The Identification of Pcl1-Interacting Proteins That Genetically Interact With Cla4 May Indicate a Link Between G₁ Progression and Mitotic Exit

Corresponding author: George F. Sprague, Jr., University of Oregon, Eugene, OR 97403-1229., gsprague{at}molbio.uoregon.edu (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes. Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes. However, loss of both proteins is lethal, suggesting some functional overlap. To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4 strain. These mutations define >60 genes. Recently, both Ste20 and Cla4 have been implicated in mitotic exit. Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4. We further show that the Pho85-coupled G₁ cyclins Pcl1 and Pcl2 contribute to this Pho85 role. We performed a two-hybrid screen with Pcl1. Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1. Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1·Pho85 complexes. NCP1, HMS1, and EPA1 also genetically interact with CLA4. Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins. We propose a model in which the G₁ Pcl1·Pho85 complex regulates mitotic exit machinery.

CELLULAR morphogenesis in budding yeast requires the essential, small Rho-like GTPase Cdc42. This molecule is required at many steps during morphogenesis, from bud site selection to cytokinesis (ADAMS et al. 1990 ; JOHNSON and PRINGLE 1990 ; JOHNSON 1999 ; RICHMAN et al. 1999 ; GULLI et al. 2000 ; KOZMINSKI et al. 2000 ; RICHMAN and JOHNSON 2000 ; GLADFELTER et al. 2002 ). The ability of Cdc42 to function at numerous points during the budding process implies that its activity is regulated and that it derives specificity in some manner. Two classes of proteins directly regulate the activity of Cdc42 by modulating its GDP/GTP bound state. The guanine nucleotide exchange factor Cdc24 promotes activation of Cdc42 whereas the GTPase activating factors Rga1, Rga2, and Bem3 promote its inactivation (BENDER and PRINGLE 1989 ; ADAMS et al. 1990 ; GLADFELTER et al. 2002 ; SMITH et al. 2002 ). In addition, Cdc42 has an array of effector molecules that are able to perform subsets of its morphogenetic functions (BENTON et al. 1997 ; CHEN et al. 1997 ; MARTIN et al. 1997 ; BI et al. 2000 ). Two of these effectors, Cla4 and Ste20, are members of the p21-activated kinase (PAK) family of signaling molecules. Both Cla4 and Ste20 physically interact with and are regulated by Cdc42 (LEBERER et al. 1992 ; CVRCKOVA et al. 1995 ). In addition, both Cla4 and Ste20 contribute to cellular morphogenesis (LEBERER et al. 1992 ; CVRCKOVA et al. 1995 ).

PAK kinases are conserved among eukaryotic species (MANSER et al. 1994 ; BAGRODIA et al. 1995 ; CREASY and CHERNOFF 1995 ; MARTIN et al. 1995 ). This family of kinases regulates mitogen-activated protein (MAP) kinase signaling, cell cycle progression, and cellular morphogenesis. In budding yeast, Cla4 and Ste20 perform both distinct and overlapping cellular tasks. Cla4 was initially identified in a mutant screen for genes required for viability in the absence of Cln1 and Cln2 (two G₁ cyclins), suggesting a functional connection to G₁ progression (CVRCKOVA et al. 1995 ). Cla4 is also required for septin function during bud formation (HOLLY and BLUMER 1999 ; GULLI et al. 2000 ; LONGTINE et al. 2000 ; BOSE et al. 2001 ; GLADFELTER et al. 2002 ). Ste20, on the other hand, was identified as a component of the mating pathway and functions upstream of the MAP kinase cascade (LEBERER et al. 1992 ). Ste20 was subsequently shown to signal upstream of two other MAP kinase cascades, one involved in filamentation and the other in growth on high salt (ROBERTS and FINK 1994 ; MOSCH et al. 1996 ; ROBERTS et al. 1997 ; O'ROURKE and HERSKOWITZ 1998 ; RAITT et al. 2000 ). In addition to these distinctive functions, Cla4 and Ste20 are thought to function redundantly in at least one instance (CVRCKOVA et al. 1995 ). This interpretation follows from the observation that the loss of either Cla4 or Ste20 is viable, but the loss of both proteins leads to a block in cell cycle progression. These double mutants are able to replicate their DNA but fail to direct bud growth properly and to undergo anaphase efficiently (CVRCKOVA et al. 1995 ). Consistent with these results, both Cla4 and Ste20 have recently been shown to contribute to mitotic exit (HOFKEN and SCHIEBEL 2002 ; CHIROLI et al. 2003 ).

