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

Corresponding author: Kazuma Tanaka, Institute for Genetic Medicine, Hokkaido University Graduate School of Medicine, N15 W7, Kita-ku, Sapporo, Hokkaido, 060-0815, Japan., k-tanaka{at}med.hokudai.ac.jp (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

THE actin cytoskeleton is essential in a wide variety of cellular functions, including cell morphogenesis, cell polarity, cytokinesis, cell adhesions, and endocytosis (BRETSCHER 1991 ; BOTSTEIN et al. 1997 ). Reorganization of the actin cytoskeleton is dynamically regulated both spatially and temporally. The mechanism by which the actin cytoskeleton assembles to mediate these functions, however, remains a fundamental puzzle in cell biology.

The budding yeast Saccharomyces cerevisiae is an excellent model system for the study of actin cytoskeleton dynamics because yeast has a relatively simple actin cytoskeleton and offers powerful experimental tools for genetic manipulation. Throughout the yeast cell cycle, highly regulated reorganizations of the actin cytoskeleton underlie spatial control of cell surface growth, thereby determining cell morphology. Cell surface extension is preceded by the polarized organization of two actin filament-containing structures: cortical actin patches and actin cables (ADAMS and PRINGLE 1984 ; KILMARTIN and ADAMS 1984 ). The formation or reorganization of cortical actin patches is regulated by cortical patch-like protein structures that include Sla1p, Sla2p, Abp1p, Sac6p, Las17p/Bee1p, Vrp1p, Myo3p, Myo5p, Rvs167p, and proteins of the Arp2/3 complex (PRUYNE and BRETSCHER 2000 ). These proteins are also involved in the uptake step of endocytosis through actin cytoskeleton regulation (WENDLAND et al. 1998 ). Actin cables extend along the mother-bud axis and are required for polarized growth (PRUYNE et al. 1998 ).

Myo3/5p are the yeast type I myosins, which are highly conserved actin-activated ATPases that function in endocytosis, membrane trafficking, contractility, and cell motility (MOOSEKER and CHENEY 1995 ). Although deletion of either MYO3 or MYO5 does not result in an obvious growth phenotype, a double knockout is synthetically lethal or nearly so, suggesting the functional redundancy of these genes (GELI and RIEZMAN 1996 ; GOODSON et al. 1996 ). Typically, the tails of unconventional myosins participate in molecular interactions to specify the role of the motor domain. Myo3/5p contain three domains within their tails that are homologous to other members of this protein family: a putative membrane-binding domain (TH1), an alanine- and proline-rich domain (TH2), and an src homology 3 domain (SH3 or TH3; GOODSON and SPUDICH 1995 ).

SH3 domains are present in a variety of proteins associated with the actin cytoskeleton reorganization and with signal transduction. This domain mediates protein-protein interactions through binding to proline-rich stretches (MUSACCHIO et al. 1994 ). The SH3 domains of Myo3/5p interact with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP; EVANGELISTA et al. 2000 ; LECHLER et al. 2000 ), and Vrp1p, a homolog of mammalian WASP-interacting protein (WIP; ANDERSON et al. 1998 ; EVANGELISTA et al. 2000 ). The COOH-terminal acidic regions within Myo3/5p and Las17p interact with the Arp2/3 complex to stimulate actin polymerization (EVANGELISTA et al. 2000 ; LECHLER et al. 2000 ). Vrp1p also interacts with Las17p, suggesting that Vrp1p mediates an efficient interaction between Myo3/5p and Las17p (NAQVI et al. 1998 ). In addition, Vrp1p sustains the interaction of the Myo5p tail region with actin filaments, suggesting a role for Vrp1p in localized Myo3/5p-induced actin polymerization (GELI et al. 2000 ).

To explore the function and regulation of the yeast type I myosins, we searched for proteins that interact with the tail region of Myo3p, using a two-hybrid screening method. We identified a novel protein, Myosin tail region-interacting protein (Mti1p), which binds to the Myo3/5p SH3 domains. Subsequent analyses demonstrated that Mti1p is a binding partner of Myo3/5p. Interestingly, the mti1 null mutation suppressed the vrp1 mutant phenotypes, suggesting that Mti1p and Vrp1p antagonistically regulate the type I myosin functions.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, and genetic techniques:
Yeast strains used in this study with their relevant genotypes are listed in Table 1. Unless otherwise specified, strains were grown in YPDA rich media [1% yeast extract (Difco Laboratories, Detroit), 2% bacto-peptone (Difco), 2% glucose, and 0.01% adenine]. Strains carrying plasmids were selected in synthetic medium (SD) containing the required nutritional supplements (SHERMAN 1991 ). Prior to tetrad analysis, diploid cells were cultured in presporulation medium (0.8% yeast extract, 0.3% bacto-peptone, and 10% glucose) for 24 hr at 25°. The cells were then sporulated in sporulation medium (0.1% yeast extract, 0.05% glucose, and 1% potassium acetate) at a cell density of 1.5 x 10⁷ cells/ml for 1 week at 25°. Standard genetic manipulations of yeast were performed as described (SHERMAN and HICKS 1991 ). Yeast transformations were performed using the lithium acetate method (ELBLE 1992 ). PCR-based gene deletion and epitope tagging of yeast genomic DNA were carried out as previously described (LONGTINE et al. 1998 ). The vrp1 and las17 disruption mutants were constructed on our strain background as follows. The regions containing the disruption marker and the flanking sequences were PCR amplified using either T65-1D (vrp1::LEU2) or DDY1438 (las17 ::URA3) genomic DNA as a template. The resulting DNA fragment was then introduced into YEF473. Proper gene disruption was verified by PCR. Escherichia coli strains DH5 and XL1-Blue were used for the construction and amplification of plasmids.

