Vol. 11, No. 10
MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 4876-4884 0270-7306/91/104876-09$02.00/0 Copyright © 1991, American Society for Microbiology
Protein Phosphatase 2A in Saccharomyces cerevisiae: Effects Cell Growth and Bud Morphogenesis
on
HANS RONNE,* MONIKA CARLBERG, GUO-ZHEN HU, AND JAN OLOF NEHLIN
Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, Box 595, 5-751 24 Uppsala, Sweden Received 17 December 1990/Accepted 2 July 1991
We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.
related phosphatases in the budding yeast Saccharomyces
The eukaryotic cell has a large number of protein kinases that are involved in signal transduction, metabolic regulation, and cell cycle control (24). This profusion of kinases is matched by a rapidly expanding number of protein phosphatases. Most of these belong to a superfamily of serine/ threonine phosphatases that includes the type 1, type 2A, and type 2B enzymes (8, 9, 11). The type 1 and 2A phosphatases are more closely related to each other than to the type 2B enzyme (9). The type 2A-related phosphatases comprise mammalian protein phosphatase 2A (PP2A) and its homologs in other organisms, but also more distantly related proteins, such as the yeast PPH1/SIT4 (2), rabbit PPX (11), and Drosophila PPV phosphatases (11). The type 1 and 2A phosphatases have broad substrate specificities and are thought to be involved in a number of cellular processes (8). In particular, they have been implicated in cell cycle control (12). Thus, a type 1 phosphatase is necessary for completion of mitosis in both fungi and Drosophila sp. (3, 5, 15, 39), and experiments in Xenopus sp. have shown that PP2A negatively regulates cdc2 kinase, a key effector in the cell cycle (17, 28). In fission yeast cells, PP2A is an essential enzyme encoded by two duplicated genes (25). Disruption of one of the genes reduces the cell size, which was interpreted as evidence of premature mitosis (25). PP2A has also been implicated in the control of cyclin degradation (30). Further evidence that PP2A plays an important role in growth control comes from experiments with simian virus 40 and polyomavirus. Thus, PP2A binds to the middle T and small t antigens, and it is thought that this interaction is necessary for their functions in transformation and viral replication (40, 52). PP2A also regulates the function of simian virus 40 large T antigen in DNA replication (27, 41, 51). Finally, it was recently shown that the PP2Arelated PPHIISIT4 gene performs an important function in the G1 phase of the cell cycle (47). We describe below the cloning and characterization of three genes encoding PP2A*
cerevisiae.
MATERIALS AND METHODS
Plasmids. The URA3 LEU2-d 2,um plasmid pHR81 and the genomic library made in this vector have been described elsewhere (37). For regulated expression of PP2A, we cloned a 1,320-bp BstBI fragment containing the PPH22 gene into the XbaI site of pJN92, which is pHR81 with a GALJ-TPK2 hybrid promoter in the BamHI site. This promoter has GAL] DNA from -820 to -75 fused to TPK2 DNA from -214 to -2, followed by the BamHI-HindIII part of the pUC18 polylinker. Numbers are relative to the start codon of each gene.
Yeast strains. The strains used are described in Table 1. Strains deficient for the PPH genes were made by one-step disruptions (42) in the W303 background (48). For PPH21 we used the BglII site at base 621, for PPH22 we used the EcoRV site at base 521, and for PPH3 we used the EcoRV site at base 299 (see Fig. 2). We also made disruptions in which the PPH21 and PPH22 open reading frames were deleted. Thus, the pph21-81 deletion extends from the EcoRI site at base 112 to the Sacl site at 896, and pph22-81 extends from the NsiI site at base 25 to the EcoNI site at 772. The GAL1 :PPH22 allele, used for regulated expression of PP2A, has a deletion of the PPH22 promoter from the XbaI site at -279 to the BstBI site at -10. The deleted DNA was replaced by the GALI-TPK2 hybrid promoter from pJN92, using the pop-in/pop-out method (4). Other methods. The methods used for yeast genetics and molecular cloning have been described previously (37, 38). Yeast cells were grown in YPD or YPGal (1% yeast extract, 2% peptone, 2% glucose or galactose). PPH3 was cloned from the pHR81 library (37) by low-stringency hybridization (32), using a PPH22 probe corresponding to positions - 162 to 649 (see Fig. 2C). The tree in Fig. 3 was computed from distance data, using the Fitch-Margoliash procedure (18) as previously described (32). Only positions for which all
Corresponding author. 4876
PROTEIN PHOSPHATASE 2A IN S. CEREVISIAE
VOL. 11, 1991 TABLE 1. Yeast strainsa Strain
H310 .
