The Novel Adaptor Protein, Mti1p, and Vrp1p, a Homolog of Wiskott-Aldrich Syndrome Protein-Interacting Protein

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The Novel Adaptor Protein, Mti1p, and Vrp1p, a Homolog of Wiskott-Aldrich Syndrome Protein-Interacting Protein (WIP), May Antagonistically Regulate Type I Myosins in Saccharomyces cerevisiae

Junko Mochida, Takaharu Yamamoto, Konomi Fujimura-Kamada and Kazuma Tanaka1 Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, 060-0815, Japan Manuscript received July 31, 2001 Accepted for publication January 7, 2002

ABSTRACT Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch- like structures and the ATP-independent interaction between the Myo5p tail region and actin ﬁlaments. We have identiﬁed and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p- GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.

HE actin cytoskeleton is essential in a wide variety Rvs167p, and proteins of the Arp2/3 complex (Pruyne Tof cellular functions, including cell morphogenesis, and Bretscher 2000). These proteins are also involved cell polarity, cytokinesis, cell adhesions, and endocytosis in the uptake step of endocytosis through actin cytoskel- (Bretscher 1991; Botstein et al. 1997). Reorganiza- eton regulation (Wendland et al. 1998). Actin cables tion of the actin cytoskeleton is dynamically regulated extend along the mother-bud axis and are required for both spatially and temporally. The mechanism by which polarized growth (Pruyne et al. 1998). the actin cytoskeleton assembles to mediate these func- Myo3/5p are the yeast type I myosins, which are highly tions, however, remains a fundamental puzzle in cell conserved actin-activated ATPases that function in en- biology. docytosis, membrane trafﬁcking, contractility, and cell The budding yeast Saccharomyces cerevisiae is an excel- motility (Mooseker and Cheney 1995). Although dele- lent model system for the study of actin cytoskeleton tion of either MYO3 or MYO5 does not result in an dynamics because yeast has a relatively simple actin cy- obvious growth phenotype, a double knockout is syn- toskeleton and offers powerful experimental tools for thetically lethal or nearly so, suggesting the functional genetic manipulation. Throughout the yeast cell cycle, redundancy of these genes (Geli and Riezman 1996; highly regulated reorganizations of the actin cytoskele- Goodson et al. 1996). Typically, the tails of unconven- ton underlie spatial control of cell surface growth, tional myosins participate in molecular interactions to thereby determining cell morphology. Cell surface ex- specify the role of the motor domain. Myo3/5p contain tension is preceded by the polarized organization of three domains within their tails that are homologous two actin ﬁlament-containing structures: cortical actin to other members of this protein family: a putative mem- patches and actin cables (Adams and Pringle 1984; brane-binding domain (TH1), an alanine- and proline- Kilmartin and Adams 1984). The formation or reorga- rich domain (TH2), and an src homology 3 domain nization of cortical actin patches is regulated by cortical (SH3 or TH3; Goodson and Spudich 1995). patch-like protein structures that include Sla1p, Sla2p, SH3 domains are present in a variety of proteins associ- Abp1p, Sac6p, Las17p/Bee1p, Vrp1p, Myo3p, Myo5p, ated with the actin cytoskeleton reorganization and with signal transduction. This domain mediates protein-protein interactions through binding to proline-rich 1Corresponding author: Division of Molecular Interaction, Institute for stretches (Musacchio et al. 1994). The SH3 domains Genetic Medicine, Hokkaido University Graduate School of Medicine, N15 W7, Kita-ku, Sapporo, Hokkaido, 060-0815, Japan. of Myo3/5p interact with both Las17p, a homolog of E-mail: [email protected] mammalian Wiskott-Aldrich syndrome protein (WASP;

Genetics 160: 923–934 (March 2002) 924 J. Mochida et al.