Molecular biological techniques:
Standard molecular biological techniques, described by SAMBROOK et al. 1989 , were used for the construction of plasmids, PCR amplification, and DNA sequencing. PCR amplification was performed using a GeneAmp PCR system 9700 (Perkin-Elmer, Norwalk, CT). DNA sequences were obtained using an ABI PRISM 310 DNA sequencer (Applied Biosystems, Foster City, CA). The DNA sequences of all constructs containing amplified PCR products were confirmed using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems), according to the manufacturer's protocol. Site-directed mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as per the manufacturer's instructions. The plasmids used in this study are listed in Table 2. Schemes for the construction of plasmids and the sequences of PCR primers are available upon request.

Two-hybrid screening:
Two-hybrid screening was performed as described by JAMES et al. 1996 . To maximize transformation efficiency, large-scale transformations were carried out according to the protocol described by AGATEP et al. 1998 . PJ69-4A, carrying the pGBD-C1-MYO3-SH3-AD plasmid, was independently transformed with three yeast genomic DNA libraries (Y2HL-C1, -C2, and -C3), plated on SD-Trp-Leu-His plates, and incubated for 6–8 days at 30°. The plates were then replica plated onto fresh SD-Trp-Leu-Ade plates for stringent selection of positive clones. After an additional 6–8 days incubation, 151, 86, and 45 colonies were picked up from the 4 x 10⁶, 4.3 x 10⁷, and 2.2 x 10⁷ initial transformants of Y2HL-C1, -C2, and -C3, respectively. These colonies were patched onto SD-Trp-Leu plates and grown at 30° for 3 days. Clones were then tested using a 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside filter assay to measure ß-galactosidase activity (VOJTEK et al. 1993 ). Plasmids were isolated from clones that turned blue (8, 29, and 16 positive clones from each genomic DNA library) and were reintroduced into PJ69-4A cells carrying the pGBD-C1-MYO3-SH3-AD plasmid. Transformants were retested both for growth on SD-Trp-Leu-His and SD-Trp-Leu-Ade plates and for the 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside filter assay. Positive clones were then sequenced. We also performed a two-hybrid screening, as described above, using the pGBD-C1-MYO3-TH1-TH2 plasmid as the bait. Quantification of ß-galactosidase activity was performed using o-nitrophenyl ß-D-galactopyranoside as a substrate (GUARENTE 1983 ). ß-Galactosidase activity is expressed in Miller units (MILLER 1972 ).

Microscopic observations:
To visualize Mti1p, the 3' end of the chromosomal MTI1 gene was tagged with the sequence encoding green fluorescent protein (GFP) as described in LONGTINE et al. 1998 . YKT142 cells were grown to early logarithmic phase in YPDA medium, harvested, and resuspended in SD medium. Cells were mounted on microslide glass and observed immediately using a GFP bandpass filter set (excitation, 460–500 nm; dichroic mirror, 505 nm; emission, 510–560 nm). To visualize the actin cytoskeleton, exponentially growing cells were fixed for 15 min by the direct addition of commercial 37% formaldehyde stock (Wako Pure Chemical Industries, Osaka, Japan) to a 5% final concentration in medium. Fixed cells were stained for 30 min at room temperature with 1 µM tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma Chemical, St. Louis). Following five washes in phosphate-buffered saline, cells were mounted in 90% glycerol containing n-propyl gallate (Wako). Cells were observed using a G-2A TRITC filter set (excitation, 510–560 nm; dichroic mirror, 575 nm; emission, 590 nm) on a Nikon ECLIPSE E800 microscope (Nikon Instec, Tokyo, Japan). The microscope setup contained an HB-10103AF super high-pressure mercury lamp and a 1.4NA 100x Plan Apo oil immersion objective (Nikon Instec) with either the appropriate fluorescence-filter sets (Nikon Instec) or differential interference contrast (DIC) optics. The images presented in this article were acquired using a cooled digital CCD camera (C4742-95-12NR; Hamamatsu Photonics K. K., Hamamatsu, Japan) and AQUACOSMOS software (Hamamatsu Photonics K. K.). This microscopic imaging system was also used to measure the size of yeast cells. Overnight cultures were inoculated into fresh SDA-U [0.17% yeast nitrogen base without amino acids (Difco), 0.5% casamino acid (Difco), 2% glucose, 0.03% tryptophan, and 0.01% adenine] to a cell density of 0.125 OD₆₀₀/ml. Cells, grown at 35° for 3 hr, were collected and observed under DIC optics. Random fields were recorded as described above. To determine cell size, the cross-sectional area of both mother and unbudded cells was measured using AQUACOSMOS software. Abnormally large cells were defined as cells measuring >1.5-fold greater in area than the average of wild-type cells.

