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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 Copyright 2002 by the Genetics Society of America 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-pro- tein 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 transforma- tion 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-galac- topyranoside 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
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