To investigate the roles of Cla4, mutations were identified that, like ste20, are lethal in the absence of Cla4 (cla4; MITCHELL and SPRAGUE 2001 ). Remarkably, at least 62 genes are individually required for viability under this condition. The molecular mechanisms responsible for these synthetic genetic interactions are poorly understood. To shed light on these mechanisms, we have chosen individual mutations for careful characterization. One such mutation led to the identification of PHO85 as being required for viability in the absence of Cla4. PHO85 encodes a nonessential cyclin-dependent kinase involved in many cellular processes including phosphate metabolism and cell cycle progression (TOH-E et al. 1988 ; HUANG et al. 1996 ; O'NEILL et al. 1996 ; TIMBLIN et al. 1996 ; TENNYSON et al. 1998 ; MCBRIDE et al. 2001 ; CARROLL and O'SHEA 2002 ). PHO85 derives specificity by coupling with specific cyclins that direct interactions with particular substrates (HUANG et al. 1998 ; TENNYSON et al. 1998 ; WANG et al. 2001B ). We found that the loss of Pho85, or the simultaneous loss of two Pho85 G₁ cyclin partners, Pcl1 and Pcl2, is lethal when combined with the loss of Cla4, consistent with previous observations (LENBURG and O'SHEA 2001 ; HUANG et al. 2002 ).

To explore the significance of the genetic interactions between CLA4 and PCL1, we performed a two-hybrid screen using Pcl1 as the bait. We identified three interacting proteins: Ncp1 (an NADP-cytochrome P450 reductase), Hms1 (a transcription factor), and Yjr072C [an essential putative ATPase of unknown function, which we have dubbed Epa1 (essential Pcl1-interacting ATPase; LORENZ and HEITMAN 1998 ; VENKATESWARLU et al. 1998 ; BAIROCH and APWEILER 2000 )]. These two-hybrid interactions were validated by glutathione S-transferase (GST) pull-down experiments and by in vitro kinase assays that demonstrated the ability of Pcl1·Pho85 complexes to specifically phosphorylate these three Pcl1-interacting proteins. NCP1, HMS1, and EPA1 were individually shown to genetically interact with CLA4. Epa1 shows sequence similarity to minD, a bacterial septation regulator, suggesting a potential role for Epa1 during mitotic exit. Pho85, Hms1, and Ncp1 are required for the proper localization of Cdc14, itself a member of the mitotic exit network. Hence, we propose that Pcl1·Pho85 regulates the mitotic exit machinery.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast manipulations:
Strains used in this study are listed in Table 1. Standard media and yeast manipulations were used (SAMBROOK et al. 1989 ; BURKE et al. 2000 ).

Plasmid construction:
To create pSL2805, YJR072C/EPA1 was cloned into YEp351 using recombination-based subcloning as described previously (MA et al. 1987 ). In brief, the YEp351 plasmid was cleaved using BamHI, gel purified, and then transformed into yeast along with PCR products containing sequence homologous to YEp351 and to the gene of interest. YJR072C/EPA1 was amplified using the primers (5'-CAG CTA TGA CCA TGA TTA CGA ATT CGA GCT CGG TAC CCG GCA ATC TTC ATA TGC AAA CCC-3') and (5'-GTG CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CGA GCT CTA AAT CTG TTG GCC-3'). Plasmids used to express maltose-binding fusion proteins were as follows. YJR072C/EPA1 and NCP1 were subcloned into the bacterial expression vector pMAL-c2G (New England Biolabs, Beverly, MA) at the XbaI site, thus generating pSL2814 and pSL2816, respectively. The YJR072C/EPA1 gene was amplified using the primers (5'-GCG CTC TAG AAT GAG TCT CAG CAC AAT CAT-3') and (5'-GCG CTC TAG AGG CCA AAA CTG TTT TGC CGG-3'), which include engineered XbaI sites at the 5' and 3' ends for cloning purposes. The NCP1 gene was amplified using the primers (5'-GCG CTC TAG AAT GCC GTT TGG AAT AGA CAA-3') and (5'-GCG CTC TAG AGG ATT TGA CGT GAA GAA CGG-3'), which include engineered XbaI sites at the 5' and 3' ends for cloning purposes. HMS1 was subcloned into the EcoRI site of pMAL-c2G to obtain pSL2815. The HMS1 gene was amplified using the primers (5'-CCC GGA ATT CAT GCC AAA TTT TCA AAA ACC-3') and (5'-CCC GGA ATT CCT TCC AAG CTG TTC TGG CGG-3'), which include engineered EcoRI sites at the 5' and 3' ends for subcloning. pEG-GST and pEG-GST-PCL1 were kind gifts of M. Snyder. These URA3-based plasmids express either GST or GST-Pcl1 under a galactose-regulated promoter. To make the Pcl1 bait, pSL2796, PCL1 was cloned into the BamHI site of pGBDU-C(1) using recombination-based subcloning. PCL1 was amplified by PCR using the primers (5'-AAA GGT CAA AGA CAG TTG ACT GTA TCG CCG GAA TTC CCC ATG TGT GAA TAC AGC AAG GCT-3') and (5'-TTT TCA GTA TCT ACG ATT CAT AGA TCT CTG CAG GTC GAC AAA CCC ATG TTG ACT CAT GAT-3'). Two-hybrid plasmids expressing fusions to the Gal4 activation domain discovered during the two-hybrid screen are described in detail below. Briefly, they were as follows: AD-Yjr072c/Epa1, pSL2793; AD-Ncp1, pSL2794; and AD-Hms1, pSL2795. The empty library plasmid, pGAD-C1, has been described previously (JAMES et al. 1996 ). The four low-copy LEU2-based plasmids containing PHO85, pSL2820, pSL2821, pSL2822, and pSL2823, were isolated from the p366 library (ATCC). Low-copy ELP2-LEU2, pSL2825, was made by recombination-based subcloning into the BamHI site of pRS315. The empty LEU2 vector is pRS315. The CLA4-URA3 plasmid, pSL2674 (also named pRS316ADE8CLA4 in previous publications), has been described previously (GIETZ et al. 1992 ). All plasmids generated during the course of this study were confirmed by DNA sequencing.