Fluid-phase endocytosis:
Lucifer yellow-carbohydrazide (Sigma) accumulation analysis was performed as described by DULIC et al. 1991 . Lucifer yellow uptake was carried out for 1 hr at 30°. Samples were observed by fluorescence microscopy using a B-2E/C fluorescein isothiocyanate bandpass filter set (excitation, 465–495 nm; dichroic mirror, 505 nm; emission, 515–555 nm) and DIC optics as described above.

Assay for the binding of GST-Myo3/5p-SH3-AD with HA-Mti1p:
Recombinant Myo3p-SH3-AD and Myo5p-SH3-AD were expressed as glutathione S-transferase (GST)-fusion proteins in E. coli DH5 and purified with glutathione Sepharose beads (Pharmacia Biotech, Uppsala, Sweden), according to the manufacturer's instructions. Overnight culture of YEF473 carrying the pKO11-MTI1 plasmid was inoculated into 200 ml of SGalA-U (0.17% yeast nitrogen base without amino acids, 0.5% casamino acid, 3% galactose, 0.2% sucrose, 0.03% tryptophan, and 0.01% adenine) to a cell density of 0.2 OD₆₀₀/ml. Cells were incubated at 30° for 4 hr to induce expression of hemagglutinin (HA) epitope-tagged Mti1p. Cells were collected by centrifugation and lysed by mixing for 5 min at 4° with 0.5-mm glass beads in 1.2 ml of IP buffer (50 mM Tris-HCl at pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml pepstatin). Protein extracts were clarified by centrifugation for 10 min at 10,000 x g. The supernatants were incubated with 20 µg of either an anti-HA antibody (HA.11; BABCO, Richmond, CA) or a mouse IgG for 2 hr at 4°. Next, 100 µl of protein-G Sepharose 4 Fast Flow (Pharmacia Biotech) pretreated with bovine serum albumin was added. Following rotation of these mixtures for 1 hr at 4°, the protein-G Sepharose beads were pelleted and washed six times with 1 ml of IP buffer. The immunoprecipitates were separated by SDS-PAGE and subsequently electroblotted onto a polyvinylidene difluoride membrane. The membrane was blocked overnight at 4° in TBST (50 mM Tris-HCl at pH 7.5, 200 mM NaCl, and 0.05% Tween 20) containing 5% skimmed milk. The membrane was incubated for 2 hr at room temperature in TBST containing 5% skimmed milk and 1 µM GST-Myo3p-SH3-AD, GST-Myo5p-SH3-AD, or GST. The bound GST fusion proteins were detected by immunoblot analysis using anti-GST antibody.

Co-immunoprecipitation of Myo5p-myc with Mti1p-HA:
YKT313 and YKT475 cells were grown in 2 liters of YPDA at 30° to a cell density of 2 OD₆₀₀/ml. Cells were collected by centrifugation and washed with phosphate-buffered saline. The cells were resuspended in an equal volume of IP buffer and disrupted using a French pressure at 1000 psi. Protein extracts were clarified by centrifugation for 1 hr at 100,000 x g. The supernatants (3 ml each) were incubated with 20 µg of either the anti-HA antibody or the control mouse IgG for 2 hr at 4°. The precipitation using the protein-G Sepharose beads was performed as described above. The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblot analysis using anti-myc (9E10; Sigma) and anti-HA antibodies.

