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Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo

2000; Springer Nature; Volume: 19; Issue: 21 Linguagem: Inglês

10.1093/emboj/19.21.5672

1460-2075

Jeannie Wu, Tatiana Tolstykh, Jookyung Lee, Kimberly Boyd, Jeffry B. Stock, James R. Broach,

Microtubule and mitosis dynamics

Article1 November 2000free access Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo Jeannie Wu Jeannie Wu Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Tatiana Tolstykh Tatiana Tolstykh Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Jookyung Lee Jookyung Lee Present address: Department of Microbiology and Immunology, State University of New York, Health Sciences Center at Brooklyn, Brooklyn, NY, 11203 USA Search for more papers by this author Kimberly Boyd Kimberly Boyd Present address: Department of Biology, Cabrini College, Radnor, PA, 19087 USA Search for more papers by this author Jeffry B. Stock Jeffry B. Stock Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author James R. Broach Corresponding Author James R. Broach Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Jeannie Wu Jeannie Wu Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Tatiana Tolstykh Tatiana Tolstykh Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Jookyung Lee Jookyung Lee Present address: Department of Microbiology and Immunology, State University of New York, Health Sciences Center at Brooklyn, Brooklyn, NY, 11203 USA Search for more papers by this author Kimberly Boyd Kimberly Boyd Present address: Department of Biology, Cabrini College, Radnor, PA, 19087 USA Search for more papers by this author Jeffry B. Stock Jeffry B. Stock Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author James R. Broach Corresponding Author James R. Broach Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Author Information Jeannie Wu1, Tatiana Tolstykh1, Jookyung Lee2, Kimberly Boyd3, Jeffry B. Stock1 and James R. Broach 1 1Department of Molecular Biology, Princeton University, Princeton, NJ, 08544 USA 2Present address: Department of Microbiology and Immunology, State University of New York, Health Sciences Center at Brooklyn, Brooklyn, NY, 11203 USA 3Present address: Department of Biology, Cabrini College, Radnor, PA, 19087 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5672-5681https://doi.org/10.1093/emboj/19.21.5672 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B′ regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme. Introduction Reversible protein phosphorylation plays a major role in regulating most cellular processes: as many as 25% of all cellular proteins are subject to reversible phosphorylation (Chelsky et al., 1985). Much of the specificity of protein phosphorylation resides in the kinases responsible for transferring the phosphate group from ATP to the target proteins, as witnessed by the rich diversity of cellular kinases (Hunter, 1994; Plowman et al., 1999). However, specificity is also imparted by phosphoprotein phosphatases, whose complexity, achieved in part through combinatorial subunit interactions, may rival that of protein kinases (Virshup, 2000). Phosphoprotein phosphatases play critical roles in the timing, extent and localization of protein phosphorylation and in regulating those cellular processes modulated by phosphorylation. Phosphoprotein phosphatase 2A (PP2A) is one of the four major classes of eukaryotic serine/threonine phosphoprotein phosphatases (Cohen, 1989; Wera and Hemmings, 1995). It affects such diverse cellular processes as metabolism, signal transduction, cell cycle progression, apoptosis, transcription, DNA replication and protein synthesis (Virshup, 2000). It exists predominantly as a heterotrimeric protein, consisting of a catalytic subunit (C), a regulatory subunit (B) and a scaffold protein (A) on which the catalytic and regulatory subunit sit (Cohen, 1989; Wera and Hemmings, 1995; Groves et al., 1999). Mammalian cells contain multiple isoforms of the C and A subunits. In addition, mammalian cells contain a number of regulatory subunits, which fall into three distinct and non-homologous families, B, B′ and B″, each of which is represented by several isoforms (Kamibayashi et al., 1994; McCright and Virshup, 1995; Csortos et al., 1996; Zolnierowicz et al., 1996). Each regulatory subunit is capable of binding an AC dimer and probably imparts a particular substrate specificity to the C subunit (Hubbard and Cohen, 1993; Molloy et al., 1998). While the heterotrimeric forms of PP2A probably account for the main biological activities of the phosphatase, cell extracts contain catalytically active AC dimers and free C subunits and these may