Evangelista et al. 2000; Lechler et al. 2000), and to the manufacturer’s protocol. Site-directed mutagenesis was Vrp1p, a homolog of mammalian WASP-interacting pro- performed using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as per the manufacturer’s instruc- tein (WIP; Anderson et al. 1998; Evangelista et al. tions. The plasmids used in this study are listed in Table 2. 2000). The COOH-terminal acidic regions within Myo3/ Schemes for the construction of plasmids and the sequences 5p and Las17p interact with the Arp2/3 complex to of PCR primers are available upon request. stimulate actin polymerization (Evangelista et al. 2000; Two-hybrid screening: Two-hybrid screening was performed Lechler et al. 2000). Vrp1p also interacts with Las17p, as described by James et al. (1996). To maximize transformation efficiency, large-scale transformations were carried out suggesting that Vrp1p mediates an efficient interaction according to the protocol described by Agatep et al. (1998). between Myo3/5p and Las17p (Naqvi et al. 1998). In PJ69-4A, carrying the pGBD-C1-MYO3-SH3-AD plasmid, was addition, Vrp1p sustains the interaction of the Myo5p independently transformed with three yeast genomic DNA tail region with actin filaments, suggesting a role for libraries (Y2HL-C1, -C2, and -C3), plated on SD-Trp-Leu-His Њ Vrp1p in localized Myo3/5p-induced actin polymeriza- plates, and incubated for 6–8 days at 30 . The plates were then replica plated onto fresh SD-Trp-Leu-Ade plates for stringent tion (Geli et al. 2000). selection of positive clones. After an additional 6–8 days incu- To explore the function and regulation of the yeast bation, 151, 86, and 45 colonies were picked up from the 4 ϫ type I myosins, we searched for proteins that interact 106, 4.3 ϫ 107, and 2.2 ϫ 107 initial transformants of Y2HL- with the tail region of Myo3p, using a two-hybrid screen- C1, -C2, and -C3, respectively. These colonies were patched Њ ing method. We identified a novel protein, Myosin tail onto SD-Trp-Leu plates and grown at 30 for 3 days. Clones were then tested using a 5-bromo-4-chloro-3-indolyl-␤-d-galac- region-interacting protein (Mti1p), which binds to the topyranoside filter assay to measure ␤-galactosidase activity Myo3/5p SH3 domains. Subsequent analyses demon- (Vojtek et al. 1993). Plasmids were isolated from clones that strated that Mti1p is a binding partner of Myo3/5p. turned blue (8, 29, and 16 positive clones from each genomic Interestingly, the mti1 null mutation suppressed the vrp1 DNA library) and were reintroduced into PJ69-4A cells car- mutant phenotypes, suggesting that Mti1p and Vrp1p rying the pGBD-C1-MYO3-SH3-AD plasmid. Transformants were retested both for growth on SD-Trp-Leu-His and SD-Trp- antagonistically regulate the type I myosin functions. Leu-Ade plates and for the 5-bromo-4-chloro-3-indolyl-␤-d-galactopyranoside filter assay. Positive clones were then sequenced. We also performed a two-hybrid screening, as described above, MATERIALS AND METHODS using the pGBD-C1-MYO3-TH1-TH2 plasmid as the bait. ␤ Strains, media, and genetic techniques: Yeast strains used Quantification of -galactosidase activity was performed using o-nitrophenyl ␤-d-galactopyranoside as a substrate (Guarente in this study with their relevant genotypes are listed in Table ␤ 1. Unless otherwise specified, strains were grown in YPDA rich 1983). -Galactosidase activity is expressed in Miller units (Mil- ler 1972). media [1% yeast extract (Difco Laboratories, Detroit), 2% Ј bacto-peptone (Difco), 2% glucose, and 0.01% adenine]. Microscopic observations: To visualize Mti1p, the 3 end of Strains carrying plasmids were selected in synthetic medium the chromosomal MTI1 gene was tagged with the sequence (SD) containing the required nutritional supplements (Sher- encoding green fluorescent protein (GFP) as described in man 1991). Prior to tetrad analysis, diploid cells were cultured Longtine et al. (1998). YKT142 cells were grown to early in presporulation medium (0.8% yeast extract, 0.3% bacto- logarithmic phase in YPDA medium, harvested, and resus- peptone, and 10% glucose) for 24 hr at 25Њ. The cells were pended in SD medium. Cells were mounted on microslide then sporulated in sporulation medium (0.1% yeast extract, glass and observed immediately using a GFP bandpass filter set 0.05% glucose, and 1% potassium acetate) at a cell density (excitation, 460–500 nm; dichroic mirror, 505 nm; emission, of 1.5 ϫ 107 cells/ml for 1 week at 25Њ. Standard genetic 510–560 nm). To visualize the actin cytoskeleton, exponen- manipulations of yeast were performed as described (Sher- tially growing cells were fixed for 15 min by the direct addition man and Hicks 1991). Yeast transformations were performed of commercial 37% formaldehyde stock (Wako Pure Chemical using the lithium acetate method (Elble 1992). PCR-based Industries, Osaka, Japan) to a 5% final concentration in me- gene deletion and epitope tagging of yeast genomic DNA were dium. Fixed cells were stained for 30 min at room temperature carried out as previously described (Longtine et al. 1998). with 1 ␮m tetramethylrhodamine isothiocyanate (TRITC)- The vrp1 and las17 disruption mutants were constructed on phalloidin (Sigma Chemical, St. Louis). Following five washes our strain background as follows. The regions containing the in phosphate-buffered saline, cells were mounted in 90% disruption marker and the flanking sequences were PCR am- glycerol containing n-propyl gallate (Wako). Cells were ob- plified using either T65-1D (vrp1::LEU2) or DDY1438 (las17 served using a G-2A TRITC filter set (excitation, 510–560 ⌬::URA3) genomic DNA as a template. The resulting DNA nm; dichroic mirror, 575 nm; emission, 590 nm) on a Nikon fragment was then introduced into YEF473. Proper gene dis- ECLIPSE E800 microscope (Nikon Instec, Tokyo, Japan). The ruption was verified by PCR. Escherichia coli strains DH5␣ and microscope setup contained an HB-10103AF super high-pres- XL1-Blue were used for the construction and amplification sure mercury lamp and a 1.4NA 100ϫ Plan Apo oil immersion of plasmids. objective (Nikon Instec) with either the appropriate fluores- Molecular biological techniques: Standard molecular bio- cence-filter sets (Nikon Instec) or differential interference logical techniques, described by Sambrook et al. (1989), were contrast (DIC) optics. The images presented in this article used for the construction of plasmids, PCR amplification, and were acquired using a cooled digital CCD camera (C4742-95- DNA sequencing. PCR amplification was performed using a 12NR; Hamamatsu Photonics K. K., Hamamatsu, Japan) and GeneAmp PCR system 9700 (Perkin-Elmer, Norwalk, CT). AQUACOSMOS software (Hamamatsu Photonics K. K.). This DNA sequences were obtained using an ABI PRISM 310 DNA microscopic imaging system was also used to measure the size sequencer (Applied Biosystems, Foster City, CA). The DNA of yeast cells. Overnight cultures were inoculated into fresh sequences of all constructs containing amplified PCR products SDA-U [0.17% yeast nitrogen base without amino acids were confirmed using an ABI PRISM BigDye terminator cycle (Difco), 0.5% casamino acid (Difco), 2% glucose, 0.03% tryp- sequencing ready reaction kit (Applied Biosystems), according tophan, and 0.01% adenine] to a cell density of 0.125 OD600/ml. A Novel Regulator of Type I Myosins 925

TABLE 1 S. cerevisiae strains used in this study

Straina Genotype Reference or source PJ69-4A MATa ura3-52 his3⌬-200 trp1-901 leu2-3,112 gal4⌬ gal80⌬LYS2::GAL1-HIS3 James et al. (1996) GAL2-ADE2 met2::GAL7-lacZ ⌬ L40 MATa lys2-801 his3 -200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 URA3:: Hollenberg et al. (1995) (lexAop)8-lacZ DDY318 MAT␣ lys2-801 ura3-52 his3⌬-200 leu2-3,112 sac6⌬::LEU2 Gift from Dr. Drubin DDY322 MAT␣ ura3-52 his3⌬-200 leu2-3,112 abp1⌬::LEU2 Gift from Dr. Drubin DDY949 MATa ura3-52 his3⌬-200 trp1-1 leu2-3,112 rvs167⌬::TRP1 Gift from Dr. Drubin DDY546 MAT␣ lys2-801 ura3-52 his3⌬-200 leu2-3,112 sla2⌬1::URA3 Gift from Dr. Drubin DDY582 MATa ura3-1 his3-11,15 trp1-1 leu2-3,112 ade2-101 rhoϩcap2-⌬1::HIS3 Gift from Dr. Drubin DDY1438 MATa lys2-801 ura3-52 his3⌬-200 leu2-3,112 las17⌬::URA3 Gift from Dr. Drubin RH2075 MAT␣ lys2 ura3 his4 leu2 bar1 end5-1 Naqvi et al. (1998) T65-1D MAT␣ ura3-52 leu2-3,112 ade1 ileϪ MEL1 vrp1::LEU2 Zoladek et al. (1995) TZ33 MAT␣ lys2-1 ura3-1 his4-519 leu2-3,112 ade2-1 SUP11 mod5-1 vrp1-1 Zoladek et al. (1995) YMW211U MATa lys2-801 ura3-52 his3⌬-200 trp1-⌬63 leu2-1 ade2-101 arp2-1 Madania et al. (1999) (H330L)::URA3 YMW221U MATa lys2-801 ura3-52 his3⌬-200 trp1-⌬63 leu2-1 ade2-101 arp2-2 Madania et al. (1999) (G19D)::URA3 YEF473 MATa/␣ lys2-801/lys2-801 ura3-52/ura3-52 his3⌬-200/his3⌬-200 trp1⌬-63/ Longtine et al. (1998) trp1⌬-63 leu2⌬-1/leu2⌬-1 YKT38 MATa lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 This study YKT112 MATa lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 myo3⌬::TRP1 This study myo5⌬::KanMX6 YKT130 MATa lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 vrp1::LEU2 This study YKT131 MAT␣ lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 vrp1::LEU2 This study YKT218 MATa lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 sla1⌬::KanMX6 This study YKT229 MATa lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 las17⌬::URA3 This study YKT241 MATa lys2-801 ura3-52 his3⌬-200 trp1⌬63 leu2⌬-1 mti1⌬::HIS3MX6 This study vrp1::LEU2 YKT143 MATa lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 mti1⌬::HIS3MX6 This study YKT144 MAT␣ lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 mti1⌬::HIS3MX6 This study YKT190 MAT␣ lys2-801 ura3-52 his3⌬-200 trp1⌬-63 leu2⌬-1 mti1⌬::KanMX6 This study YKT142 MATa/␣ lys2-801/lys2-801 ura3-52/ura3-52 his3⌬-200/his3⌬-200 trp1⌬-63/ This study trp1⌬-63 leu2⌬-1/leu2⌬-1 MTI1-EGFP:KanMX6/MTI1-EGFP:KanMX6 YKT261 MATa/␣ lys2-801/lys2-801 ura3-52/ura3-52 his3⌬-200/his3⌬-200 trp1⌬-63/ This study trp1⌬-63 leu2⌬-1/leu2⌬-1 MTI1-3HA:HIS3MX6/MTI1 YKT113 MATa/␣ lys2-801/lys2-801 ura3-52/ura3-52 his3⌬-200/his3⌬-200 trp1⌬-63/ This study trp1⌬-63 leu2⌬-1/leu2⌬-1 myo3⌬::TRP1/MYO3 MYO5-3HA:HIS3MX6/MYO5 YKT313 MATa/␣ lys2-801/lys2-801 ura3-52/ura3-52 his3⌬-200/his3⌬-200 trp1⌬-63/ This study trp1⌬-63 leu2⌬-1/leu2⌬-1 MYO5-13myc:KanMX6/MYO5 YKT475 MATa/␣ lys2-801/lys2-801 ura3-52/ura3-52 his3⌬-200/his3⌬-200 trp1⌬-63/ This study trp1⌬-63 leu2⌬-1/leu2⌬-1 MYO5-13myc:kanMX/MYO5 MTI1-HA:HIS3/MTI1 a YKT strains are isogenic derivatives of YEF473.