Other procedures:
To extract proteins from yeast cells under denaturing conditions, cells were treated with 250 mM NaOH and 5% trichloroacetic acid. The resulting precipitates were subjected to SDS-PAGE.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of Mti1p as a protein that binds to Myo3p:
We attempted to identify proteins that bind to Myo3p by a two-hybrid screening method. Myo3p-SH3-AD, containing the SH3 domain and the adjacent COOH-terminal acidic region, was used as bait (Fig 1A). Thirty-five clones demonstrated positive interactions with Myo3p-SH3-AD. Of these clones, two contained regions of the Ubp7p deubiquitinating enzyme (amino acid positions 184–513 and 174–549). We will report on the interaction between Myo3p-SH3-AD and Ubp7p elsewhere. Additional clones contained regions of Bnr1p (amino acid positions 316–802) and Vrp1p (amino acid positions 125–371). Both regions contained a proline-rich sequence that may serve as a binding site for the Myo3p SH3 domain. The remainder of the clones contained related regions separated by 56 bp in the same locus, encompassing YJL020C and YJL021C (Fig 1B). The YJL020C and YJL021C region was also cloned as a protein that bound to Myo3p-TH1-TH2, 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-AD with a higher affinity than with Myo3p-TH1-TH2 (data not shown). Our DNA sequencing of the region between YJL020C and YJL021C revealed four cytosine residues missing from the Saccharomyces Genome Database (SGD). The nucleotide sequence from 2178 to 2191 of the YJL020C open reading frame was cataloged as 5'-AGTACCCAGTACCC-3' in the SGD, whereas the corresponding region within our sequence was 5'-AGTACCCCCAGTACCCCC-3'. 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 5' end of YJL020C (HA-YJL020C) or the 3' end of YJL021C (YJL021C-HA). The calculated molecular weights of Yjl020p [771 amino acids (aa)], Yjl021p (365 aa), and Yjl020p + Yjl021p (1157 aa) are 86, 41, and 128 kD, respectively. Immunoblot analysis with an anti-HA antibody detected a protein of 190 kD, larger than the expected molecular weight of Yjl020p + Yjl021p expressed as a single protein, in both HA-YJL020C-expressing and YJL021C-HA-expressing strains (Fig 1C). Therefore, the region encompassing the YJL020C and YJL021C sequences encodes a single protein, dubbed Mti1p. The increased molecular weight of Mti1p from that expected may result from multiple proline residues, which may cause retardation of mobility in SDS-PAGE analysis (LEE et al. 1988 ). The expression level of Mti1p was roughly estimated by immunoblot analysis using Myo5p-HA levels as a standard. The relative cellular content of Mti1p-HA ranged from approximately one-tenth to one-hundredth of that of Myo5p-HA (see the legend to Fig 1C).

View larger version (23K): In this window In a new window Download PPT slide   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. Arrowheads indicate bands corresponding to the HA-tagged proteins. An asterisk indicates a 130-kD protein that nonspecifically reacted with the anti-HA antibody.

The Mti1p amino acid sequence predicts that Mti1p contains an SH3 domain in the NH₂-terminal region (Fig 2). In the central region, Mti1p contains multiple proline-rich motifs that may bind to the Myo3p SH3 domain. PxxxxPxxP (P is proline, x is any amino acid) represents a minimal consensus sequence for both Myo3/5p SH3 ligands (EVANGELISTA et al. 2000 ). Mti1p has 10 potential Myo3/5p SH3 ligands within the Myo3p-binding region of Mti1p. Database searches revealed that the protein Spac23a1.17p, found in the fission yeast Schizosaccharomyces pombe, possesses regional homologies to Mti1p (Fig 2). Spac23a1.17p also contains an SH3 domain in the NH₂-terminal region. Mti1p and Spac23a1.17p also share a glutamic and aspartic 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 (Fig 2). This high degree of homology suggests that the function of Mti1p is conserved between S. cerevisiae and S. pombe.

View larger version (15K): In this window In a new window Download PPT slide   Figure 2. Domain comparison of the Saccharomyces cerevisiae Mti1p (Sc-Mti1p) and the Schizosaccharomyces pombe Spac23a1.17 (Sp-Mti1p) proteins. Amino acid sequence identity is shown for each homologous region. , SH3 domain; , glutamic and aspartic acids-rich region; , proline-rich region; and , COOH-terminal conserved region.

A region of Mti1p containing the proline-rich and COOH-terminal conserved regions specifically interacts with Myo3/5p-SH3-AD:
Mti1p (665–1157) and Mti1p (799–1157), the original isolates of the two-hybrid screening, interacted with Myo5p-SH3-AD, containing the SH3 domain and the COOH-terminal acidic region of Myo5p (Fig 3). We examined various fragments of Mti1p for interactions with Myo3/5p-SH3-AD. The full-length Mti1p bound to Myo3/5p-SH3-AD, but at reduced levels. Mti1p (599–1157) interacted with Myo3/5p-SH3-AD to a similar extent as Mti1p (665–1157) and Mti1p (799–1157); Mti1p (599–892), missing the COOH-terminal conserved region, could not interact with Myo3/5p-SH3-AD. Mti1p (893–1157), lacking the proline-rich region, retained weak interactions with Myo3/5p-SH3-AD. These results suggest that both the proline-rich and COOH-terminal conserved regions of Mti1p are required for efficient interactions with Myo3/5p-SH3-AD. We confirmed that all of the MTI1 fragments used in Fig 3 were comparably expressed, by immunoblot analysis using an antibody against GAL4-activating domain (data not shown). Vrp1p also interacts with Myo3/5p-SH3-AD (ANDERSON et al. 1998 ). Mti1p interacted with Myo3/5p-SH3-AD to a similar extent as Vrp1p.