contribute to the biological function of the phosphatase (Cohen, 1989). C subunits also form heterodimers with a subunit, called α4 in mammalian cells and Tap42 in budding yeast (Saccharomyces cerevisiae), whose association with C may inhibit its catalytic activity (Di Como and Arndt, 1996; Murata et al., 1997; Chen et al., 1998; Jiang and Broach, 1999). In yeast, association of Tap42 with the C subunit is essential for viability (Di Como and Arndt, 1996; Beck and Hall, 1999). Thus, PP2A consists of a large collection of distinct entities with different ternary structures, subunit compositions and substrate recognition properties. Genetic analysis of PP2A in S.cerevisiae has helped clarify the diverse roles PP2A that plays in the cell. Two highly homologous genes, PPH21 and PPH22, redundantly encode the C subunit of PP2A (Sneddon et al., 1990). Inactivation of both genes severely retards growth and analysis of temperature-sensitive alleles has identified a requirement for PP2A in actin cytoskeletal organization and progression through mitosis (Ronne et al., 1991; Lin and Arndt, 1995; Evans and Stark, 1997). Inactivation of PPH3, a gene with homology to PPH21/22, in a pph21 pph22 background is lethal, suggesting that Pph3 is a partially redundant isoform of C (Ronne et al., 1991). CDC55 and RTS1 encode proteins homologous to the mammalian B and B′ regulatory subunits, respectively (Healy et al., 1991; Shu et al., 1997). The yeast genome does not encode a protein homologous to the B″ subunit. Inactivation of CDC55 renders growth cold sensitive and causes defects in cytokinesis and in the spindle checkpoint, while inactivation of RTS1 causes temperature-sensitive growth, reduced ability to use non-fermentable carbon sources and cell cycle arrest in G2. Thus, the two regulatory subunits appear to affect different cellular processes, presumably as a result of targeting the phosphatase to distinct substrates. Inactivation of the A subunit, encoded by TPD3, results in a phenotype that approximates the sum of the phenotypes induced by inactivation of the individual regulatory subunits (van Zyl et al., 1992). Tap42 competes in vivo with A and B for association with the C subunit (Jiang and Broach, 1999). The association of Tap42 with C is required for mitotic growth and this association is disrupted by nutrient starvation or treatment of cells with the macrolide drug rapamycin (Di Como and Arndt, 1996; Schmidt et al., 1998). Thus, yeast PP2A participates in a number of different cellular processes, dictated in part by the regulatory subunits with which it associates. The C subunit of mammalian PP2A is subject to at least three post-translational modifications: methyl esterification of the C-terminal leucine, phosphorylation of a conserved tyrosine located two residues from the C-terminus and phosphorylation of an as yet unidentified threonine (Chen et al., 1992, 1994; Guo and Damuni, 1993; Lee and Stock, 1993; Xie and Clarke, 1994). Phosphorylation of either the tyrosine or the threonine site inhibits phosphatase activity in vitro. On the other hand, carboxyl methylation of the C subunit does not affect phosphatase activity in vitro (Tolstykh et al., 2000). However, purified PP2A carboxyl methyltransferase methylates the C subunit only in the context of an AC dimer and the resulting dimer containing the methylated C subunit has a higher affinity for a B regulatory subunit than does a dimer with an unmethylated C subunit (Tolstykh et al., 2000). Consistent with this observation, a tagged version of a mammalian PP2A C subunit containing a mutation of the conserved C-terminal leucine showed reduced participation in ABC heterotrimers in vivo but normal participation as an AC heterodimer (Chung et al., 1999). These observations suggest that carboxyl methylation could affect the association of the C subunit with regulatory subunits in vivo and, as a result, alter targeting of the phosphatase towards certain substrates relevant for its normal biological activity. To examine this hypothesis, we have identified the gene encoding the budding yeast PP2A carboxyl methyltransferase and characterized strains lacking its activity. We have found that the PP2A heterotrimer is destabilized in strains lacking methyltransferase activity and that the phenotypes of such strains are quite similar to those of strains lacking the PP2A regulatory subunits. These observations confirm the biological role of PP2A carboxyl methylation and raise the possibility that this reversible modification participates in regulation of cellular growth. Results Isolation of cDNA for the human PP2A carboxyl methyltransferase By conventional chromatographic techniques we