Cells, grown at 35Њ for 3 hr, were collected and observed under teins in E. coli DH5␣ and purified with glutathione Sepharose DIC optics. Random fields were recorded as described above. beads (Pharmacia Biotech, Uppsala, Sweden), according to To determine cell size, the cross-sectional area of both mother the manufacturer’s instructions. Overnight culture of YEF473 and unbudded cells was measured using AQUACOSMOS soft- carrying the pKO11-MTI1 plasmid was inoculated into 200 ml ware. Abnormally large cells were defined as cells measuring of SGalA-U (0.17% yeast nitrogen base without amino acids, Ͼ1.5-fold greater in area than the average of wild-type cells. 0.5% casamino acid, 3% galactose, 0.2% sucrose, 0.03% trypto- Fluid-phase endocytosis: Lucifer yellow-carbohydrazide (Sig- phan, and 0.01% adenine) to a cell density of 0.2 OD600/ml. ma) accumulation analysis was performed as described by Cells were incubated at 30Њ for 4 hr to induce expression of Dulic et al. (1991). Lucifer yellow uptake was carried out for hemagglutinin (HA) epitope-tagged Mti1p. Cells were col- 1hrat30Њ. Samples were observed by fluorescence microscopy lected by centrifugation and lysed by mixing for 5 min at 4Њ using a B-2E/C fluorescein isothiocyanate bandpass filter set with 0.5-mm glass beads in 1.2 ml of IP buffer (50 mm Tris- (excitation, 465–495 nm; dichroic mirror, 505 nm; emission, HCl at pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, 515–555 nm) and DIC optics as described above. 50 ␮g/ml aprotinin, 5 ␮g/ml leupeptin, 1 mm phenylmethyl- Assay for the binding of GST-Myo3/5p-SH3-AD with sulfonyl fluoride, and 5 ␮g/ml pepstatin). Protein extracts HA-Mti1p: Recombinant Myo3p-SH3-AD and Myo5p-SH3-AD were clarified by centrifugation for 10 min at 10,000 ϫ g.The were expressed as glutathione S-transferase (GST)-fusion pro- supernatants were incubated with 20 ␮g of either an anti-HA 926 J. Mochida et al.

