro7 sro77 myo1 cells were more hypersensitive to LA than sro7 sro77 cells (data not shown). These results indicate that Sro7p plays a positive role in the actin cytoskeleton that is independent of Myo1p. The reduced activity of Myo2p in the myo2-66 cells is rescued by a high dose of Sro7p, and cells require an elevated amount of Myo2p in the absence of Sro7 function (Figure 8). These genetic interactions between SRO7 and MYO2 suggest that Sro7p plays a positive role in the Myo2 pathway. Myo2p is concentrated in the bud during bud growth, and it is critical for polarized cell growth and for vectorial transport of secretory vesicles to the bud (JOHNSTON et al. 1991 ; LILLIE and BROWN 1994 ). Therefore, it is possible that the Sro7 function that acts positively in the actin cytoskeleton is involved in recruiting sufficient activity of the Myo2 pathway for bud growth.

Model of Sro7 function:
Cell separation and bud growth both require transport of the materials used to construct cell walls and membranes. Based on mutant phenotypes, Myo1p and Myo2p are believed to be the motile factors that are responsible for transport in cell separation and bud growth, respectively. Based on all the genetic interactions described in this article, we propose a model in which Sro7p is involved in targeting and sorting different myosins to their intrinsic destination (Figure 9), i.e., Myo1p exclusively for cell separation and Myo2p for bud growth.

View larger version (23K): In this window In a new window Download PPT slide   Figure 9. A model deduced from the interactions between Sro7p and myosins. Black arrows indicate positive involvement. T bars indicate inhibitory activities. A gray arrow indicates positive involvement by protein-protein interaction.

Sro7p was detected over most of the cell surface. This suggests that Sro7p is not restricted to function only in a complex with Myo1p. Concerning Myo2p, genetic analysis suggests that Sro7 function plays a positive role in the Myo2 pathway for bud growth. On the other hand, at the bud neck, Sro7p inhibits a Myo1p-independent cell separation that is most likely promoted by Myo2p, suggesting that Sro7p takes inhibitory action against Myo2p. We assume that Sro7p covers the cell membrane to prevent the myosin pathways from having random access to the membrane. That is, depending on a signal that localizes Myo1p to the bud neck, Myo1p can have access to the bud neck and the Myo1p-Sro7p complex acts positively on the access. At the same time, the motile factor for the Myo1p-independent cell separation is inhibited by Sro7p covering the bud neck. For targeting the Myo2 pathway to the bud tip that is covered by Sro7p, we assume the existence of another regulatory factor that permits Myo2p to the bud tip. By this model, the genetic interactions between SRO7 and MYO2 (Figure 8) are interpreted as follows: The overproduction of Sro7p excludes Myo2p activity strictly from the cell surface, except for wherever the access of Myo2p is permitted and results in increased Myo2p activity at the bud tip to promote cell growth. In contrast, lack of Sro7 function disperses Myo2p activity at the bud tip and causes a growth defect that is suppressed by increased dosage of Myo2p.

Rho3p is likely to be involved in targeting the exocytosis machinery to the site for surface extension, because in cells that produce constitutively active mutant Rho3p, we found that Sec8, a component of the exocytosis machinery (TERBUSH and NOVICK 1995 ), was recruited to be colocalized with Rho3p (J. IMAI, A. TOH-E and Y. MATSUI, unpublished results). The suppression of the rho3 defect by SRO7 might be the result of Sro7p activity involved in polarized secretion, as suggested by the genetic interaction between SRO7 and MYO2. Sro7p and Sro77p are also described as the proteins that interact with Sec9p, a yeast SNAP-25 homologue, in Saccharomyces Genome Database [(http://genome-www.stanford.edu/Saccharomyces/) SGDID; L0004193 and L0004194]. Sec9p is a component of t-SNARE that is the acceptor of v-SNARE on secretory vesicles, as well as an essential terminus to fuse secretory vesicles to the plasma membrane during exocytosis (BRENNWALD et al. 1994 ). In this context, the functional relationship between Sro7p/Sro77p and myosins is an attractive finding because the molecular basis between secretory mechanism and actin cytoskeleton still has not been well determined. The relationship strongly suggests that Sro7p and Sro77p play a pivotal role in targeting the myosin pathways to their cell surface proper destination and then targeting the transport system of secretory vesicles.

The characterization of the yeast counterpart of l(2)gl will undoubtedly be of much interest to future research on the molecular mechanism of the regulation of motile factors in targeting the machinery of exocytosis, which governs cell differentiation and malignant transformation.

	ACKNOWLEDGMENTS

We thank Dr. G. C. JOHNSTON for the myo2-66 strain and MYO2 plasmid, Dr. K. KOYAMA and Dr. Y. NAKAMURA for the cDNA clone of LLGL, Dr. S. TSUKAMOTO for the information on LA, Dr. I. HERSKOWITZ and Dr. R. P. JANSEN for helpful discussions, Dr. P. BRENNWALD for communicating their results before publication, and R. MATSUI for technical assistance. Part of this work was supported by a grant for scientific work from Monbusho; M.K. is a recipient of the Fellowship of Japan Society for the Promotion of Science for Japanese Junior Scientists.

Manuscript received December 22, 1997; Accepted for publication May 12, 1998.

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