previously purified PP2A methyltransferase (PPMT) to homogeneity from extracts of bovine brain (Lee and Stock, 1993). We digested the resulting 47 kDa protein with chymotrypsin or endoproteinase Lys-C and obtained sequence data from the N-terminus of several of the resulting fragments. A search of the DDBJ/EMBL/GenBank expressed sequence tag (EST) database revealed nine human and one mouse entry that encoded sequences highly homologous to one or more of the peptides. From the overlap of the human cDNA entries, we assembled a 300 bp fragment with a 98 amino acid contiguous peptide sequence of the putative human methyltransferase. Using this assembled sequence, we recovered and sequenced four human cDNA clones, one of which appeared to contain the entire PPMT open reading frame. The sequence of the gene was identical to that recently reported by De Baere et al. (1999) and the predicted protein sequence of human PPMT derived from the DNA sequence contains the peptide fragments we identified. To confirm that the recovered cDNA clone encoded human PPMT, we expressed the full-length cDNA in Escherichia coli under the control of an inducible LacZ promoter and assayed PPMT activity in extracts of cells expressing this cDNA. Extracts from induced cells but not from uninduced cells were capable of transferring [3H]methyl from S-adenosyl methionine to bovine PP2A AC dimer (data not shown). This confirmed that the human cDNA clone we isolated on the basis of homology to peptides derived from bovine PPMT encodes human PPMT. Identification of yeast genes encoding PP2A carboxyl methyltransferase and methylesterase BLAST analysis identified two genes in S.cerevisiae (YDR435c and YOL141w), two in Schizosaccharomyces pombe (emb|CAA21793| and emb|CAA19576|), one in Caenorhabditis elegans (emb|CAA84295| and WormPD B0285.4) and one in Drosophila melanogaster (gb|AAF53483|) and an additional human cDNA (dbj|BAA25473|) that encode proteins with extensive homology to human PPMT. The larger of the two genes from S.cerevisiae (YOL141w) and from S.pombe (CAA19576), as well as the second human gene, encode proteins that show homology to the entire human PPMT protein in their N-termini but extend for an additional 300 amino acids C-terminal to the region of homology. The C-terminal extensions of the three proteins show homology to each other (data not shown). The sequence relationships among these different genes are represented in Figure 1. On the basis of the homology to human PPMT and the data presented below, we have designated YDR435c and YOL141w from S.cerevisiae as the phospho protein phosphatase 2A methyltransferase genes, PPM1 and PPM2, respectively. Figure 1.Sequence relationship of putative human, S.cerevisiae, S.pombe, D.melanogaster and C.elegans PP2A carboxyl methyltransferases. Predicted amino acid sequences of human methyltransferases (hsmt1, AAF18267; hsmt2, BAA25473) and those of related proteins predicted from the genomic sequences of S.cerevisiae (PPM1, YDR435c; PPM2, YOL141w), S.pombe (spmt1, CAA21793; spmt2, CAA19576), D.melanogaster (dmmt, AAF53483) and C.elegans (cemt, CAA84295) are aligned using the PILEUP program from the University of Wisconsin GCG. Download figure Download PowerPoint Lee et al. (1996) identified and purified to homogeneity a bovine methylesterase (PPME) specific for methylated PP2A C subunit. Ogris et al. (1999) subsequently found that this protein co-precipitated with a mutant form of the PP2A C subunit and proceeded to clone the gene encoding the methylesterase. By BLAST analysis, we identified a single open coding region in S.cerevisiae (YHR075C) with significant homology to the bovine enzyme. Our subsequent analysis, described below, confirmed that this gene promotes PPME activity. Accordingly, we have designated this gene PPE1. To determine whether either of the two identified S.cerevisiae genes homologous to bovine PPMT encodes a protein with PPMT activity, we created null alleles of PPM1 and PPM2 by replacing the entire open reading frame of each gene with a selectable marker. We then generated isogenic strains in a W303 background that contained a deletion of one or the other of these genes. We tested extracts of these strains for PPMT activities by incubating them with purified bovine AC dimer and S-[3H]adenosyl methionine, and assessing the extent of radioactivity incorporated into the C subunit as described in Materials and methods. As shown in Figure 2A, wild-type yeast extract contains a significant level of PPMT activity, as do strains deleted for PPM2. However, extracts of ppm1 strains have no detectable PPMT activity. From these data we conclude that PPM1 encodes the entirety of yeast