TABLE 2 Plasmids used in this study

Plasmid Characteristic and source a ␮ pGBD-C1 DBDGAL4, TRP1,2 m(James et al. 1996) ␮ pGBD-C1-MYO3-SH3-AD DBDGAL4-MYO3 (1121–1271), TRP1,2 m ␮ pGBD-C1-MYO3-TH1-TH2 DBDGAL4-MYO3 (757–1123), TRP1,2 m a ␮ pBTM116-HA DBDLexA-HA, TRP1,2 m(Fujiwara et al. 1998) ␮ pBTM116-HA-MYO3-SH3-AD DBDLexA-HA-MYO3 (1121–1271), TRP1,2 m ␮ pBTM116-HA-MYO5-SH3-AD DBDLexA-HA-MYO5 (1087–1219), TRP1,2 m a ␮ pGAD-C1, -C2 and -C3 ADGAL4, LEU2,2 m(James et al. 1996) ␮ pGAD-C1-MTI1 (665–1157) ADGAL4-MTI1 (665–1157), LEU2,2 m; isolated in this study from the yeast genomic library Y2HL-C1 provided by Dr. James ␮ pGAD-C3-MTI1 (799–1157) ADGAL4-MTI1 (799–1157), LEU2,2 m; isolated in this study from the yeast genomic library Y2HL-C3 provided by Dr. James ␮ pGAD-C1-MTI1 (1–1157) ADGAL4-MTI1 (1–1157), LEU2,2 m ␮ pGAD-C1-MTI1 (1–600) ADGAL4-MTI1 (1–600), LEU2,2 m ␮ pGAD-C2-MTI1 (599–1157) ADGAL4-MTI1 (599–1157), LEU2,2 m ␮ pGAD-C1-MTI1 (599–892) ADGAL4-MTI1 (599–892), LEU2,2 m ␮ pGAD-C1-MTI1 (893–1157) ADGAL4-MTI1 (893–1157), LEU2,2 m ␮ pACTII-HK ADGAL4, LEU2,2 m(Ozaki et al. 1996) ␮ pACTII-HK-VRP1 (1–200) ADGAL4-VRP1 (1–200), LEU2,2 m ␮ pBTM116-HA-MTI1 (665–1157) DBDLexA-HA-MTI1 (665–1157), TRP1,2 m ␮ pGAD-C1-MYO3-SH3-AD ADGAL4-MYO3 (1121–1271), LEU2,2 m ␮ pGAD-C1-MYO3-SH3 ADGAL4-MYO3 (1121–1186), LEU2,2 m ␮ pGAD-C1-MYO3-AD ADGAL4-MYO3 (1182–1271), LEU2, 2 m ␮ pGAD-C1-MYO5-SH3-AD ADGAL4-MYO5 (1087–1219), LEU2,2 m ␮ pGAD-C1-MYO5-SH3-AD (W1123S) ADGAL4-MYO5 (1087–1219, W1123S), LEU2,2 m ␮ pGAD-C1-MYO5-SH3 ADGAL4-MYO5 (1087–1152), LEU2,2 m ␮ pGAD-C1-MYO5-AD ADGAL4-MYO5 (1148–1219), LEU2,2 m ␮ pGAD-C1-ABP1 (1–592) ADGAL4-ABP1 (1–592), LEU2,2 m ␮ pGAD-C1-RVS167 (276–482) ADGAL4-RVS167 (276–482), LEU2,2 m ␮ pGAD-C1-SLA1 (1–444) ADGAL4-SLA1 (1–444), LEU2,2 m pGEX-4T-1 GST (Pharmacia) pGEX-4T-1-MYO3-SH3-AD GST-MYO3 (1121–1271) pGEX-4T-1-MYO5-SH3-AD GST-MYO5 (1087–1291) pRS316 URA3, CEN6 (Sikorski and Hieter 1989) pRS316-MTI1gap The 5Ј-flanking region (547 bp) and 3Ј-flanking region (386 bp) of the MTI1 gene were amplified by PCR. The PCR products were cloned into the appropriate sites of pRS316. pRS316-MTI1 MTI1, URA3, CEN6; pRS316-MTI1gap was linearized by restriction enzyme digestion and was introduced into YEF473. The plasmid was isolated from Uraϩ colonies. pRS316-VRP1gap The 5Ј-flanking region (260 bp) and 3Ј-flanking region (419 bp) of the VRP1 gene were cloned into the appropriate sites of pRS316. pRS316-VRP1 VRP1, URA3, CEN6; pRS316-VRP1gap was linearized by restriction enzyme digestion, and was introduced into YEF473. The plasmid was isolated from Uraϩ colonies. pRS316-VRP1-1 vrp1-1, URA3, CEN6; pRS316-VRP1gap was linearized by restriction enzyme digestion and was introduced into TZ33. The plasmid was isolated from Uraϩ colonies. pRS316-END5-1 end5-1, URA3, CEN6; pRS316-VRP1gap was linearized by restriction enzyme digestion and was introduced into RH2075. The plasmid was isolated from Uraϩ colonies. ϫ pKO11 PGAL1-HA 2-TTDH3, URA3, CEN6 (Kamei et al. 1998) ϫ pKO11-YJL020C (pKO11-MT11) PGAL1-HA 2-MTI1-TTDH3, URA3, CEN6

a DBDGAL4 and DBDLexA are the DNA-binding domains of Gal4p and LexA, respectively. ADGAL4 is the transcriptional activating domain of Gal4p.

antibody (HA.11; BABCO, Richmond, CA) or a mouse IgG 200 mm NaCl, and 0.05% Tween 20) containing 5% skimmed for2hrat4Њ. Next, 100 ␮l of protein-G Sepharose 4 Fast Flow milk. The membrane was incubated for 2 hr at room tempera- (Pharmacia Biotech) pretreated with bovine serum albumin ture in TBST containing 5% skimmed milk and 1 ␮m GST- was added. Following rotation of these mixtures for 1 hr at Myo3p-SH3-AD, GST-Myo5p-SH3-AD, or GST. The bound 4Њ, the protein-G Sepharose beads were pelleted and washed GST fusion proteins were detected by immunoblot analysis six times with 1 ml of IP buffer. The immunoprecipitates were using anti-GST antibody. separated by SDS-PAGE and subsequently electroblotted onto Co-immunoprecipitation of Myo5p-myc with Mti1p-HA: a polyvinylidene difluoride membrane. The membrane was YKT313 and YKT475 cells were grown in 2 liters of YPDA at Њ Њ blocked overnight at 4 in TBST (50 mm Tris-HCl at pH 7.5, 30 to a cell density of 2 OD600/ml. Cells were collected by A Novel Regulator of Type I Myosins 927 centrifugation and washed with phosphate-buffered saline. acid positions 184–513 and 174–549). We will report The cells were resuspended in an equal volume of IP buffer and on the interaction between Myo3p-SH3-AD and Ubp7p disrupted using a French pressure at 1000 psi. Protein extracts were clarified by centrifugation for 1 hr at 100,000 ϫ g. The elsewhere. Additional clones contained regions of supernatants (3 ml each) were incubated with 20 ␮g of either Bnr1p (amino acid positions 316–802) and Vrp1p the anti-HA antibody or the control mouse IgG for 2 hr at 4Њ. (amino acid positions 125–371). Both regions contained The precipitation using the protein-G Sepharose beads was a proline-rich sequence that may serve as a binding performed as described above. The immunoprecipitates were site for the Myo3p SH3 domain. The remainder of the separated by SDS-PAGE and analyzed by immunoblot analysis using anti-myc (9E10; Sigma) and anti-HA antibodies. clones contained related regions separated by 56 bp in Other procedures: To extract proteins from yeast cells un- the same locus, encompassing YJL020C and YJL021C der denaturing conditions, cells were treated with 250 mm (Figure 1B). The YJL020C and YJL021C region was also NaOH and 5% trichloroacetic acid. The resulting precipitates cloned as a protein that bound to Myo3p-TH1-TH2, were subjected to SDS-PAGE. which contains a region between the IQ motifs and SH3 domain of Myo3p, in a subsequent two-hybrid screening. This clone, however, interacted with Myo3p-SH3- RESULTS AD with a higher affinity than with Myo3p-TH1-TH2 Isolation of Mti1p as a protein that binds to Myo3p: (data not shown). Our DNA sequencing of the region We attempted to identify proteins that bind to Myo3p between YJL020C and YJL021C revealed four cytosine by a two-hybrid screening method. Myo3p-SH3-AD, residues missing from the Saccharomyces Genome Data- containing the SH3 domain and the adjacent COOH- base (SGD). The nucleotide sequence from 2178 to terminal acidic region, was used as bait (Figure 1A). 2191 of the YJL020C open reading frame was cataloged as Thirty-five clones demonstrated positive interactions 5Ј-AGTACCCAGTACCC-3Ј in the SGD, whereas the corre- with Myo3p-SH3-AD. Of these clones, two contained sponding region within our sequence was 5Ј-AGTACCC regions of the Ubp7p deubiquitinating enzyme (amino CCAGTACCCCC-3Ј. The sequence data comprising this region were submitted to GenBank (accession no. AF373805). These changes connect YJL020C and YJL021C to make a single open reading frame that encodes a protein of 1157 amino acids. To determine if YJL020C and YJL021C encode a single protein, a series of HA epitope tags were introduced into either the 5Ј end of YJL020C (HA-YJL020C) or the 3Ј end of YJL021C (YJL021C-HA). The calculated molecular weights of Yjl020p [771 amino acids (aa)], Yjl021p (365 aa), and Yjl020p ϩ Yjl021p (1157 aa) are and 128 kD, respectively. Immunoblot analysis ,41 ,86ف ,kD 190ف with an anti-HA antibody detected a protein of