View larger version (19K): In this window In a new window Download PPT slide   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 ß-galactosidase activity assay as described in MATERIALS AND METHODS. Each value represents the average and standard deviation for three independent determinations.

Next, we examined the region(s) of Myo3/5p required for efficient interactions with Mti1p (Table 3). Substitution of a conserved residue within the SH3 domain (W1123S) abolished the interaction of Myo5p-SH3-AD with Mti1p (665–1157), supporting the requirement of the SH3 domain for this interaction. Myo3/5p-SH3, lacking the COOH-terminal acidic region, did not interact with Mti1p (665–1157), although this construct provided a positive interaction in a more sensitive growth assay on a His^- plate than the ß-galactosidase assay (data not shown). This result indicates that both the SH3 domain and the COOH-terminal acidic region of Myo3/5p are required for interactions with Mti1p. It is known that various proteins containing SH3 domains, including Abp1p, Rvs167p, and Sla1p, associate with the actin cytoskeleton. We examined possible interactions of Mti1p with these proteins. Abp1p (1–592), Rvs167p (276–482), and Sla1p (1–444) did not interact with Mti1p (665–1157), suggesting that Myo3/5p are specific binding partners for Mti1p (Table 3). We confirmed that all of the MYO3/MYO5, ABP1, RVS167, and SLA1 fragments used in Table 3 were comparably expressed, by immunoblot analysis using the antibody against GAL4-activating domain (data not shown).

Localization of Mti1p:
To examine the intracellular localization of Mti1p, GFP was fused to the COOH terminus of Mti1p. This genomic MTI1-GFP gene was nearly functional as MTI1-GFP possessed a much weaker synthetic growth defect with the sac6 mutation than the mti1 null mutation (see below, data not shown). Mti1p-GFP was localized to patch-like structures present around the cortical region. These patch-like structures were rich in growing regions, including the bud tip and cell division site (Fig 4A). This pattern of Mti1p-GFP localization is similar to those of Myo5p (ANDERSON et al. 1998 ), Las17p (MADANIA et al. 1999 ), and Vrp1p (VADUVA et al. 1997 ). Co-localization experiments of Mti1p-GFP with Myo5p were not possible as we have been unable to observe Mti1p-GFP by immunofluorescence using commercial anti-GFP antibody. We also attempted to observe an Mti1p molecule tagged with the HA epitope, but again failed to detect it. The expression levels of Mti1p may be below the limits of detection by this method (see Fig 1C). Following staining of filamentous actin with TRITC-phalloidin in Mti1p-GFP-expressing cells, the majority of Mti1p-GFP patches co-localized with cortical actin patches (Fig 4B). The localization of Myo5p also overlaps with cortical actin patch staining (ANDERSON et al. 1998 ). Our results suggest that Mti1p is a component of cortical patch-like structures including the Myo3/5p, Vrp1p, and Las17p proteins.

View larger version (43K): In this window In a new window Download PPT slide   Figure 4. Co-localization of Mti1p-GFP with cortical actin patches. (A) Cellular localization of Mti1p-GFP. Exponentially growing cells expressing Mti1p-GFP (YKT142) were visualized using a GFP bandpass filter. (B) Mti1p-GFP partially co-localized with cortical actin patches. Fixed YKT142 cells were stained with TRITC-phalloidin and were visualized with both a TRITC filter (Actin) and a GFP bandpass filter (Mti1p-GFP). Bar, 5 µm.

Direct interaction and co-immunoprecipitation of Myo3/5p with Mti1p:
To examine direct interactions between Mti1p and Myo3/5p, we performed an overlay assay using recombinant proteins. HA-Mti1p was expressed under the GAL1 promoter in yeast and purified by immunoprecipitation using an anti-HA antibody. The purified HA-Mti1p was subjected to SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. The membrane was subjected to the overlay assay using recombinant Myo3/5p-SH3-AD fused to GST. Both GST-Myo3p-SH3-AD and GST-Myo5p-SH3-AD bound to the HA-Mti1p, but GST alone did not (Fig 5A), indicating that Myo3/5p bind directly to Mti1p.

View larger version (44K): In this window In a new window Download PPT slide   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 subsequently electroblotted onto a polyvinylidene difluoride membrane. The membrane was probed with GST, GST-Myo3p-SH3-AD, or GST-Myo5p-SH3-AD, which was detected with an antibody against GST. (B) Co-immunoprecipitation of Myo5p-myc with Mti1p-HA. Extracts prepared from YKT313 (MYO5-myc/MYO5 MTI1/MTI1) or YKT475 (MYO5-myc/MYO5 MTI1-HA/MTI1) cells were used for immunoprecipitation with either an anti-HA antibody or a control mouse IgG. Immunoprecipitates were subjected to SDS-PAGE, followed by immunoblot analysis using antibodies against myc (top) and HA (bottom). An arrow indicates a probable degradation product of Mti1p-HA. The results shown are representative of three independent experiments.