Yeast two-hybrid screen:
A yeast two-hybrid screen was performed using the Phil James strains and reagents (JAMES et al. 1996 ). The PCL1 "bait," pSL2796, was introduced into the yeast strain PJ69-4A (GIETZ et al. 1992 ). The subsequent strain was transformed with the genomic libraries Y2HL-C1, Y2HL-C2, and Y2HL-C3; 6 x 10⁶, 5 x 10⁵, and 1 x 10⁶ transformants were screened from each library, respectively (GIETZ et al. 1992 ). Transformants were initially screened for the ability to grow on medium lacking histidine and supplemented with 4.8 mM 3AT. A total of 1200 positives were identified in the primary screen. These transformants were then tested for the ability to grow on medium lacking adenine; 56 activated both reporter genes. Only 3 of these still activated the reporter genes after plasmid rescue and retransformation into the PJ69-4A strain containing the PCL1 bait. These 3 library plasmids required the PCL1 bait to activate the reporter genes. Sequence analysis of the 3 positives revealed three unique activation domain fusions. Positive 1, pSL2793, had the GAL4 activation domain fused to the YJR072C/EPA1 coding sequence at position 529. Positive 284, pSL2794, had the GAL4 activation domain fused to the NCP1 coding sequence at position 517. Positive 686, pSL2795, had the GAL4 activation domain fused to the HMS1 coding sequence at position 183.

Bacterial protein purification:
Maltose-binding protein (MBP) fusions, MBP-Epa1, MBP-Hms1, and MBP-Ncp1 (expressed from pSL2814, pSL2815, and pSL2816, respectively), were individually expressed in Escherichia coli (gold cells) and purified over separate amylose columns (SMITH and JOHNSON 1988 ). MBP alone was obtained from New England Biolabs.

GST pull-down experiments:
To confirm a physical interaction between Pcl1 and Epa1, Hms1, or Ncp1, GST pull-down experiments were performed. Plasmids expressing GAL-promoter driven GST-Pcl1 or GST alone (pEG-GST-PCL1 and pEG-GST, respectively, both generous gifts from M. Snyder), were transformed into yeast (ESM1362) to obtain strains SY4108 and -4109, respectively. Cells were grown to midlog phase in selective medium lacking uracil and containing 2% raffinose as the carbon source. GST fusion protein expression was induced by growing cultures for 4 hr in 2% galactose. Cells were then harvested, converted to spheroplasts (BOWERS et al. 2000 ), and lysed in IP buffer (50 mM Tris, pH 8.0; 1 mM EDTA; 50 mM NaCl; 1% NP40; 5 µg of aprotinin/ml; 5 µg of antipain/ml; 5 µg of leupeptin/ml; 5 µg of pepstatin A/ml; and 1 mM phenylmethylsulfonyl fluoride) for 15 min. Following centrifugation for 5 min at 13,000 rpm, lysates were incubated with glutathione-Sepharose (Amersham Pharmacia) for 1 hr with gentle agitation. The GST complexes were washed several times with IP buffer and then incubated with 2 µg of bacterially purified fusion protein for 2 hr on ice. The GST complexes were rewashed several times with IP buffer, and the final pellets were suspended in 30 µl of 2x Laemmli buffer (LAEMMLI 1970 ), boiled for 5 min, centrifuged for 1 min, and then resolved on SDS-PAGE gels. Western analysis was performed as described below.

Kinase assays:
GST fusion protein expression was induced in strains containing GAL-promoter-driven GST-Pcl1 or GST alone as described above. Cells were then harvested, spheroplasted (BOWERS et al. 2000 ), and lysed in IP buffer for 15 min. Following centrifugation for 5 min at 13,000 rpm, lysates were incubated with glutathione-Sepharose for 1 hr with gentle agitation. GST complexes were washed once with IP buffer, once with RIPA buffer (150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, and 50 mM Tris-HCl, pH 8.0), and twice with kinase reaction buffer (50 mM Tris-HCl, pH 7.5; 40 mM magnesium chloride; 1 mM dithiothreitol; 0.5 mM sodium orthovanadate; 5 µg aprotinin/ml; and 5 µg leupeptin/ml). Kinase assays were carried out in 30 µl of kinase buffer containing 1 µM MBP-Hms1, MBP-Ncp1, MBP-Epa1, or MBP alone and ATP (1000 Ci/mol) for 30 min at 30°. These reactions were terminated with 30 µl of 2x Laemmli buffer followed by 5 min of boiling. Terminated reactions were then subjected to electrophoresis through an 8% polyacrylamide gel and transferred to nitrocellulose. Phosphorylated proteins were detected using a STORM 860 phosphodetector system (Amersham Biosciences, Piscataway, NJ) and quantified using Imagequant V1.11 (Molecular Dynamics, Sunnyvale, CA). Blots were then subjected to Western analysis as described below (see Western analysis), except blots were developed using ECL plus (Amersham, Arlington Heights, IL), detected using the STORM 860 scanner, and quantified using Imagequant V1.11 software.