Figure 1.—YJL020C and YJL021C encode a single protein that binds to Myo3p. (A) Domain structure of Myo3p. A bar indicates the region used as bait for two-hybrid screening. IQ, IQ motifs; SH3, Src homology 3 domain; AD, acidic domain. (B) A schematic diagram of the YJL020C and YJL021C genomic locus. Arrows indicate the direction of translation. The boxes designate the Myo3p-SH3-AD-interacting clones identiﬁed by two-hybrid screening. (C) Expression of the HA-Yjl020p and Yjl021p-HA constructs in yeast. HA-Yjl020p was expressed under the control of the GAL1 promoter from a plasmid, whereas Yjl021p-HA was expressed under the control of the native promoter from the genomic locus. Left, cell lysates from YEF473 (control), YKT261 (YJL021C-HA), and YKT113 (MYO5-HA) were subjected to SDS-PAGE, followed by immunoblot analysis using an anti-HA antibody. For YEF473 and YKT261, 1.2 OD600 units of cell culture were used for protein extraction; for YKT113, 0.012 OD600 unit of cell culture was mixed with 1.2 OD600 units of YEF473 cell culture and the mixture was used for protein extraction. Right, cells of YEF473 carrying the pKO11 (control) or pKO11-YJL020C (HA-YJL020C) plasmids were grown in SGalA-U medium to induce the expression of HA-Yjl020p. Total isolated protein was subjected to SDS-PAGE, followed by immunoblot analysis using an anti-HA antibody. Ar- rowheads indicate bands corresponding to the HA-tagged proteins. An asterisk indicates a 130-kD protein that nonspeciﬁ- cally reacted with the anti-HA antibody. 928 J. Mochida et al.

acids-rich region (28.3 and 31.7% glutamic and aspartic acids for Mti1p and Spac23a1.17p, respectively), a proline-rich region (32.5 and 37.9% proline for Mti1p and Spac23a1.17p, respectively), and a COOH-terminal conserved region (Figure 2). This high degree of homology suggests that the function of Mti1p is conserved between S. cerevisiae and S. pombe. Figure 2.—Domain comparison of the Saccharomyces cerevisiae A region of Mti1p containing the proline-rich and Mti1p (Sc-Mti1p) and the Schizosaccharomyces pombe Spac23a1.17 COOH-terminal conserved regions specifically interacts (Sp-Mti1p) proteins. Amino acid sequence identity is shown for each homologous region. , SH3 domain; , glutamic with Myo3/5p-SH3-AD: Mti1p (665–1157) and Mti1p and aspartic acids-rich region; ᭿, proline-rich region; and , (799–1157), the original isolates of the two-hybrid COOH-terminal conserved region. screening, interacted with Myo5p-SH3-AD, containing the SH3 domain and the COOH-terminal acidic region of Myo5p (Figure 3). We examined various fragments larger than the expected molecular weight of Yjl020p ϩ of Mti1p for interactions with Myo3/5p-SH3-AD. The Yjl021p expressed as a single protein, in both HA-YJL020C- full-length Mti1p bound to Myo3/5p-SH3-AD, but at expressing and YJL021C-HA-expressing strains (Figure reduced levels. Mti1p (599–1157) interacted with 1C). Therefore, the region encompassing the YJL020C Myo3/5p-SH3-AD to a similar extent as Mti1p (665– and YJL021C sequences encodes a single protein, 1157) and Mti1p (799–1157); Mti1p (599–892), missing dubbed Mti1p. The increased molecular weight of the COOH-terminal conserved region, could not inter- Mti1p from that expected may result from multiple pro- act with Myo3/5p-SH3-AD. Mti1p (893–1157), lacking line residues, which may cause retardation of mobility the proline-rich region, retained weak interactions with in SDS-PAGE analysis (Lee et al. 1988). The expression Myo3/5p-SH3-AD. These results suggest that both the level of Mti1p was roughly estimated by immunoblot proline-rich and COOH-terminal conserved regions of analysis using Myo5p-HA levels as a standard. The rela- Mti1p are required for efficient interactions with Myo3/ tive cellular content of Mti1p-HA ranged from approxi- 5p-SH3-AD. We confirmed that all of the MTI1 frag- mately one-tenth to one-hundredth of that of Myo5p- ments used in Figure 3 were comparably expressed, by HA (see the legend to Figure 1C). immunoblot analysis using an antibody against GAL4- The Mti1p amino acid sequence predicts that Mti1p activating domain (data not shown). Vrp1p also inter- contains an SH3 domain in the NH2-terminal region acts with Myo3/5p-SH3-AD (Anderson et al. 1998). (Figure 2). In the central region, Mti1p contains multi- Mti1p interacted with Myo3/5p-SH3-AD to a similar ex- ple proline-rich motifs that may bind to the Myo3p SH3 tent as Vrp1p. domain. PxxxxPxxP (P is proline, x is any amino acid) Next, we examined the region(s) of Myo3/5p required represents a minimal consensus sequence for both for efficient interactions with Mti1p (Table 3). Substitution Myo3/5p SH3 ligands (Evangelista et al. 2000). Mti1p of a conserved residue within the SH3 domain (W1123S) has 10 potential Myo3/5p SH3 ligands within the abolished the interaction of Myo5p-SH3-AD with Mti1p Myo3p-binding region of Mti1p. Database searches re- (665–1157), supporting the requirement of the SH3 vealed that the protein Spac23a1.17p, found in the fis- domain for this interaction. Myo3/5p-SH3, lacking the sion yeast Schizosaccharomyces pombe, possesses regional COOH-terminal acidic region, did not interact with homologies to Mti1p (Figure 2). Spac23a1.17p also con- Mti1p (665–1157), although this construct provided a tains an SH3 domain in the NH2-terminal region. Mti1p positive interaction in a more sensitive growth assay on and Spac23a1.17p also share a glutamic and aspartic a HisϪ plate than the ␤-galactosidase assay (data not

Figure 3.—Two-hybrid interactions between Myo3/ 5p-SH3-AD and regions of Mti1p. DNA fragments encoding various regions of MTI1 were cloned into pGAD vectors (GAL4 transcriptional activating domain plasmids). The resultant plasmids were introduced into L40 cells expressing a fusion of Myo3/5p-SH3-AD with the LexA DNA-binding domain. The interaction of Myo3/ 5p-SH3-AD with Mti1p was examined for each transformant by quantitative ␤-galactosidase activity assay as described in materials and methods. Each value represents the average and standard deviation for three independent determinations. A Novel Regulator of Type I Myosins 929

