Protein Phosphatase-1 Binding to Scd5p is Important for Regulation of Actin Organization and Endocytosis in Yeast

SCD5, an essential gene, encodes a protein important for endocytosis and actin organization in yeast. Previous two-hybrid screens showed that Scd5p interacts with Glc7p, a yeast Ser/Thr-specific protein phosphatase-1 (PP1) that participates in a variety of cellular processes. PP1 substrate specificity in vivo is regulated by association with different regulatory or targeting subunits, many of which have a consensus PP1-binding site ((V/I)XF, with a basic residue at the -1 or -2 position). Scd5p contains two of these potential PP1-binding motifs: KVDF (amino acids 240-243) and KKVRF (amino acids 272-276). Deletion analysis mapped the PP1-binding domain to a region of Scd5p containing these motifs. Therefore, the consequence of mutating these two potential PP1-binding sites was examined. Although mutation of KVDF had no effect, alteration of KKVRF dramatically reduced Scd5p interaction with Glc7p and resulted in temperature-sensitive growth. Furthermore, this mutation caused defects in fluid phase and receptor-mediated endocytosis and actin organization. Overexpression of GLC7 suppressed the temperature-sensitive growth of the KKVRF mutant and partially rescued the actin organization phenotype. These results provide evidence that Scd5p is a PP1 targeting subunit for regulation of actin organization and endocytosis or that Scd5p is a PP1 substrate, which regulates the function of Scd5p in these processes.


INTRODUCTION
Protein phosphatase-1 (PP1 1 ) is one of the major Ser/Thr protein phosphatases of eukaryotic cells (1). It is highly conserved and is involved in a wide variety of cellular processes, including glycogen and protein synthesis, cell cycle regulation, muscle contraction, and calcium transport (1,2). While the catalytic enzyme has broad substrate specificities in vitro, many lines of evidence have shown that regulatory or targeting subunits direct PP1 to physiological substrates or subcellular locations to perform specific dephosphorylation in vivo (2,3).
Several mammalian and yeast PP1-binding proteins contain a consensus PP1-binding motif (V/I-x-F) with a basic residue at the -1 or -2 position (R/K-V/I-x-F or R/K-x-V/I-x-F) 4 competitive. However, the V/I-x-F motif exists in more than 10% of all known proteins. Most of these are unlikely to interact with PP1, so the importance of this motif for PP1 binding and function has required confirmation by mutational analysis of the V/I-x-F sequence, such as has been done for yeast Gac1p (20) and Reg1p (21), and mammalian PTG (22), NIPP1 (23), and Nrb I (24).
A number of protein interaction screens have identified additional PP1 binding proteins in yeast (10,25,26). Among these is Scd5p, an 872 amino acid protein, which we have recently shown plays a critical role in actin cytoskeleton organization and endocytosis (27). Interestingly, Scd5p contains two potential PP1-binding motifs: KVDF and KKVRF (amino acids 240-243 and 272-276, respectively). In this report we map the Glc7p/PP1 binding site to a region of Scd5p containing these motifs. Mutational analysis indicates that the second putative PP1-binding site (KKVRF) is crucial for Glc7p interaction. Furthermore, this PP1-binding site mutation causes temperature sensitive growth and defects in actin cytoskeleton organization and endocytosis.