Western analysis:
Protein preparations were subjected to electrophoresis through an 8% polyacrylamide gel and transferred to nitrocellulose. To detect GST, the blots were probed with a 1:200 dilution of polyclonal GST antibody (Molecular Probes, Eugene, OR) and then with a 1:3000 dilution of Bio-Rad (Richmond, CA) goat anti-rabbit IgG horseradish peroxidase conjugate. To detect MBP fusions, the blots were probed with a 1:10,000 dilution of polyclonal MBP antibody (New England Biolabs) and subsequently with a 1:3000 dilution of Bio-Rad goat anti-rabbit antibodies. Proteins were visualized using ECL plus.

ATPase assays:
NADH-based indirect ATPase assays were performed as described previously (TSUNODA et al. 2001 ). Bacterially purified proteins—maltose-binding protein alone, MBP-Ncp1, and MBP-Epa1—were individually added to 1 ml of reaction mixture containing 25 mM Tris, pH 7.5, 25 mM KCl, 2 mM MgCl₂, 5 mM KCN, 2 mM phosphophenolpyruvate, 5 mM ATP, 0.5 mM NADH, 30 units of L-lactic acid dehydrogenase, and 30 units of pyruvate kinase. These reactions proceeded at 30° for 3 min. The absorbance at 340 nm was followed spectrophotometrically during the 3-min incubation; changes in absorbance reflect ATP hydrolysis. Each sample was assayed in triplicate.

Cdc14 localization:
Cdc14 localization was performed essentially as previously described (HOFKEN and SCHIEBEL 2002 ). Briefly, yeast strains containing CDC14-GFP (ESM1362, a generous gift from E. Schiebel) and strains additionally deleted for pho85, hms1, or ncp1 (SY4111-4113) were synchronized with -factor, released from G₁ arrest, and grown at 10°. Cells were observed using a Zeiss Axioplan II microscope with Nomarski optics or differential interference contrast optics with a fluorescence microscopy filter. The fraction of cells exhibiting nucleolar Cdc14-GFP was quantitated. A total of 100 cells were observed for each sample and all samples were analyzed in triplicate.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

PHO85 is required for viability in the absence of CLA4:
The contribution of Cla4 during cell cycle progression remains poorly understood. To gain insight into this cellular role, mutations were identified that, like the loss of STE20, were lethal in the absence of CLA4 (MITCHELL and SPRAGUE 2001 ). Several alleles of elp2 (a transcription elongation factor) were identified (MITCHELL and SPRAGUE 2001 ). In the process of cloning ELP2, four low-copy PHO85-containing plasmids that bypassed the elp2cla4 lethal phenotype were obtained (Fig 1). The basis of the ELP2 and CLA4 synthetic interaction is under investigation and has been described elsewhere; here we focus on PHO85 (GOEHRING et al. 2003 ). PHO85 encodes a nonessential cyclin-dependent kinase involved in many cellular tasks including phosphate metabolism and cell cycle progression (TOH-E et al. 1988 ; HUANG et al. 1996 , HUANG et al. 1998 , HUANG et al. 2002 ; O'NEILL et al. 1996 ; TIMBLIN et al. 1996 ; TENNYSON et al. 1998 ; CARROLL et al. 2001 ; LENBURG and O'SHEA 2001 ; MCBRIDE et al. 2001 ; WANG et al. 2001A , WANG et al. 2001B ; CARROLL and O'SHEA 2002 ). This kinase derives specificity by coupling with cyclin molecules; its role in G₁ progression is mediated by its association with the G₁ cyclins, Pcl1 and Pcl2 (HUANG et al. 1998 ; WILSON et al. 1999 ; WANG et al. 2001B ). The ability of the PHO85-containing plasmids to suppress an elp2 allele suggested that PHO85 and CLA4 genetically interact. Indeed, we found that, like elp2cla4 mutants, the pho85cla4 double mutants were inviable (Fig 2). In addition, we found that simultaneous loss of the Pho85 cyclin molecules Pcl1 and Pcl2 was lethal in the absence of Cla4, consistent with previous observations (Fig 2; LENBURG and O'SHEA 2001 ; HUANG et al. 2002 ).