TABLE 3 Two-hybrid interactions between Mti1p (665–1157) and regions of Myo3/5p tails

Transcriptional activating ␤-Galactosidase activity a DNA-binding domain fusion domain fusions (Miller units) Mti1p (665–1157) Vector 0.1 Ϯ 0.0 Myo3p-SH3-AD (1121–1271) 87.2 Ϯ 5.9 Myo3p-SH3 (1121–1186) 0.8 Ϯ 0.1 Myo3p-AD (1182–1271) 0.1 Ϯ 0.0 Myo5p-SH3-AD (1087–1219) 65.3 Ϯ 3.0 Myo5p-SH3-AD (1087–1219, W1123S) 0.1 Ϯ 0.0 Myo5p-SH3 (1087–1152) 0.5 Ϯ 0.1 Myo5p-AD (1148–1219) 0.1 Ϯ 0.0 Abp1p (1–592) 0.1 Ϯ 0.0 Rvs167p (276–482) 0.1 Ϯ 0.0 Sla1pb (1–444) 0.1 Ϯ 0.0 a Each value represents the average and standard deviation for three independent quantifications. b Sla1p contains three SH3 domains. Sla1p (1–444) contains all of them. shown). This result indicates that both the SH3 domain (Anderson et al. 1998). Our results suggest that Mti1p and the COOH-terminal acidic region of Myo3/5p are is a component of cortical patch-like structures includ- required for interactions with Mti1p. It is known that ing the Myo3/5p, Vrp1p, and Las17p proteins. various proteins containing SH3 domains, including Direct interaction and co-immunoprecipitation of Abp1p, Rvs167p, and Sla1p, associate with the actin Myo3/5p with Mti1p: To examine direct interactions cytoskeleton. We examined possible interactions of between Mti1p and Myo3/5p, we performed an overlay Mti1p with these proteins. Abp1p (1–592), Rvs167p assay using recombinant proteins. HA-Mti1p was ex- (276–482), and Sla1p (1–444) did not interact with pressed under the GAL1 promoter in yeast and purified Mti1p (665–1157), suggesting that Myo3/5p are specific by immunoprecipitation using an anti-HA antibody. binding partners for Mti1p (Table 3). We confirmed The purified HA-Mti1p was subjected to SDS-PAGE and that all of the MYO3/MYO5, ABP1, RVS167, and SLA1 electroblotted onto a polyvinylidene difluoride mem- fragments used in Table 3 were comparably expressed, brane. The membrane was subjected to the overlay assay by immunoblot analysis using the antibody against using recombinant Myo3/5p-SH3-AD fused to GST. Both GAL4-activating domain (data not shown). GST-Myo3p-SH3-AD and GST-Myo5p-SH3-AD bound to Localization of Mti1p: To examine the intracellular the HA-Mti1p, but GST alone did not (Figure 5A), indi- localization of Mti1p, GFP was fused to the COOH termi- cating that Myo3/5p bind directly to Mti1p. nus of Mti1p. This genomic MTI1-GFP gene was nearly To identify in vivo interactions between Mti1p and functional as MTI1-GFP possessed a much weaker syn- Myo5p, we next immunoprecipitated Mti1p-HA from thetic growth defect with the sac6 mutation than the cell extracts expressing Mti1p-HA and Myo5p, tagged mti1 null mutation (see below, data not shown). Mti1p- with a COOH-terminal myc epitope (Myo5p-myc). GFP was localized to patch-like structures present around Mti1p-HA and Myo5p-myc were expressed under the the cortical region. These patch-like structures were rich control of native promoters of MTI1 and MYO5, respec- in growing regions, including the bud tip and cell divi- tively. Immunoprecipitates were analyzed by immu- sion site (Figure 4A). This pattern of Mti1p-GFP localiza- noblot using anti-HA and anti-myc antibodies. The re- tion is similar to those of Myo5p (Anderson et al. 1998), sults indicated that Myo5p-myc co-immunoprecipitated Las17p (Madania et al. 1999), and Vrp1p (Vaduva et al. with Mti1p-HA (Figure 5B); this antibody-dependent 1997). Co-localization experiments of Mti1p-GFP with co-immunoprecipitation was specific, occurring only in Myo5p were not possible as we have been unable to cells expressing both tagged constructs. Our results sup- observe Mti1p-GFP by immunofluorescence using com- port a role for Mti1p as a Myo3/5p-binding partner. mercial anti-GFP antibody. We also attempted to ob- Genetic interaction between MTI1 and the genes in- serve an Mti1p molecule tagged with the HA epitope, volved in the regulation of the actin cytoskeleton: To but again failed to detect it. The expression levels of explore the functions of Mti1p, we constructed a strain Mti1p may be below the limits of detection by this harboring the mti1 null mutation. mti1 mutant cells grew method (see Figure 1C). Following staining of filamen- normally at 18Њ,25Њ,30Њ, and 37Њ and displayed normal tous actin with TRITC-phalloidin in Mti1p-GFP-express- morphology throughout the cell cycle (data not shown). ing cells, the majority of Mti1p-GFP patches co-localized Whereas wild-type diploid cells display a bipolar bud- with cortical actin patches (Figure 4B). The localization ding pattern, mutants with a perturbed actin cytoskele- of Myo5p also overlaps with cortical actin patch staining ton demonstrate a random budding pattern (Yang et 930 J. Mochida et al.