Affinity isolation of GST-Scd5p and GST-scd5p-∆338 fusion proteinspJSC2 (GST-SCD5), pJSC12 (GST-scd5-∆338), or pDG101 (GST-bms1-898-100) were transformed into a protease-deficient strain, BJ2168, containing YCp-HA-GLC7. Transformants were grown in 5 ml synthetic complete (SC) medium lacking uracil and leucine and containing 1.95% galactose and 0.05% glucose at 30˚C overnight and then diluted and grown in 50 ml of the same medium until cultures reached 1.0 x 10 7 cells/ml. Approximately 50 x 10 7 cells were harvested, resuspended in 1.2 ml cold lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP40, 1 mM PMSF plus a protease inhibitor cocktail (37)) and lysed by glass beads in a Braun homogenizer for 3 min. Cell extracts were transferred to an ice-cold centrifuge tube and spun down at 20,800 x g for 30 min at 4˚C. The supernatant was saved and protein concentration was by guest on March 23, 2020 http://www.jbc.org/ Downloaded from determined using the Bio-Rad protein determination kit. Extracts (420 µg) diluted to 5 ml with lysis buffer were incubated with 100 µl of a 75% (vol/vol) slurry of glutathione-Sepharose 4B beads (Pharmacia) for 3 h at 4˚C with gentle mixing. The Sepharose beads were pelleted and washed four times with 1 ml lysis buffer and one time with l ml lysis buffer without detergent.
The final bead pellets were resuspended in 100 µl 2x SDS-PAGE sample buffer and boiled for 5 min. Samples (30 µg) of crude extract were also diluted to 100 µl with sample buffer and boiled for 5 min. Crude extract and equal volumes of GST affinity-purified samples were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. Western blots were probed with affinity-purified rabbit antibodies to GST (1:1500, Santa Cruz Biotechnology) or anti-HA rat monoclonal antibodies 3F10 (1:750, Boehringer Mannheim). These were detected by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Sigma) or rabbit anti-rat IgG (1:10,000, Sigma), respectively, and enhanced chemiluminescence (Amersham).
Immunoblotting of Scd5pYeast cells were grown to log phase in 5 ml synthetic selective medium at 25˚C. Approximately 2 x 10 7 cells were harvested, washed with dH 2 O, and resuspended in 0.1 ml 2x SDS-PAGE sample buffer containing 1 mM PMSF plus a protease inhibitor cocktail (37). After lysis with glass beads, extracts (22 µl) were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. Equal loading of protein was confirmed by amido black staining of transfers. Blots were probed with rabbit anti-Scd5p antibodies (1:6,000, from Clarence Chan) and developed as described above for GST-pulldowns.
Two-hybrid analysisBait plasmids (URA3, pGBD-fusions) were transformed into YPJ96-4A and prey plasmids (LEU2, pGAD-fusions) were transformed into SL3004. YPJ96-4A and SL3004 containing these plasmids were mated pairwise on a YEPD plate for 2 days and the diploids containing both plasmids were selected on C-LEU-URA. Cells were grown in liquid C-8 LEU-URA medium to a concentration of 0.5 x 10 7 cells/ml and equal numbers of cells were spotted on C-LEU-URA and C-LEU-URA-ADE to monitor expression of the GAL2-ADE2 reporter gene. β-galactosidase assays were performed as described previously (38,39). Miller units of β-galactosidase activity were calculated from three independent cultures (40).
Endocytosis assaysThe lucifer yellow (LY) uptake assay was performed essentially as described previously (41). Cells were grown at 25˚C in YEPD to early log phase and cultures were kept at 25˚C or pre-shifted to 37˚C for 15 min before addition of LY (Sigma). After incubation for 1 h at 25˚C or 37˚C, cells were washed and observed immediately with a Zeiss Axioplan-2 fluorescence microscope equipped with DIC optics as described by (27). The 35 Salpha-factor internalization assay was performed as described previously (42).
Actin stainingYeast cells were grown in YEPD to early log phase, fixed with formaldehyde, and stained with Alexa-568-phalloidin (Molecular Probes) as described previously (43). Immunofluorescence was performed as described in (44). Cells were fixed using a methanol/acetone dehydration method and stained with anti-actin guinea pig antibodies (1:2,000; (45)), followed by incubation with Alexa-594-conjugated goat anti-guinea pig IgG as described previously (27).