To identify in vivo interactions between Mti1p and Myo5p, we next immunoprecipitated Mti1p-HA from cell extracts expressing Mti1p-HA and Myo5p, tagged with a COOH-terminal myc epitope (Myo5p-myc). Mti1p-HA and Myo5p-myc were expressed under the control of native promoters of MTI1 and MYO5, respectively. Immunoprecipitates were analyzed by immunoblot using anti-HA and anti-myc antibodies. The results indicated that Myo5p-myc co-immunoprecipitated with Mti1p-HA (Fig 5B); this antibody-dependent co-immunoprecipitation was specific, occurring only in cells expressing both tagged constructs. Our results support a role for Mti1p as a Myo3/5p-binding partner.

Genetic interaction between MTI1 and the genes involved in the regulation of the actin cytoskeleton:
To explore the functions of Mti1p, we constructed a strain harboring the mti1 null mutation. mti1 mutant cells grew normally at 18°, 25°, 30°, and 37° and displayed normal morphology throughout the cell cycle (data not shown). Whereas wild-type diploid cells display a bipolar budding pattern, mutants with a perturbed actin cytoskeleton demonstrate a random budding pattern (YANG et al. 1997 ). The mti1 homozygous diploid cells, however, exhibited a normal bipolar budding pattern (data not 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 cytokinesis (data not shown), as seen in wild-type cells. Mutations in the actin cytoskeleton, such as myo3 myo5, vrp1, las17, and sla2 mutants, possess defects in endocytosis (WENDLAND et al. 1998 ). The mti1 mutant was not deficient in fluid-phase endocytosis, as shown by lucifer yellow uptake (see below).

We next examined the genetic interaction of the mti1 mutation with a mutation of genes involved in the regulation of the actin cytoskeleton. The mti1 mutant was crossed with each mutant; the resulting diploid was sporulated and dissected for tetrad analysis. The growth characteristics of the resulting double mutants were determined from the observation of more than eight independent spore clones for each. The mti1 mutation showed a synthetic lethal interaction with sac6 at 30°, a temperature at which the sac6 mutant cells grow (Fig 6 and Table 4). The mti1 sac6 double mutant also exhibited reduced growth rates at 25°. The mti1 mutation also showed a synthetic lethal interaction with sla1 at 37°, a temperature at which the sla1 mutant cells grow on our strain background. In addition, the mti1 mutation showed a synthetic lethal interaction with sla2 at 25°. The mti1 mutation, however, did not show a synthetic lethal interaction with any of the abp1, arp2-1, arp2-2, cap2, las17, myo3 myo5, rvs167, or vrp1 mutations (Table 4). The absence of synthetic lethality of mti1 with abp1, cap2, or rvs167 may result from the reduced severity of these mutants in comparison with the sac6 and sla2 mutants; the sac6 and sla2 mutant cells do not grow at 37°, whereas abp1, cap2, and rvs167 mutant cells do. In contrast, the arp2-1, arp2-2, las17, vrp1, and myo3 myo5 mutants show a temperature-sensitive growth phenotype. The vrp1 and myo3 myo5 mutant cells show, as well as sla2 and sac6 mutant cells, a slow-growth phenotype even at 30°. The absence of synthetic lethality between mti1 and myo3 myo5 may be because Mti1p functions through Myo3/5p. Our results suggest that Mti1p is involved in the regulation of the actin cytoskeleton.

View larger version (47K): In this window In a new window Download PPT slide   Figure 6. Synthetic lethality between the mti1 and sac6 mutations. A tetra type tetrad from a diploid cell, heterozygous for mti1 and sac6, was grown at 25°. Following streaking onto a YPDA plate, the samples were incubated at 30° for 2 days. The result shown is representative of 10 independent tetrads.