View larger version (59K): In this window In a new window Download PPT slide   Figure 1. PHO85 is a low-copy suppressor of the elp2cla4 lethality. The ability of PHO85-containing plasmids to restore viability to an elp2cla4 strain is shown. The growth phenotype of an elp2cla4 [CLA4-URA3] strain was cotransformed with one of the indicated low-copy LEU2-marked plasmids: empty vector, ELP2-containing plasmid, and four different plasmids containing genomic DNA that spans the PHO85 locus. The strains were replica plated to either selective medium or selective medium containing 0.1% 5-FOA. The ability to grow on 5-FOA indicates the ability to lose the CLA4-URA3 plasmid and thus to suppress the elp2cla4 lethality. Strains and plasmids used were as follows: elp2cla4 [CLA4-URA3] strain, SY4090; CLA4-URA3 plasmid, pSL2674; empty LEU2-marked vector, pRS315; ELP2-containing LEU2-marked plasmid, pSL2825; and PHO85-containing LEU2-marked plasmids 1–4, pSL2820, pSL2821, pSL2822, and pSL2823.

View larger version (33K): In this window In a new window Download PPT slide   Figure 2. PHO85 or PCL1 and PCL2 together are required for viability in the absence of CLA4. PHO85, ELP2, or PCL1 and PCL2 together were deleted in a cla4 strain containing a CLA4-URA3 plasmid. These strains were replica plated to either rich medium or selective medium lacking uracil and containing 0.1% 5-FOA. The inability of particular strains to grow on 5-FOA demonstrates the requirement of the CLA4-URA3 plasmid for viability. Strains and plasmids were as follows: cla4 [CLA4-URA3], SY3363; cla4elp2 [CLA4-URA3], SY4086; cla4pho85 [CLA4-URA3], SY4087; cla4pcl1pcl2 [CLA4-URA3], SY4091; and CLA4-URA3 plasmid, pSL2674.

Pcl1 interacts with Ncp1, Hms1, and Yjr072C/Epa1:
Cla4 is required for normal bud formation and for mitotic exit (CVRCKOVA et al. 1995 ; HOFKEN and SCHIEBEL 2002 ). Given that Pho85 or Pcl1 and Pcl2 together are required for viability in the absence of Cla4, we considered that the targets of Pcl1·Pho85 might be involved in one or more Cla4-mediated processes. As one means to identify targets of Pcl1·Pho85 complexes, we performed a two-hybrid screen using Pcl1 as the bait. We screened 6 million library plasmids using the Phil James two-hybrid system (JAMES et al. 1996 ) and found three proteins that interact with Pcl1: Ncp1, Hms1, and Yjr072c (Fig 3). Ncp1 is an NADP-cytochrome P450 reductase involved in ergosterol biosynthesis (O'NEILL et al. 1996 ). Hms1 is an Myc-like transcription factor involved in filamentous growth (JASPERSEN et al. 1998 ). Yjr072C is an essential, novel, presumptive ATPase of previously unknown function, which we have termed Epa1 (see below).

View larger version (80K): In this window In a new window Download PPT slide   Figure 3. Pcl1 interacts with Epa1, Ncp1, and Hms1 by yeast two-hybrid. The ability of Pcl1 to interact with Epa1, Ncp1, and Hms1 by yeast two-hybrid is shown. Strains of the PJ69-4A background containing a plasmid encoding the Gal4 DNA-binding domain fused to Pcl1 (Pcl1 bait) and a library plasmid (encoding the Gal4 activation domain fusion alone or fused to one of the following proteins: Epa1, Ncp1, or Hms1) were replica plated to medium lacking both uracil and leucine. To assay for two-hybrid interaction, cells were also replica plated to medium additionally lacking histidine supplemented with 4.8 mM 3AT and to medium additionally lacking adenine. The ability to grow on medium lacking histidine supplemented with 4.8 mM 3AT indicates the ability to activate the GAL1-HIS3 reporter gene. The ability to grow on medium lacking adenine indicates the ability to activate the GAL2-ADE2 reporter gene. To ensure that the observed interactions were bait dependent, PJ69-4A strains containing only the AD-encoding library plasmids were replica plated to the following: medium lacking leucine, medium lacking leucine and histidine and supplemented with 3AT, and medium additionally lacking both leucine and adenine. The inability of strains lacking the Pcl1 bait to grow without supplemental histidine or adenine indicates that the library plasmids are unable to activate the reporter genes on their own. Two-hybrid fusion proteins were expressed from the following plasmids: Pcl1 bait, pSL2796; Gal4 activation domain (AD) alone, pGAD-C1; AD-Epa1, pSL2793; AD-Ncp1, pSL2794; and AD-Hms1, pSL2795.

As one means to validate the physical interaction of Pcl1 with Epa1, Hms1, and Ncp1, we performed GST pull-down experiments using GST-Pcl1 and GST alone. GST-Pcl1 or GST alone was purified and subsequently incubated with 1 µM of bacterially expressed and purified maltose-binding protein fusions to Epa1, Hms1, or Ncp1. We found that samples containing GST-Pcl1 pulled down MBP-Epa1, MBP-Hms1, and MBP-Ncp1, but failed to pull down MBP alone (Fig 4). The GST alone samples failed to pull down any of the fusion proteins (Fig 4). Therefore, GST-Pcl1 specifically interacts with maltose-binding fusions of Epa1, Hms1, and Ncp1.