Scd5p Interacts with Glc7p
In VivoPrevious two-hybrid screens using Glc7p as a bait identified Scd5p as an interacting protein (10,25), suggesting that Scd5p might be a regulatory subunit of yeast PP1. To test whether Scd5p interacts with Glc7p in vivo, we expressed GST fusions of Scd5p and Scd5p-∆338 (a C-terminal truncation of 338 amino acids) in wild type yeast also expressing an HA-tagged version of Glc7p. Cells were grown on galactose to induce expression of the GST fusion proteins, which were then affinity-purified from protein extracts A number of mammalian and yeast PP1-binding proteins contain a consensus PP1binding motif (R/K-V/I-x-F or R/K-x-V/I-x-F) through which they interact with the PP1 catalytic subunit, although the latter motif is most common in yeast (18,19). Interestingly, Scd5p contains two potential PP1 binding sequences: KVDF (amino acids 240-243) and KKVRF (amino acids 272-276) which are located in the Glc7p-binding region defined by the two hybrid Scd5p truncation analysis.

The Second PP1 Binding Motif (KKVRF) is Crucial for Scd5p FunctionTo determine
whether these potential PP1-binding motifs are important for Scd5p function, mutations in each or both of the sites were introduced into the full length SCD5 expressed from its own promoter on a CEN LEU2 plasmid and tested for complementation of the scd5 null allele. YCp plasmids carrying scd5-PP1 binding site mutations were transformed into SL4121, which carries a scd5-∆::TRP1 disruption but is viable because of the presence of SCD5 on a URA3 2µ plasmid.
Following plasmid shuffling on 5-FOA to force loss of the URA3 plasmid, the YCp, LEU2 plasmids became the sole source of Scd5 protein. While cells expressing only scd5-PP1∆1 grew normally at 25˚C and 37˚C, scd5-PP1∆2 cells were temperature sensitive for growth at 37˚C (Fig. 4A). Moreover, the double mutant (∆1∆2) failed to complement a scd5 null mutation at any temperature (Fig. 4A).
Immunoblot analysis showed that Scd5p-PP1∆1 was expressed at levels identical to the wild type protein and Scd5p-PP1∆2 was even slightly more abundant that normal Scd5p (Fig.   4B). We were unable to examine expression of the double mutant protein directly, since the strain is inviable. However, preliminary studies from expression of the double mutant protein in the presence of a functional copy of SCD5 suggest that the double mutation causes protein instability 2 . This would explain the lack of interaction in the two hybrid analysis and the lack of complementation of the null mutation at all temperatures. Thus, we conclude that the second PP1 binding site is most important for interaction of Glc7p with Scd5p and for Scd5p's function.

The scd5-PP1∆2 Mutation Causes Defects in Endocytosis and Actin
OrganizationRecently our laboratory has shown that Scd5p plays a critical role in endocytosis and actin cytoskeleton organization (27). Scd5p also colocalizes with cortical actin patches and physically or genetically interacts with a number of cortical actin patch components, many of which are also important for actin organization and endocytosis (27). Therefore, we examined whether the Scd5p-PP1 binding site mutations affect these processes.
We next examined the effect of the Scd5p-PP1-binding motif mutations on the actin cytoskeleton by staining cells with Alexa-564 phalloidin to visualize assembled filamentous (F) actin. In yeast, actin cables, which are bundles of actin filaments, extend from the mother cell into the bud for polarized delivery of organelles and other materials into the growing daughter cell. Cables reorient towards the mother/daughter cell neck during cytokinesis. Cortical patches appear at the site of bud emergence and then localize primarily to the growing bud. Late in the cell cycle they concentrate at the bud neck for septum formation and cytokinesis.