Figure 5.—Direct interaction and co-immunoprecipitation of Myo3/5p with Mti1p. (A) Direct interaction of recombinant HA-Mti1p with GST-Myo3/5p-SH3-AD. Extracts prepared from cells expressing HA-Mti1p were used for immunoprecipitation with either an anti-HA antibody or a control mouse IgG. Immunoprecipitates were subjected to SDS-PAGE and Figure 4.—Co-localization of Mti1p-GFP with cortical actin subsequently electroblotted onto a polyvinylidene difluoride patches. (A) Cellular localization of Mti1p-GFP. Exponentially membrane. The membrane was probed with GST, GST- growing cells expressing Mti1p-GFP (YKT142) were visualized Myo3p-SH3-AD, or GST-Myo5p-SH3-AD, which was detected using a GFP bandpass filter. (B) Mti1p-GFP partially co-local- with an antibody against GST. (B) Co-immunoprecipitation ized with cortical actin patches. Fixed YKT142 cells were of Myo5p-myc with Mti1p-HA. Extracts prepared from YKT313 stained with TRITC-phalloidin and were visualized with both (MYO5-myc/MYO5 MTI1/MTI1) or YKT475 (MYO5-myc/MYO5 a TRITC filter (Actin) and a GFP bandpass filter (Mti1p-GFP). MTI1-HA/MTI1) cells were used for immunoprecipitation Bar, 5 ␮m. with either an anti-HA antibody or a control mouse IgG. Immu- noprecipitates were subjected to SDS-PAGE, followed by immunoblot analysis using antibodies against myc (top) and HA (bottom). An arrow indicates a probable degradation product al. 1997). The mti1 homozygous diploid cells, however, of Mti1p-HA. The results shown are representative of three exhibited a normal bipolar budding pattern (data not independent experiments. shown). Staining of filamentous actin in mti1 cells revealed that cortical actin patches were localized to the polarized regions, including the bud tip and the site of Table 4). The mti1 sac6 double mutant also exhibited cytokinesis (data not shown), as seen in wild-type cells. reduced growth rates at 25Њ. The mti1 mutation also Mutations in the actin cytoskeleton, such as myo3 myo5, showed a synthetic lethal interaction with sla1 at 37Њ,a vrp1, las17, and sla2 mutants, possess defects in endocy- temperature at which the sla1 mutant cells grow on tosis (Wendland et al. 1998). The mti1 mutant was not our strain background. In addition, the mti1 mutation deficient in fluid-phase endocytosis, as shown by lucifer showed a synthetic lethal interaction with sla2 at 25Њ. yellow uptake (see below). The mti1 mutation, however, did not show a synthetic We next examined the genetic interaction of the mti1 lethal interaction with any of the abp1, arp2-1, arp2-2, mutation with a mutation of genes involved in the regu- cap2, las17, myo3 myo5, rvs167,orvrp1 mutations (Table lation of the actin cytoskeleton. The mti1 mutant was 4). The absence of synthetic lethality of mti1 with abp1, crossed with each mutant; the resulting diploid was spor- cap2,orrvs167 may result from the reduced severity of ulated and dissected for tetrad analysis. The growth these mutants in comparison with the sac6 and sla2 characteristics of the resulting double mutants were de- mutants; the sac6 and sla2 mutant cells do not grow at termined from the observation of more than eight inde- 37Њ, whereas abp1, cap2, and rvs167 mutant cells do. In pendent spore clones for each. The mti1 mutation showed contrast, the arp2-1, arp2-2, las17, vrp1, and myo3 myo5 a synthetic lethal interaction with sac6 at 30Њ, a tempera- mutants show a temperature-sensitive growth pheno- ture at which the sac6 mutant cells grow (Figure 6 and type. The vrp1 and myo3 myo5 mutant cells show, as well A Novel Regulator of Type I Myosins 931

TABLE 4 Summary of genetic interactions between mti1 and mutations in actin-related genes

Double mutant phenotype Mutation Synergistic effect sac6 sla1 sla2 Not synthetic lethal abp1 arp2-1 Figure 6.—Synthetic lethality between the mti1 and sac6 arp2-2 mutations. A tetra type tetrad from a diploid cell, heterozygous cap2 for mti1 and sac6, was grown at 25Њ. Following streaking onto las17 a YPDA plate, the samples were incubated at 30Њ for 2 days. myo3 myo5 The result shown is representative of 10 independent tetrads. rvs167 vrp1 Each mutant strain was crossed to a mti1 null mutant as sla2 and sac6 mutant cells, a slow-growth phenotype (YKT143, YKT144, or YKT190) and resultant diploid cells were even at 30Њ. The absence of synthetic lethality between sporulated and dissected for tetrad analysis to obtain a double mti1 and myo3 myo5 may be because Mti1p functions mutant. Synergistic effect indicates that synthetic lethality with through Myo3/5p. Our results suggest that Mti1p is mti1 was observed at a temperature at which each mutant can grow. The mutant strains used were DDY318 (sac6), YKT218 involved in the regulation of the actin cytoskeleton. (sla1), DDY546 (sla2), DDY322 (abp1), YMW211U (arp2-1), The mti1 mutation suppresses the phenotypes of the YMW221U (arp2-2), DDY582 (cap2), YKT229 (las17), YKT112 vrp1 mutants: During the examination of genetic inter- (myo3 myo5), DDY949 (rvs167), and YKT131 (vrp1). actions of the mti1 mutation with various mutations involved in the actin cytoskeleton, we noted an intriguing finding. The mti1 mutation partially suppressed the tem- products regulate cortical actin patch assembly, includ- perature-sensitive growth phenotype of the vrp1 null ing VRP1, LAS17, and MYO3 MYO5, are required for mutant (Figure 7A), but not that of the las17, arp2-1, growth in high salt medium (Goodson et al. 1996; arp2-2, and myo3 myo5 mutants. A single-copy plasmid Madania et al. 1999). The mti1 mutation suppressed carrying the wild-type MTI1 gene conferred a tempera- the NaCl-sensitive growth of the vrp1-1, but not the end5- ture-sensitive growth phenotype on the mti1 vrp1 mutant 1, mutant (data not shown). The mti1 mutation did not (data not shown). These results prompted us to examine suppress the depolarization of cortical actin patches in possible suppression of other vrp1 mutant phenotypes. vrp1 mutants (data not shown). These results indicate We utilized the vrp1 null mutant as well as two indepen- that the mti1 null mutation partially reduces the require- dent point mutants of VRP1; vrp1-1 results in the substi- ment of Vrp1p in the regulation of the actin cytoskel- tution of Pro for Leu at amino acid position 425 (Vaduva eton. et al. 1999) and end5-1 causes a translational frameshift after amino acid position 604 (Naqvi et al. 1998). Al- though the vrp1-1 and end5-1 mutants showed tem- DISCUSSION perature-sensitive growth phenotypes on other strain In this study, we demonstrated that Mti1p may be backgrounds, vrp1-1 and end5-1 mutants on our strain involved in the reorganization of the actin cytoskeleton. Њ background could grow at 37 , at reduced rates from the Mti1p possesses an NH2-terminal SH3 domain and a wild-type strain (data not shown). The vrp1 mutant cell central proline-rich region. Mti1p has also been identi- culture contains a significant proportion of abnormally fied as a type I myosin-binding protein by C. Boone’s large cells (Donnelly et al. 1993). This phenotype was group and named Bbc1p (Tong et al. 2002). A putative observed in the vrp1 null and end5-1 mutant cell cul- protein in S. pombe, Spac23a1.17p, exhibits homology tures, but not in the vrp1-1 mutant. The mti1 mutation to Mti1p through the entire length of the protein, in- significantly reduced the proportion of abnormally cluding the SH3 domain and proline-rich region, sug- large cells in both the vrp1 null and end5-1 mutant cell gesting a conserved role for Mti1p. cultures (Figure 7B). vrp1 mutants are also deficient in Mti1p is observed within cortical patch-like structures, the internalization step of endocytosis (Munn et al. 1995; co-localizing with cortical actin patches and involved in Zoladek et al. 1995). The mti1 mutation did not affect the assembly of actin filaments. The mti1 null mutation lucifer yellow uptake (Figure 8). At 30Њ, the mti1 mu- showed synthetic lethality with sla1, sla2, and sac6 muta- tation partially restored lucifer yellow uptake in the tions. These genetic interactions indicate the involve- vrp1-1 and end5-1 mutants (Figure 8), but not in the ment of Mti1p in actin cytoskeleton regulation. Sla1p vrp1 null mutant (data not shown). Genes whose protein is a cortical actin-associated protein containing three 932 J. Mochida et al.