View larger version (35K): In this window In a new window Download PPT slide   Figure 4. Pcl1 physically associates with Epa1, Hms1, and Ncp1 in GST pull-downs. The ability of Pcl1 to associate with Epa1, Hms1, and Ncp1 in GST pull-down experiments is shown. GST pull-down experiments were performed using GST-Pcl1 or GST alone purified from yeast and substrates MBP-Epa1, MBP-Hms1, and MBP-Ncp1 purified from bacteria as described in MATERIALS AND METHODS. The presence of a given substrate in the pull-down was detected using anti-MBP antiserum on a Western blot. An anti-GST antibody was used to detect either GST alone or GST-Pcl1. Fusion proteins were expressed from the following plasmids: GST alone, pEG-GST; GST-Pcl1, pEG-GST-PCL1; MBP-Epa1, pSL2814; MBP-Hms1, pSL2815; and MBP-Ncp1, pSL2816.

Pcl1·Pho85 complexes phosphorylate bacterially purified Epa1, Hms1, and Ncp1:
The ability of Epa1, Hms1, and Ncp1 to physically interact with Pcl1 suggests that these may be targets of Pcl1·Pho85 kinase. We tested the ability of GST-Pcl1 purified from yeast to phosphorylate the following bacterially expressed and purified substrates: MBP-Epa1, MBP-Hms1, MBP-Ncp1, and MBP (maltose-binding protein alone). We found that Pcl1·Pho85 complexes phosphorylated MBP-Epa1, MBP-Hms1, and MBP-Ncp1 but did not phosphorylate MBP alone (Fig 5). GST alone (lacking the Pcl1 moiety) failed to phosphorylate any of the substrates (Fig 5). Therefore, Pcl1·Pho85 complexes specifically phosphorylate MBP-Epa1, MBP-Hms1, and MBP-Ncp1.

View larger version (44K): In this window In a new window Download PPT slide   Figure 5. Pcl1·Pho85 complexes specifically phosphorylate Epa1, Hms1, and Ncp1. The ability of Pcl1·Pho85 complexes to phosphorylate Epa1, Hms1, and Ncp1 is shown. Kinase assays were performed using either GST alone or GST-Pcl1 purified from yeast lysates and bacterially expressed and purified MBP-Epa1, MBP-Hms1, and MBP-Ncp1 as described in MATERIALS AND METHODS. Incorporation of 32P into specific substrates was detected as described in MATERIALS AND METHODS. Total levels of each substrate were detected using anti-MBP antibodies against a Western blot. Fusion proteins were expressed from the following plasmids: GST alone, pEG-GST; GST-Pcl1, pEG-GST-PCL1; MBP-Epa1, pSL2814; MBP-Hms1, pSL2815; and MBP-Ncp1, pSL2816.

Epa1 is an ATPase with homology to the bacterial minD septation regulator:
The observation that Epa1 interacts with Pcl1 and is phosphorylated by Pcl1·Pho85 complexes suggests that Epa1 may have a role during morphogenesis. Epa1 has no previously described biological role or molecular function. SWISS-PROT and GenBank (JUNKER et al. 1999 ; MOLLER et al. 1999 ; O'DONOVAN et al. 2002 ) search algorithms revealed that Epa1 is well conserved in all eukaryotic organisms examined, but no function has been ascribed to these homologs (Fig 6).

View larger version (62K): In this window In a new window Download PPT slide   Figure 6. Epa1 shares homology with the bacterial minD septation regulator and is conserved in eukaryotes. Sequence alignments of Epa1 with (A) the bacterial minD septation regulator, (B) the Caenorhabditis elegans GOP-2 protein of unknown function, and (C) the human (AJ010842) protein of unknown function are shown. National Center for Biotechnology Information blast and SWISS-PROT database alignment algorithms were utilized to prepare these alignments (JUNKER et al. 1999 ; MOLLER et al. 1999 ; O'DONOVAN et al. 2002 ).

Interestingly, sequence analysis using SWISS-PROT also revealed sequence similarity between Epa1 and minD (Fig 6), a bacterial protein that regulates the placement of the bacterial septal ring. The sequence homology between Epa1 and minD, especially within the ATP-binding and ATPase domains of minD (not specifically shown), suggests that Epa1 might be an ATPase. To test this, we performed NADH-based indirect ATPase assays. Epa1 showed significant ATPase activity above the maltose-binding protein control (Fig 7), whereas bacterially expressed and purified Ncp1 (prepared identically to Epa1) had no ATPase activity above the maltose-binding protein background. The ATPase activity observed for Epa1 is therefore unlikely to be due to contamination by bacterial proteins and we conclude that Epa1 is an ATPase.

View larger version (26K): In this window In a new window Download PPT slide   Figure 7. Epa1 has ATPase activity. Purified MBP-Epa1, MBP-Ncp1, and MBP alone were tested for ATPase activity in NADH-based indirect ATPase assays as described in MATERIALS AND METHODS. Fusion proteins were expressed from the following plasmids: MBP-Epa1, pSL2814; MBP-Ncp1, pSL2816.