The mti1 mutation suppresses the phenotypes of the vrp1 mutants:
During the examination of genetic interactions of the mti1 mutation with various mutations involved in the actin cytoskeleton, we noted an intriguing finding. The mti1 mutation partially suppressed the temperature-sensitive growth phenotype of the vrp1 null mutant (Fig 7A), but not that of the las17, arp2-1, arp2-2, and myo3 myo5 mutants. A single-copy plasmid carrying the wild-type MTI1 gene conferred a temperature-sensitive growth phenotype on the mti1 vrp1 mutant (data not shown). These results prompted us to examine possible suppression of other vrp1 mutant phenotypes. We utilized the vrp1 null mutant as well as two independent point mutants of VRP1; vrp1-1 results in the substitution of Pro for Leu at amino acid position 425 (VADUVA et al. 1999 ) and end5-1 causes a translational frameshift after amino acid position 604 (NAQVI et al. 1998 ). Although the vrp1-1 and end5-1 mutants showed temperature-sensitive growth phenotypes on other strain backgrounds, vrp1-1 and end5-1 mutants on our strain background could grow at 37°, at reduced rates from the wild-type strain (data not shown). The vrp1 mutant cell culture contains a significant proportion of abnormally large cells (DONNELLY et al. 1993 ). This phenotype was observed in the vrp1 null and end5-1 mutant cell cultures, but not in the vrp1-1 mutant. The mti1 mutation significantly reduced the proportion of abnormally large cells in both the vrp1 null and end5-1 mutant cell cultures (Fig 7B). vrp1 mutants are also deficient in the internalization step of endocytosis (MUNN et al. 1995 ; ZOLADEK et al. 1995 ). The mti1 mutation did not affect lucifer yellow uptake (Fig 8). At 30°, the mti1 mutation partially restored lucifer yellow uptake in the vrp1-1 and end5-1 mutants (Fig 8), but not in the vrp1 null mutant (data not shown). Genes whose protein products regulate cortical actin patch assembly, including VRP1, LAS17, and MYO3 MYO5, are required for growth in high salt medium (GOODSON et al. 1996 ; MADANIA et al. 1999 ). The mti1 mutation suppressed the NaCl-sensitive growth of the vrp1-1, but not the end5-1, mutant (data not shown). The mti1 mutation did not suppress the depolarization of cortical actin patches in vrp1 mutants (data not shown). These results indicate that the mti1 null mutation partially reduces the requirement of Vrp1p in the regulation of the actin cytoskeleton.

View larger version (37K): In this window In a new window Download PPT slide   Figure 7. Disruption of MTI1 suppresses the vrp1 mutant phenotype. (A) Suppression of the temperature-sensitive growth of a vrp1 null mutant. A tetra type tetrad from a diploid heterozygous for mti1 and vrp1 was streaked onto a YPDA plate and incubated at 37° for 2 days. The result shown is representative of 10 independent tetrads. WT, YKT38; mti1, YKT143; vrp1, YKT130; mti1 vrp1, YKT241. (B) Suppression of the abnormally large cell morphology of vrp1 mutants. (Top) The morphology of YKT130 cells carrying the pRS316-VRP1 (MTI1 VRP1), pRS316 (MTI1 vrp1), or pRS316-END5-1 (MTI1 end5-1) plasmids and YKT241 cells carrying the pRS316-VRP1 (mti1 VRP1), pRS316 (mti1 vrp1), or pRS316-END5-1 (mti1 end5-1) plasmids. Cells of each strain, grown at 25°, were inoculated into fresh SDA-U medium to a cell density of 0.125 OD600/ml. Following further incubation at 35° for 3 hr, the cells were collected and observed under DIC optics. Arrowheads indicate the abnormally large cells. Bar, 5 µm. (Bottom) Percentages of abnormally large cells in each mutant. The cells were first prepared as described above, and then the cell sizes of all mother and unbudded cells in random fields were measured as described in MATERIALS AND METHODS. The numbers of cells examined were 141, 242, 204, 221, 232, and 243 for the MTI1 VRP1, MTI1 vrp1, MTI1 end5-1, mti1 VRP1, mti1 vrp1, and mti1 end5-1 mutants, respectively.

View larger version (79K): In this window In a new window Download PPT slide   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 30° were incubated with lucifer yellow at 30° for an additional 1 hr. Bar, 5 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we demonstrated that Mti1p may be involved in the reorganization of the actin cytoskeleton. Mti1p possesses an NH₂-terminal SH3 domain and a central proline-rich region. Mti1p has also been identified as a type I myosin-binding protein by C. Boone's group and named Bbc1p (TONG et al. 2002 ). A putative protein in S. pombe, Spac23a1.17p, exhibits homology to Mti1p through the entire length of the protein, including the SH3 domain and proline-rich region, suggesting a conserved role for Mti1p.

Mti1p is observed within cortical patch-like structures, co-localizing with cortical actin patches and involved in the assembly of actin filaments. The mti1 null mutation showed synthetic lethality with sla1, sla2, and sac6 mutations. These genetic interactions indicate the involvement of Mti1p in actin cytoskeleton regulation. Sla1p is a cortical actin-associated protein containing three 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 et al. 1993 ; YANG et al. 1999 ). Sac6p is an actin-bundling protein fimbrin (ADAMS et al. 1991 ). Abp1p is a cortical actin-binding protein and possesses an SH3 domain. Like the mti1 mutant, the abp1 mutant does not show any obvious phenotype alone, but also shows the synthetic lethal phenotypes with sla1, sla2, and sac6 mutations (HOLTZMAN et al. 1993 ). Although their precise functions remain unknown, Mti1p and Abp1p may share a common function.