Cells expressing scd5-PP1-∆1 exhibited normal actin structures throughout the cell cycle at 25˚C and 37˚C, similar to those observed in wild type cells (Fig. 6). The scd5-PP1-∆2 strain grown at 25˚C also displayed a relatively normal polarized distribution of cortical actin patches and cables. In contrast, actin structures were aberrant in scd5-PP1-∆2 cells shifted to 37˚C (Fig.   6). In small-and medium-budded cells many actin patches were polarized to the daughter cells, but significant depolarization to mother cells was also observed. In addition, actin cables were often misoriented and much thinner or hardly visible, as compared to those seen in the wild-type and scd5-PP1-∆1 cells. In large-budded cells increased numbers of actin patches were seen distributed throughout cells with scd5-PP1-∆2. These patches were often much larger than normal and an actin ring at the bud neck was rarely observed. In addition, actin cables were barely visible.
Cells were also stained with anti-actin antibodies, which allows visualization of both Fand G-actin. The scd5-PP1-∆2 cells often (up to 11% of cells at 37˚C) contained G-actin bars, which are thought to be aggregates of monomeric or disassembled actin (Fig. 6), whereas none of the wild-type and scd5-PP1-∆1 cells displayed the actin bar phenotype (data not shown). The overall size of scd5-PP1-∆2 cells throughout the cell cycle was also larger than normal at both 25˚C and 37˚C (see Figs. 6, 7), consistent with effects on the actin cytoskeleton and polarized growth.
Overexpression of GLC7 Suppresses scd5-PP1∆2Often when phenotypes in yeast are caused by a mutation that affects the productive interaction of two proteins, the defects can be suppressed by overexpression of the interacting partner. Thus we tested whether overexpression of PP1 can suppress the phenotypes caused by the scd5-PP1∆2 mutation. We found scd5-PP1∆2 cells carrying a vector control (YEp24) were inviable at the restrictive temperature of 37˚C, whereas growth was rescued when cells were transformed with GLC7 expressed from a multicopy plasmid (Fig. 7A). While LY uptake was still defective in the scd5-PP1-∆2 mutant overexpressing GLC7 (not shown), the actin organization phenotype was also partially suppressed by YEpGLC7 (Fig. 7B). Nearly 50% of mutant cells overexpressing GLC7 displayed highly polarized actin patches and normally oriented actin cables, similar to wild-type cells with YEpGLC7 (Fig. 7B). In addition, significant numbers of large budded cells had actin at the site of cytokinesis in the scd5-PP1-∆2 strain overexpressing GLC7. These overexpression studies provide further evidence that Scd5p interaction with PP1 is important for Scd5p function.

DISCUSSION
Scd5p interacts with the yeast PP1 homologue, Glc7p, through its PP1-binding motifMutational analysis of a number of PP1 regulatory proteins has shown that alteration of the highly conserved V/I and/or F residues in their PP1 binding motif disrupts or severely weakens interaction with PP1 and prevents specific functional targeting of the phosphatase (20)(21)(22)(23)(24). In this paper we provide evidence that Scd5p interacts with the GLC7-encoded PP1 in vivo and that, like other PP1 regulatory proteins, a V/I-x-F binding motif in Scd5p is important for PP1 association and the biological function of Scd5p.
Mutational analysis further revealed that the KKVRF signal is crucial for interaction of Scd5p with Glc7p, while the KVDF motif is not likely to bind to the PP1 hydrophobic channel in the Cterminus of PP1. Although KVDF contains the highly conserved V/I and F residues, it contains an acidic residue within the core sequence. Peptide panning experiments to identify PP1 binding sequences found that the most frequent residues in the second position of the V/I-x-F motif were His or Arg (19). In addition, previous work has shown that phosphorylation of serine at this site prevents binding of G M to mammalian PPlc (46). This is also predicted by the crystal structure (18), suggesting that Asp and possibly Glu are not favorable either. However, a KVEF motif found in mammalian aurora kinase appears to be important for PP1 binding, suggesting glutamic acid would be compatible in some contexts (47). Nevertheless our data suggest that the Asp residue in the KVDF sequence of Scd5p prevents interaction with residues comprising the by guest on March 23, 2020 http://www.jbc.org/ Downloaded from hydrophobic pocket of Glc7p, or, alternatively, the KVDF sequence may be inaccessible on the protein surface of Scd5p for binding to the Glc7p hydrophobic channel.