Ncp1, Hms1, and Epa1 contribute to a shared essential role with Cla4:
To test whether the Pcl1-interacting proteins partake in the shared essential role with Cla4, we took two experimental approaches. First, we deleted HMS1 or NCP1 in a strain genomically deleted for CLA4, but containing a CLA4-URA3 plasmid. EPA1 could not be deleted in this background because it is essential. We then asked whether the double-mutant strains could survive in the absence of the CLA4-URA3 plasmid. We observed that both ncp1cla4 and hms1cla4 double-mutant strains were inviable as revealed by their inability to grow on medium containing 5-fluoroorotic acid (5-FOA; Fig 8). Both double-mutant strains had mislocalized septin rings (data not shown). This is the same phenotype observed in a ste20cla4 mutant (CVRCKOVA et al. 1995 ). Therefore, it appears that Ncp1 and Hms1 share an essential role with Cla4.

View larger version (89K): In this window In a new window Download PPT slide   Figure 8. NCP1, HMS1, and EPA1 show genetic interactions with CLA4. Genetic interactions between CLA4 and HMS1, NCP1, and EPA1 are depicted. (A) The HMS1 and NCP1 genes were deleted in a cla4 strain containing a CLA4-URA3 plasmid. These strains were replica plated to either rich medium or medium containing 0.1% 5-FOA. The inability of strains to grow on 5-FOA demonstrates the requirement of the CLA4 for viability and thus the lethality of the hms1cla4 and ncp1cla4 strains. (B) High-copy LEU2-marked EPA1 was transformed into cla4 strains deleted for a second gene, as indicated; these strains carried a copy of CLA4 on a URA3-marked plasmid. The ability to grow on medium containing 0.1% 5-FOA indicates the ability to lose the CLA4-URA3 plasmid and therefore suppress the synthetic lethal phenotype. Strains and plasmids were as follows: high-copy LEU2-marked EPA1, pSL2805; CLA4-URA3 plasmid, pSL2674; cla4 [CLA4-URA3], SY3363; cla4pcl1pcl2 [CLA4-URA3], SY4091; cla4ncp1 [CLA4-URA3], SY4089; cla4hms1 [CLA4-URA3], SY4088; cla4elp2 [CLA4-URA3], SY4086; cla4pho85 [CLA4-URA3], SY4087; and cla4ste20 [CLA4-URA3], SY3748.

As one means to determine whether EPA1 shares a morphogenetic role with CLA4, a high-copy construct containing EPA1 was tested for the ability to suppress several CLA4 synthetic lethal phenotypes. In fact, EPA1 is able to bypass the lethality of an elp2cla4 strain, which supports the idea that EPA1 shares some function(s) with CLA4 (Fig 8).

Pho85 and two Pcl1-interacting targets may have roles during mitotic exit:
Cla4 has functions during both G₁ progression and mitotic exit (CVRCKOVA et al. 1995 ; BENTON et al. 1997 ; HOLLY and BLUMER 1999 ; GULLI et al. 2000 ). Since Pho85 and its Pcl1-interacting targets, Epa1, Hms1, and Ncp1, all genetically interact with Cla4, we considered that they might function during either of these processes. Physical interaction data has suggested a role for Epa1 and Hms1 during mitotic exit. Specifically, Ho et al. identified a physical interaction between Epa1 and Dbf2, a kinase that is a crucial regulator in the mitotic exit network (TOYN and JOHNSTON 1994 ; HO et al. 2002 ). The same screen also detected an interaction between Hms1 and the Cdc14 phosphatase, another crucial member of the mitotic exit network (HO et al. 2002 ).

To investigate the potential contribution of PHO85, HMS1, and NCP1 to mitotic exit, we deleted these genes in a strain containing green fluorescent protein (GFP)-tagged CDC14. Previous studies have reported a marked decrease in Cdc14 nucleolar localization just prior to mitotic exit, which suggests that Cdc14 is the key effector of the mitotic exit transition in yeast (JASPERSEN et al. 1998 ; VISINTIN et al. 1998 , VISINTIN et al. 1999 ; JASPERSEN and MORGAN 2000 ; LEE et al. 2001 ). Additionally, a diminishment in Cdc14 nucleolar localization indicates progression through mitotic exit (HOFKEN and SCHIEBEL 2002 ). We found that Pho85, Hms1, and Ncp1 are individually required for the proper relocalization of Cdc14. Specifically, Cdc14 remains localized to the nucleolus nearly 100% of the time in cells lacking pho85, hms1, or ncp1 (Fig 9). This is in sharp contrast to otherwise wild-type cells and strongly suggests a role for these three proteins during mitotic exit. Because EPA1 is required for viability, we could not perform the same analysis in an epa1 mutant. However, sequence analysis presented here has identified a similarity between Epa1 and a bacterial septation regulator, raising the interesting possibility that this protein may be required for septation in yeast (Fig 6).