TABLE 2. Tetrad data
Relevant genotype a pph2l-81:: URA3 a pph22-81::HIS3 a pph2l-81::HIS3 GAL1:PPH22 a pph2-b81::HIS3 GALI :PPH22
H314 . H328 . pph3: :LEU2 H336 . H340 ..a pph21-81::LEU2 GALI:PPH22 a pph2l-81::HIS3 H341 . H346 ..a pph2l::HIS3 pph22::LEU2 a All strains also have the ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-I ura3-1 markers of W303-1A (48).
Ascus type
Interval
pph2l-pph22 pph2l-cdc9 pph22-cdc9 pph2l-trpl pph3-trpl pph3-rad55 a
sequences could be aligned were used, which correspond to residues 20 to 294 in human PP2A (1). 4',6'-Diamidino-2phenylindole (DAPI) staining and photography were carried out as previously described (32). Nucleotide sequence accession numbers. The EMBL accession numbers of the sequences are X58856 (PPH21), X58857 (PPH22), and X58858 (PPH3). RESULTS Cloning of three yeast genes encoding PP2A-related phosphatases. In a search for high-copy-number suppressors of the GAL] promoter (37, 38), we cloned a plasmid containing the 5' half of the TFPI gene (43). The suppressor activity was mapped to a GAL4 site in TFPI, which inhibits GAL] expression by promoter competition (37). During the characterization of this plasmid, we found that a nearby gene encodes a protein highly similar to mammalian PP2A. Since results for Xenopus cells suggest that PP2A plays an important role in cell cycle control (17, 28), we decided to investigate its function in yeast cells. Initial studies showed that disruption of the PP2A gene had no effect. This is usually the case when proteins are encoded by duplicated genes. Indeed, Southern analysis revealed a second, closely related gene, which we cloned by colony hybridization. Disruption of the second PP2A gene was also without effect. In contrast, disruption of both genes caused a very severe growth defect, but some doubledisrupted cells were still alive. This prompted us to search for more genes. By low-stringency hybridization, we cloned a third gene which is only distantly related to the other two. Disruption of the third gene did not affect growth, but spores disrupted for all three genes failed to grow. We conclude that PP2A performs an important function in yeast cells. The first two of our cloned genes can both provide this function and sustain normal growth, but the third gene is only marginally capable of doing so. One Saccharomyces gene encoding a PP2A-related phosphatase has already been described. This is SIT4, which was renamed PPHI (for protein phosphatase) when its sequence became known (2). The first two of our cloned genes are highly similar to each other and encode true homologs of mammalian PP2A (see below). We will refer to them as PPH21 and PPH22. These two genes were also recently cloned by Sneddon et al. (44), and one of them, PPH22, was cloned by Sutton et al. (47). The third gene, which is only distantly related to the mammalian PP2A genes, we will call PPH3. Mapping of the PPH genes. All three PPH genes were located to chromosome 4 in Southern blots of yeast DNA separated on a contour-clamped homogeneous electric field gel (7). We proceeded to map the genes by tetrad analysis.
4877
Parental P Parentl
Nonparental
ditype 80
ditype 5
139 109 37 64 126
1 3 17 15 0
Tetratype Tertp 92 35 63 123 288 0
Map distance
(cM), 35.5 11.5 23.1 79.2 57.0 0
Calculated as described by Ma and Mortimer (33).