While alteration of the KKVRF motif in Scd5p severely impaired interaction with Glc7p, Scd5p appears to have additional contacts on the Glc7p protein surface, since changing KKVRF to AKAAA did not completely disrupt binding to Glc7p or Scd5p function at the permissive temperature and resulted in a temperature sensitive growth phenotype. However, the mutation did cause some phenotypic consequences at 24˚C, as reduced α-factor uptake and the actin bar phenotype were already observed at this temperature, even though the protein was completely stable. We note that some PP1-binding proteins, such as M 110 (48,49), AKAP220 (50), and PP1 inhibitors (51)(52)(53), bind PP1 through V/I-x-F motifs, but also make additional contacts on the protein surface of PP1, which increase stability of the association or regulate activity and specificity. In addition, in vivo other proteins might form a complex with Scd5p and PP1 to stabilize their interaction. Although we found that Scd5p mutated at both the KVDF and KKVRF motifs was not able to rescue the scd5∆ mutation, the lack of physical interaction with Glc7p by two hybrid analysis or functional complementation by the double mutant protein most likely results from instability of the protein 2 .

Role of Scd5p-PP1 interaction in endocytosis and actin cytoskeleton organization In
addition to causing temperature sensitive growth, scd5-PP1∆2 dramatically blocked both receptor-mediated and fluid-phase endocytosis and had a significant effect on actin organization. Therefore, the critical role of Scd5p in endocytosis and actin organization most likely requires its interaction with and targeting of Glc7p. Supporting this, we found that overexpression of Glc7p could partially rescue Scd5p phenotypes resulting from mutation of the KKVRF PP1 binding motif. In addition, previous studies have shown that some glc7 mutations cause a cortical actin defect (54). A role for PP1 in regulating actin organization has been demonstrated in animal cells as well. For example, neurabin I (Nrb I) binds F-actin and recruits PP1 to control cell morphology (24,55). When the PP1-binding motif in Nrb I was altered, the mutant protein failed to bind PP1 and to induce filopodia formation (24). PP1 regulatory proteins target PP1 to distinct subcellular locations or promote association with specific substrates to reverse or counter the consequences of regulatory phosphorylation by kinases (1-3). One example in yeast is the regulation of a glycogen synthase by a Gac1p/PP1 holoenzyme in glycogen synthesis. Gac1p targets PP1 to a glycogen synthase (Gsy2p) and reverses phosphorylation and inactivation of Gsy2p by cyclin-dependent kinase Pho85p (7,(56)(57)(58)(59). By analogy, Scd5p may direct PP1 to dephosphorylate actin patch-associated components for regulation of actin organization and endocytosis. Our recent work (27) has shown that Scd5p partially colocalizes with actin patches and physically associates with actin patch proteins, such as Sla2p/End4p and Rvs167p (60,61), both of which are also important for endocytosis (62,63).
Interestingly, Scd5p also contains sites that could be targets of regulatory phosphorylation, including a central repeat that has motifs related to those found in other Prk1p substrates. Thus, Scd5p, itself, could be regulated by ARKs or other kinases. This raises the possibility that Scd5p is also a PP1 substrate, and PP1's binding reverses regulatory phosphorylation on Scd5p. This would be similar to the role of Glc7p binding to Reg1p in association with Snf1 kinase for glucose repression. Glc7p dephosphorylates both Snf1p and Reg1p during a regulatory cascade that turns off Snf1p in high glucose conditions (68).  (A) Suppression of scd5-PP1∆2 temperature sensitive growth. SL4417 (scd5-∆::TRP1 + pJSC9 (scd5-PP1∆2)) was transformed with YEp24 or YEpGLC7 and streaked for growth on YEPD for