Figure 8.—Fluid-phase endocytosis was restored in vrp1-1 and end5-1 mutants by the additional disruption of MTI1. Lucifer yellow uptake was examined in YKT130 cells carrying the pRS316 (MTI1 vrp1), pRS316-VRP1-1 (MTI1 vrp1-1), or pS316-END5-1 (MTI1 end5-1) plasmids and in YKT241 cells carrying the pRS316-VRP1 (mti1 VRP1), pRS316-VRP1-1 (mti1 vrp1-1), or pRS316-END5-1 (mti1 end5-1) plasmids. Cells grown at 30Њ were incubated with lucifer yellow at 30Њ for an additional 1 hr. Bar, 5 ␮m.

SH3 domains (Holtzman et al. 1993). Sla2p is a cortical actin-binding protein containing a COOH-terminal region homologous to the COOH terminus of talin; the protein also shows structural similarity to the mammalian huntingtin-interacting protein 1 (Hip1; Holtzman Figure 7.—Disruption of MTI1 suppresses the vrp1 mutant et al. 1993; Yang et al. 1999). Sac6p is an actin-bundling phenotype. (A) Suppression of the temperature-sensitive protein fimbrin (Adams et al. 1991). Abp1p is a cortical growth of a vrp1 null mutant. A tetra type tetrad from a diploid actin-binding protein and possesses an SH3 domain. heterozygous for mti1 and vrp1 was streaked onto a YPDA Like the mti1 mutant, the abp1 mutant does not show any Њ plate and incubated at 37 for 2 days. The result shown is obvious phenotype alone, but also shows the synthetic representative of 10 independent tetrads. WT, YKT38; mti1, YKT143; vrp1, YKT130; mti1 vrp1, YKT241. (B) Suppression lethal phenotypes with sla1, sla2, and sac6 mutations of the abnormally large cell morphology of vrp1 mutants. (Holtzman et al. 1993). Although their precise func- (Top) The morphology of YKT130 cells carrying the pRS316- tions remain unknown, Mti1p and Abp1p may share a VRP1 (MTI1 VRP1), pRS316 (MTI1 vrp1), or pRS316-END5-1 common function. (MTI1 end5-1) plasmids and YKT241 cells carrying the pRS316- Our findings that Mti1p (a) specifically and directly VRP1 (mti1 VRP1), pRS316 (mti1 vrp1), or pRS316-END5-1 (mti1 end5-1) plasmids. Cells of each strain, grown at 25Њ, were binds to the SH3 domains of Myo3/5p, (b) is localized inoculated into fresh SDA-U medium to a cell density of 0.125 to cortical patch-like structures, and (c) co-immunopre- Њ OD600/ml. Following further incubation at 35 for 3 hr, the cipitates with Myo5p strongly support the role of Mti1p cells were collected and observed under DIC optics. Arrow- as a Myo3/5p-binding partner. The SH3 domains of ␮ heads indicate the abnormally large cells. Bar, 5 m. (Bottom) Myo3/5p also interact with both Vrp1p (Anderson et al. Percentages of abnormally large cells in each mutant. The cells were first prepared as described above, and then the cell 1998) and Las17p (Evangelista et al. 2000). Therefore, sizes of all mother and unbudded cells in random fields were Mti1p may compete with Vrp1p and/or Las17p for inter- measured as described in materials and methods. The num- actions with Myo3/5p. Vrp1p plays an important role bers of cells examined were 141, 242, 204, 221, 232, and 243 in Myo3/5p-dependent actin assembly. The SH3 do- for the MTI1 VRP1, MTI1 vrp1, MTI1 end5-1, mti1 VRP1, mti1 mains of Myo3/5p bind to at least two proline-rich re- vrp1, and mti1 end5-1 mutants, respectively. gions (1–200 and 211–437) of Vrp1p and one proline- rich region (213–222) of Las17p (Evangelista et al. 2000). In addition, the COOH-terminal region (480– A Novel Regulator of Type I Myosins 933

816) of Vrp1p interacts with the NH2-terminal region Little is currently known about the temporal and spa- (2–266) of Las17p (Naqvi et al. 1998). The acidic do- tial regulation of polymerization, assembly, and disas- mains of Myo3/5p and Las17p directly activate the actin- sembly of the actin cytoskeleton. In addition to cell polymerizing activity of the Arp2/3 complex (Madania cycle-dependent actin regulation, yeast change their et al. 1999; Winter et al. 1999; Evangelista et al. 2000; physiological state by reorganizing the actin cytoskele- Lechler et al. 2000). Therefore, Vrp1p works to assemble ton in response to various stimuli, including mating the actin polymerizing machinery, efficiently bringing to- pheromones, heat stress, salt stress, changes in nutrient gether Myo3/5p, Las17p, and the Arp2/3 complex. composition, and so on. Myo3/5p interact with a variety The mti1 mutation partially suppresses the vrp1 mu- of adaptor proteins through their SH3 domains. These tant phenotype, including temperature-sensitive growth, complex molecular interactions may underlie the com- abnormal morphology, defects in endocytosis, and salt- plexity of actin cytoskeleton regulation and function. sensitive growth, caused by defects in cortical actin patch The novel functional properties of the MTI1 gene found assembly. These results suggest that Mti1p and Vrp1p in this study may reflect the complexity of this actin antagonistically regulate the functions of the type I myo- cytoskeleton reorganization. sins. The mechanism by which the mti1 mutation sup- We thank Drs. David Drubin, John Pringle, Anita Hopper, Barbara presses the vrp1 mutation is obscure at present, but one Winsor, Yoshimi Takai, Philip James, and Howard Riezman for yeast possibility is that Mti1p plays a negative regulatory role strains, plasmids, and antibodies. We thank Dr. Charles Boone for in Myo3/5p-dependent actin polymerization by compet- critical reading of our manuscript. We are grateful to Dr. Masahiko ing with Vrp1p for binding to the Myo3/5p SH3 do- Watanabe for the DNA sequencer. We also thank Dr. Erfei Bi for advice on methods of yeast sporulation. We thank Hirofumi Toi, mains. In the vrp1 mutant, Mti1p may also inhibit Naruhiro Matsuo, Hiroyasu Watanabe, and Nao Hamamoto for assis- Las17p to interact with the Myo3/5p SH3 domains. The tance with plasmid construction. We thank Eriko Itoh and Aiko Ishioh two-hybrid analysis suggests the COOH-terminal acidic for technical assistance. This work was supported by grants-in-aid for regions of Myo3/5p are required for efficient interac- Scientific Research from the Ministry of Education, Science, Sports, tions with Mti1p, possibly indicating an additional func- and Culture, Japan, to T.Y. and K.T. and grants from the Naito Founda- tion, the Akiyama Foundation, and the Suhara Memorial Foundation tion for Mti1p. 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