PPMT. Figure 2.PPM1 and PPE1 encode a PP2A carboxyl methyltransferase and a PP2A carboxyl methylesterase, respectively. (A) Extracts from W303-1A (PPM1 PPM2), Y2752 (ppm1), Y2739 (ppm2) and Y2740 (ppm1 ppm2) were incubated at 30°C for 30 min with or without bovine AC dimers and [3H]AdoMet, and then subjected to SDS–PAGE. The C subunit was analyzed for incorporation of [3H]methyl esters as described in Materials and methods. The data shown are means ± SD of duplicates from two separate experiments. (B) Extracts from Y2762 (pph21 pph22, lanes 1–4), Y2760 (pph21 pph22 ppm1, lane 5) or Y2761 (pph21 pph22 ppe1, lane 6) expressing triple HA-tagged Pph22 containing the indicated mutations were separated by SDS–PAGE and then transferred to PDVF membranes. The membranes were probed with a monoclonal antibody against HA, monoclonal antibody 6A3 (which recognizes both methylated and unmethylated C subunit) and monoclonal antibody 4D9 (which recognizes only methylated C subunit). Extracts were run as separate sets of tracks for each antibody. Download figure Download PowerPoint To confirm the assignment of PPMT activity to PPM1 and PPME activity to PPME1, we examined the methylation state of the PP2A C subunit in wild-type and various mutant yeast cells. We have previously raised monoclonal antibodies against an amidated peptide corresponding to the C-terminal domain of the C subunit of PP2A (Tolstykh et al., 2000). One of these monoclonal antibodies, 6A3, recognizes the C subunit of both the methylated and unmethylated protein. However, a second monoclonal antibody, 4D9, binds only to the carboxyl methylated C subunit. These reagents can thus be used to assess the in vivo methylation state of the PP2A C subunit. Accordingly, we constructed isogenic wild-type, ppm1Δ and ppe1Δ strains expressing a hemagglutinin (HA)-tagged version of either Pph21 or Pph22 and performed western analysis on extracts of these strains. We also performed western analysis on extracts of wild-type cells expressing HA-tagged versions of the C subunit carrying various mutations affecting the C-terminal domain of the protein. The results of this analysis are shown in Figure 2B. Both the methylation-specific and the -nonspecific monoclonal antibodies recognized the HA-tagged PP2A C subunit present in extracts of wild-type cells. The methylation-nonspecific antibody bound to mutant protein carrying a leucine to alanine substitution of the C-terminal amino acid, but the methylation-specific antibody did not. This is consistent with the apparent requirement of a terminal leucine residue in order for the C subunit to be methylated by PPMT. Furthermore, as evident from the figure, protein present in a ppm1 strain showed no cross-reactivity with the methylation-specific antibody, while protein present in a ppe1 strain showed increased cross-reactivity with the antibody relative to that seen in the PPM1 PPE1 strain. As evident from lanes reacted with anti-HA antibodies, the PP2A C subunit was expressed at equal levels in all the strains. These results indicate that methylation of the C subunit of PP2A is reduced in ppm1 strains and enhanced in ppe1 strains, consistent with the hypothesis that PPM1 encodes the yeast PPMT and PPE1 the yeast PPME. Given the degree of increased cross-reactivity of the 4D9 antibody with PP2A C in ppe1 cells compared with wild-type cells, we conclude that at least 50% of the C subunit in exponentially growing cells is unmethylated. Methyltransferase activity is required for stable association of the PP2A C subunit with its regulatory subunits in vivo In vitro experiments using purified PP2A components have shown that C subunit methylation enhances the binding of the B subunit to the AC dimer (Tolstykh et al., 2000). To examine in vivo the effect on heterotrimer stability of C subunit carboxyl methylation, we assessed the stability of PP2A heterotrimers in a strain lacking methyltransferase activity. We immunoprecipitated HA-tagged C subunit from extracts of PPM1 and ppm1 strains using anti-HA antibody under non-denaturing conditions. We then fractionated the immunoprecipitates by gel electrophoresis, transferred the fractionated proteins to a PVDF membrane, and probed the membrane with antibodies specific for Tpd3, Cdc55 or HA. As seen in Figure 3A and as noted previously (Jiang and Broach, 1999), a significant fraction, but by no means all, of the A and B subunits in extracts of the PPM1 strain co-precipitated with Pph21 and Pph22. In contrast, but consistent with previous in vitro results (Tolstykh et al., 2000), significantly less Cdc55 co-precipitated with Pph21 or Pph22 from extracts of the ppm1Δ strain. Unexpectedly, though, we observed