Our findings that Mti1p (a) specifically and directly binds to the SH3 domains of Myo3/5p, (b) is localized to cortical patch-like structures, and (c) co-immunoprecipitates with Myo5p strongly support the role of Mti1p as a Myo3/5p-binding partner. The SH3 domains of Myo3/5p also interact with both Vrp1p (ANDERSON et al. 1998 ) and Las17p (EVANGELISTA et al. 2000 ). Therefore, Mti1p may compete with Vrp1p and/or Las17p for interactions with Myo3/5p. Vrp1p plays an important role in Myo3/5p-dependent actin assembly. The SH3 domains of Myo3/5p bind to at least two proline-rich regions (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 (480–816) of Vrp1p interacts with the NH₂-terminal region (2–266) of Las17p (NAQVI et al. 1998 ). The acidic domains of Myo3/5p and Las17p directly activate the actin-polymerizing activity of the Arp2/3 complex (MADANIA et al. 1999 ; WINTER et al. 1999 ; EVANGELISTA et al. 2000 ; LECHLER et al. 2000 ). Therefore, Vrp1p works to assemble the actin polymerizing machinery, efficiently bringing together Myo3/5p, Las17p, and the Arp2/3 complex.

The mti1 mutation partially suppresses the vrp1 mutant phenotype, including temperature-sensitive growth, abnormal morphology, defects in endocytosis, and salt-sensitive growth, caused by defects in cortical actin patch assembly. These results suggest that Mti1p and Vrp1p antagonistically regulate the functions of the type I myosins. The mechanism by which the mti1 mutation suppresses the vrp1 mutation is obscure at present, but one possibility is that Mti1p plays a negative regulatory role in Myo3/5p-dependent actin polymerization by competing with Vrp1p for binding to the Myo3/5p SH3 domains. In the vrp1 mutant, Mti1p may also inhibit Las17p to interact with the Myo3/5p SH3 domains. The two-hybrid analysis suggests the COOH-terminal acidic regions of Myo3/5p are required for efficient interactions with Mti1p, possibly indicating an additional function for Mti1p. Mti1p may regulate the interactions of the Myo3/5p acidic regions with the Arp2/3 complex. To examine the effect of Mti1p stoichiometry on actin cytoskeleton assembly, we overexpressed Mti1p under the GAL1 promoter. The overexpression of Mti1p, however, did not affect cell growth or polarization of cortical actin patches (our unpublished observations), suggesting that Mti1p does not simply downregulate actin polymerization. Vrp1p also mediates the interaction of the Myo3/5p SH3 domains with actin filaments (GELI et al. 2000 ). The type I myosins possess an ATP-independent actin-binding site in their tail regions, mapped to the TH2 domain in protozoan type I myosins (BRZESKA et al. 1988 ; DOBERSTEIN and POLLARD 1992 ; JUNG and HAMMER 1994 ). In yeast type I myosins, this ATP-independent actin-binding site maps to the SH3 domain. Vrp1p is required for the myosin tail-actin interaction (GELI et al. 2000 ). This function of Vrp1p accounts for the requirement of Vrp1p for the localization of Myo5p to the polarized region in an actin cytoskeleton-dependent manner (ANDERSON et al. 1998 ). It is currently unknown whether Vrp1p interacts with actin filaments directly or indirectly, through either Las17p or an as yet unknown protein. The mti1 mutation, however, may restore the interaction of the type I myosin SH3 domains with actin filaments in the vrp1 mutant cells. The NH₂-terminal SH3 domain of Mti1p may also be involved in protein-protein interactions. This domain interacts with the proline-rich region of Vrp1p, but not with that of Mti1p, by two-hybrid analysis (our unpublished observations). This Mti1p-Vrp1p interaction may also interfere with the interaction of Vrp1p with the Myo3/5p SH3 domains.

Little is currently known about the temporal and spatial regulation of polymerization, assembly, and disassembly of the actin cytoskeleton. In addition to cell cycle-dependent actin regulation, yeast change their physiological state by reorganizing the actin cytoskeleton in response to various stimuli, including mating pheromones, heat stress, salt stress, changes in nutrient composition, and so on. Myo3/5p interact with a variety of adaptor proteins through their SH3 domains. These complex molecular interactions may underlie the complexity of actin cytoskeleton regulation and function. The novel functional properties of the MTI1 gene found in this study may reflect the complexity of this actin cytoskeleton reorganization.

	ACKNOWLEDGMENTS

We thank Drs. David Drubin, John Pringle, Anita Hopper, Barbara Winsor, Yoshimi Takai, Philip James, and Howard Riezman for yeast strains, plasmids, and antibodies. We thank Dr. Charles Boone for critical reading of our manuscript. We are grateful to Dr. Masahiko Watanabe for the DNA sequencer. We also thank Dr. Erfei Bi for advice on methods of yeast sporulation. We thank Hirofumi Toi, Naruhiro Matsuo, Hiroyasu Watanabe, and Nao Hamamoto for assistance with plasmid construction. We thank Eriko Itoh and Aiko Ishioh for technical assistance. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan, to T.Y. and K.T. and grants from the Naito Foundation, the Akiyama Foundation, and the Suhara Memorial Foundation to K.T.

Manuscript received July 31, 2001; Accepted for publication January 7, 2002.

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