View larger version (12K): In this window In a new window Download PPT slide   Figure 9. Pho85, Hms1, and Ncp1 affect Cdc14 localization. Cells containing CDC14-GFP and deleted for PHO85, HMS1, or NCP1 were synchronized with -factor, released from G1 arrest, and grown at 10°. The fraction of cells exhibiting nucleolar Cdc14-GFP was observed (n > 100). Strains were as follows: wild type, ESM1362; pho85, SY4111; hms1, SY4112; and ncp1, SY4113.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The ability of p21-activated kinases to contribute to morphogenesis has been well documented (DANIELS and BOKOCH 1999 ; CONNOLLY et al. 2002 ; PUTO et al. 2003 ; SCHNEEBERGER and RAABE 2003 ). In budding yeast, Cla4 and Ste20 may have a common role during cellular morphogenesis. This interpretation is derived from the observation that the single mutants are viable, but the double mutant is inviable and exhibits defects in G₁ function, in septin function, in cytokinesis, and in bud morphology (CVRCKOVA et al. 1995 ; BENTON et al. 1997 ; HOLLY and BLUMER 1999 ; GULLI et al. 2000 ). In addition, Cla4 and Ste20 have both been shown to individually contribute to mitotic exit (HOFKEN and SCHIEBEL 2002 ; CHIROLI et al. 2003 ).

To gain insight into Cla4's roles, we set out to identify mutations that confer lethality in the absence of CLA4. We found that PHO85 is such a gene. Loss of two genes encoding Pho85 cyclin partners, PCL1 and PCL2, was also lethal with the simultaneous loss of CLA4. Because Pho85 is a cyclin-dependent kinase, we sought to identify potential targets with the view that such targets would speak to Cla4 function. This effort identified three proteins: Ncp1, Hms1, and Epa1. We have found that all three of these molecules physically interact with Pcl1 and are specifically phosphorylated by Pcl1·Pho85 complexes. Furthermore, we show that Ncp1, Hms1, and Epa1 genetically interact with Cla4. Ncp1 and Hms1 are required for viability in the absence of Cla4 whereas high-copy EPA1 bypasses the lethality of an elp2cla4 strain. The roles of Ncp1, Hms1, and Epa1 during morphogenesis may hint at the precise morphogenetic function that Cla4 and Pcl1·Pho85 impinge upon.

Several lines of evidence suggest that Cla4 and Pho85 signaling may intersect during mitotic exit. HOFKEN and SCHIEBEL 2002 have demonstrated that Cla4 is required for the proper localization of the Cdc14 phosphatase, a key effector of mitotic exit. Here we show that Pho85, Hms1, and Ncp1 are individually required for the proper localization of Cdc14, implicating these proteins in mitotic exit. Consistent with this, Hms1 physically associates with Cdc14 (HO et al. 2002 ). Interestingly, the third Pcl1·Pho85 target identified by our work, Epa1, has previously been shown to physically associate with another critical component of the septation initiation network in budding yeast, the Dbf2 kinase (HO et al. 2002 ). In light of these data, the observed homology between Epa1 and the bacterial septation regulator, MinD, is particularly intriguing. One possibility is that Epa1 is involved in septation in yeast. In addition to the homology with MinD, SWISS-PROT and GenBank search algorithms revealed that Epa1 is well conserved in all eukaryotic organisms examined (Fig 6). Further investigation is needed to determine whether these eukaryotic proteins, including Epa1, play a role in septation or cytokinesis.

The ability of components expressed during the G₁ phase of the cell cycle to regulate late mitotic events was initially suggested by CVRCKOVA et al. 1995 . They found that the loss of Cla4 is lethal with the loss of two G₁ cyclins, Cln1 and Cln2, and that Cla4 had a mitotic function. CVRCKOVA et al. 1995 suggested that the Cln1 and Cln2 G₁ function may affect a later mitotic function that acts in parallel with Cla4. Here we present a possible direct link between G₁ components and the late mitotic machinery. The G₁ Pcl1·Pho85 complex phosphorylates Epa1, Hms1, and Ncp1, each of which genetically interacts with Cla4. Moreover, each of these proteins has been suggested by some combination of function, homology, or protein interaction to play a role in mitotic exit. We therefore propose that Pcl1·Pho85 may regulate mitotic exit machinery, and this role may be essential in the absence of Cla4. One explanation for the genetic interactions between Cla4 and the G₁ machinery could be that Cla4 instills the competence to respond to a G₁ signal that regulates late mitotic events.

	FOOTNOTES

¹ Present address: Institute for Cancer Genetics, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
² Present address: Howard Hughes Medical Institute, Division of Basic Science, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109.
³ These authors contributed equally to this work.
⁴ Present address: Wellcome Trust/Cancer Research, UK Institute for Developmental Biology, Tennis Court Rd., Cambridge CB2 1QR, United Kingdom.

	ACKNOWLEDGMENTS

We thank David Mitchell, April Goehring, Phil Kinsey, Scott Givan, Lucia Liverio, Greg Smith, Paul Cullen, Monique Dail, Karen Sprague, Tom Stevens, Bruce Bowerman, Judith Eisen, and Kathy Chicas-Cruz for providing advice, strains, and/or plasmids. This work was supported by research (GM-30027) and training (GM-07413) grants from the National Institutes of Health.

Manuscript received August 28, 2003; Accepted for publication November 19, 2003.

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