a comparable diminution in the amount of Tpd3 that co-precipitated with Pph21 or Pph22 from extracts of the ppm1Δ strain. We confirmed this observation by immunoprecipitating Tpd3 from extracts of the ppm1Δ strain and finding less co-precipitation of Pph21 or Pph22 than obtained from extracts of the wild-type strain. Thus, we conclude that the absence of PP2A carboxyl methyltransferase activity destabilized not only the PP2A ABC heterotrimer but also the AC heterodimer in vivo. Figure 3.Loss of Ppm1 activity affects the interaction of PP2A regulatory subunits with the C subunits. Extracts from Y2480 [pph22:: (HA)3PPH22] and Y2734 [ppm1 pph22:: (HA)3PPH22] cells were immunoprecipitated with anti-HA epitope monoclonal antibodies. Samples of the precipitates ('IP'), extracts before precipitation ('Ext') and extracts after precipitation ('Sup') were separated by SDS–PAGE and then transferred to PVDF membranes. The membranes were probed separately with anti-HA, anti-Tpd3, anti-Cdc55 (A) and anti-Tap42 (B) polyclonal antibodies. Download figure Download PowerPoint A portion of the PP2A C subunit is associated with Tap42 in exponentially growing yeast cells. This association is diminished in stationary phase cells and in cells treated with rapamycin, and is increased in cells deleted for Tpd3 (Di Como and Arndt, 1996; Jiang and Broach, 1999). Accordingly, we investigated whether loss of Ppm1 activity affected the extent of association of Tap42 with the PP2A C subunit. As shown in Figure 3B, a slightly larger amount of Tap42 co-precipitates with PP2A C from extracts of the ppm1Δ strain than from an isogenic wild-type strain. Quantification of signals on the western blot suggests that the increase is ∼50%. While this increase is small, we obtained essentially the same results with different extracts of several different strains, so we can conclude that the interaction of Tap42 with PP2A C does not depend on Ppm1 activity and may even be enhanced in its absence. Phenotypes of ppm1 strains are similar to those of cdc55 and rts1 strains Because of the reduced association of the PP2A regulatory subunits with the C subunits in ppm1 strains in vivo, we examined whether such strains were phenotypically similar to those lacking these regulatory subunits. cdc55 mutants were originally identified as isolates exhibiting cytokinesis and morphological defects at low temperature, and subsequently as mutants defective in cell cycle arrest in response to spindle damage (Healy et al., 1991; Minshull et al., 1996; Wang and Burke, 1997). These two defects may both derive from a role of Cdc55 in promoting dephosphorylation of Cdc28, since certain cdc20 and cdc28 alleles suppress both the checkpoint and morphological defects caused by cdc55 mutations (Minshull et al., 1996; Wang and Burke, 1997). More recently, we have shown that Cdc55 antagonizes the Tor signaling pathway, in that cdc55 mutants exhibit rapamycin resistance as well as an enhanced association in vivo between the Tap42 and the C subunit of PP2A (Jiang and Broach, 1999). RTS1 was initially identified by homology to a mammalian B′ subunit and subsequent characterization revealed deletion mutants to be temperature sensitive for growth, deficient for growth on non-fermentable carbon sources and defective in the G2–M transition in the cell cycle (Shu et al., 1997). Accordingly, we tested ppm1 strains for many of these phenotypes associated with inactivation of CDC55 or RTS1. As shown in Figure 4, ppm1Δ strains showed no growth defects on rich media. While cdc55 strains do not exhibit a growth defect at reduced temperatures, as measured by plating efficiency, they are morphologically distinctive, mainly because of a defect in cytokinesis. We saw no such morphological defect for ppm1Δ strains in the W303 background or in the S288C background, in which the cdc55 morphological phenotype is more pronounced (data not shown). As noted above, rts1 strains were reported to be deficient in growth on non-fermentable carbon sources. However, we observed no growth defect of any of the mutants on YEP media with glycerol, ethanol or glycerol plus ethanol as carbon source (data not shown). Figure 4.Genetic interactions between Ppm1 and PP2A B regulatory subunit genes. Exponentially growing cultures of W303-1A (wild type), Y2752 (ppm1), Y2739 (ppm2), Y2740 (ppm1 ppm2), Y2483 (cdc55), Y2745 (ppm1 cdc55), Y2741 (ppm2 cdc55), Y2742 (ppm1 ppm2 cdc55), Y2736 (rts1), Y2737 (ppm1 rts1), Y2738 (ppm2 rts1) and Y2744 (ppm1 ppm2 rts1) were 10-fold serially diluted and spotted on to YEPD plates. Growth is shown after 2 days at 30 or 37°C. Download figure Download PowerPoint The macrolide drug rapamycin inhibits yeast growth by inhibiting those essential activities of Tor kinase mediated at least in part through Tap42. cdc55 strains are resistant to rapamycin, since Cdc55 is required to reverse Tor-mediated phosphorylation of Tap42. As shown in Figure 5A, the ppm1 strain can grow in the presence of 100 nM rapamycin, a concentration to which the isogenic wild-type and ppm2 strains are sensitive but on which cdc55 strains can grow. Thus, ppm1 strains mirror this phenotype of cdc55 strains. Figure 5.ppm1 cells are resistant to and supersensitive to benomyl. Exponentially growing cultures of wild-type cells, Y2752 (ppm1), Y2739 (ppm2), Y2483 (cdc55), Y2736 (rts1), Y2745 (ppm1 cdc55) and Y2737 (ppm1 rts1) were 10-fold serially diluted and spotted on to YEPD plates containing 100 nM rapamycin (A) or 12 μg/ml benomyl (B). Growth is shown after incubation at 30°C for 2 days without drug or 4 days in the presence of rapamycin and benomyl. Download figure Download PowerPoint To test whether ppm1 strains exhibit a checkpoint defect similar to that caused by loss of CDC55, we examined the benomyl sensitivity of such strains. Strains defective in the spindle checkpoint pathway are more sensitive to low levels of benomyl, a microtubule-depolymerizing agent, probably due to an increased proportion of cells that transit mitosis in the absence of an intact spindle (Hoyt et al., 1991; Li and Murray, 1991). As shown in Figure 5B, both cdc55 and ppm1 strains show substantially lower plating efficiency on benomyl than does the wild-type strain. rts1 strains were also more sensitive to benomyl, which was surprising since Rts1 had not previously been implicated in spindle checkpoint function. However, in a systematic study of phenotypes of yeast strains carrying insertions in individual genes, K.H.Cheung, A.Kumar, X.Liu and P.Ross-Macdonald (http://ygac.med.yale.edu/) list rts1::Tn strains as benomyl supersensitive, confirming our results. To confirm the checkpoint deficiency of ppm1 strains, we examined the loss of viability of a ppm1 strain as a function of cell cycle progression in the presence of nocodazole, another microtubule-depolymerizing agent. Cells of the test strain were arrested at G1 by treatment with α-factor and then released from the G1 block in the presence of nocodazole. Samples were removed at various times after release and analyzed for position in the cell cycle and for viability. Cells with an intact spindle checkpoint arrest in mitosis and retain viability, whereas cells defective for the spindle checkpoint proceed through mitosis in the absence of a spindle and lose viability. Accordingly, loss of viability coincides with transit through mitosis. As evident from Figure 6, both the wild-type strain and the ppm2 strain arrest with a G2 content of DNA and retain viability during the course of the experiment. In contrast, the viability of cdc55, rts1 and ppm1 strains declines steeply, coincident with transit through mitosis. From these results we conclude that the ppm1 and rts1 mutants show a checkpoint defect similar to that of cdc55 mutants. Figure 6.ppm1, cdc55 and rts1 are required for spindle assembly checkpoint activity. Exponentially growing cultures of W303-1A (wild type), Y2752 (ppm1), Y2739 (ppm2), Y2483 (cdc55) and Y2736 (rts1) were incubated in the presence of α-factor for 3 h at 30°C and then transferred into fresh YEPD medium containing 12 μg/ml nocodazole. At different times after release from cell cycle arrest, cells were removed, sonicated, counted and then plated on to YEPD plates. Viable colonies were counted after 2 days. Squares, wild type; diamonds, cdc55; circles, rts1; upward pointing triangles, ppm1; downward pointing triangles, ppm2. Download figure Download PowerPoint PPE1 overexpression causes phenotypes similar to those of ppm1 strains If the in vivo level of methylation of PP2A results from a dynamic equilibrium between the activities of PPMT and PPME, we would expect that overexpression of PPE1 should induce phenotypes similar to those associated with loss of PPM1. As shown in Figure 7, this is the case. A strain containing PPE1 on a high-copy-number vector exhibits rapamycin resistance equivalent to that of a ppm1 strain. This strain is also supersensitive to benomyl (data not shown). We have not seen any obvious phenotypes associated with loss of Ppe1 despite the fact that such strains exhibit enhanced methylation of PP2A (Figure 2B). ppe1 strains grow normally at all temperatures on fermentable and non-fermentable carbon sources, and show wild-type sensitivities to nocodazole, benomyl and rapamycin (data not shown). Thus, while reduced methylation of PP2A, whether achieved by reduced PPMT activity or