PPH21 and PPH22 are both on the left arm of chromosome 4. Thus, PPH22 is 23 centimorgans (cM) telomeric to cdc9, while PPH21 is 12 cM centromeric to cdc9, near RPBI and ARF2 (Table 2). In fact, the 3' end of the ARF2 gene (45) is present in our cloned DNA (Fig. 1). Interestingly, the closest neighbor of PPH22 on the genetic map is ARFJ (35). The fact that PPH21 and PPH22 map near the duplicated ARF genes suggests that a duplication of several genes may have occurred, like that which created the CYCI and CYC7 regions (34). However, the restriction maps of PPH22 and ARFI do not extend far enough to establish a possible physical linkage of these two genes. The PPH3 gene was mapped to the right arm of chromosome 4, 57 cM from trpl. This is close to the rad5S locus, and further crosses revealed that PPH3 is tightly linked to radS5 (Table 2). A comparison of the restriction maps (31) shows that the 3' end of PPH3 is adjacent to the 5' end of RAD55 (Fig. 1). Interestingly, the PP2A-related PPHIISIT4 gene is also located on chromosome 4 (2). Sequences of the PPH genes. The sequences of the three genes are shown in Fig. 2. PPH21 and PPH22 are highly similar within the coding regions. In particular, the 5' parts of the genes are almost identical in sequence, whereas the 3' parts are more divergent. There are also a few limited similarities between the upstream regions, but none of these correspond to known regulatory sites (50). Between PPH22 and TFPI there is a small open reading frame (Fig. 1), which would encode a 32-kDa protein with no significant similarity to known sequences. Disruption of the open reading frame produced no obvious phenotype, and Northern (RNA) analysis failed to reveal a transcript. PPH3 differs considerably in sequence from PPH21 and PPH22. The upstream region of PPH3 has one possible CXC IB O S E
X
Ikb IIh11UL1IKL~ 1Ik PPH21
ARF2
CECCBE S V KXHOB VCE H
EH
ll11111 11111 lI I 11 1 1 PPH22 K
H
TFP1
EL H S VCHLE
IINII 11111111
MOLECULAR AND CELLULAR BIOLOGY, OCt. 1991, p. 4876-4884 0270-7306/91/104876-09$02.00/0 Copyright © 1991, American Society for Microbiology
Protein Phosphatase 2A in Saccharomyces cerevisiae: Effects Cell Growth and Bud Morphogenesis
on
HANS RONNE,* MONIKA CARLBERG, GUO-ZHEN HU, AND JAN OLOF NEHLIN
Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, Box 595, 5-751 24 Uppsala, Sweden Received 17 December 1990/Accepted 2 July 1991
We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.
related phosphatases in the budding yeast Saccharomyces
The eukaryotic cell has a large number of protein kinases that are involved in signal transduction, metabolic regulation, and cell cycle control (24). This profusion of kinases is matched by a rapidly expanding number of protein phosphatases. Most of these belong to a superfamily of serine/ threonine phosphatases that includes the type 1, type 2A, and type 2B enzymes (8, 9, 11). The type 1 and 2A phosphatases are more closely related to each other than to the type 2B enzyme (9). The type 2A-related phosphatases comprise mammalian protein phosphatase 2A (PP2A) and its homologs in other organisms, but also more distantly related proteins, such as the yeast PPH1/SIT4 (2), rabbit PPX (11), and Drosophila PPV phosphatases (11). The type 1 and 2A phosphatases have broad substrate specificities and are thought to be involved in a number of cellular processes (8). In particular, they have been implicated in cell cycle control (12). Thus, a type 1 phosphatase is necessary for completion of mitosis in both fungi and Drosophila sp. (3, 5, 15, 39), and experiments in Xenopus sp. have shown that PP2A negatively regulates cdc2 kinase, a key effector in the cell cycle (17, 28). In fission yeast cells, PP2A is an essential enzyme encoded by two duplicated genes (25). Disruption of one of the genes reduces the cell size, which was interpreted as evidence of premature mitosis (25). PP2A has also been implicated in the control of cyclin degradation (30). Further evidence that PP2A plays an important role in growth control comes from experiments with simian virus 40 and polyomavirus. Thus, PP2A binds to the middle T and small t antigens, and it is thought that this interaction is necessary for their functions in transformation and viral replication (40, 52). PP2A also regulates the function of simian virus 40 large T antigen in DNA replication (27, 41, 51). Finally, it was recently shown that the PP2Arelated PPHIISIT4 gene performs an important function in the G1 phase of the cell cycle (47). We describe below the cloning and characterization of three genes encoding PP2A*
cerevisiae.
MATERIALS AND METHODS
Plasmids. The URA3 LEU2-d 2,um plasmid pHR81 and the genomic library made in this vector have been described elsewhere (37). For regulated expression of PP2A, we cloned a 1,320-bp BstBI fragment containing the PPH22 gene into the XbaI site of pJN92, which is pHR81 with a GALJ-TPK2 hybrid promoter in the BamHI site. This promoter has GAL] DNA from -820 to -75 fused to TPK2 DNA from -214 to -2, followed by the BamHI-HindIII part of the pUC18 polylinker. Numbers are relative to the start codon of each gene.
Yeast strains. The strains used are described in Table 1. Strains deficient for the PPH genes were made by one-step disruptions (42) in the W303 background (48). For PPH21 we used the BglII site at base 621, for PPH22 we used the EcoRV site at base 521, and for PPH3 we used the EcoRV site at base 299 (see Fig. 2). We also made disruptions in which the PPH21 and PPH22 open reading frames were deleted. Thus, the pph21-81 deletion extends from the EcoRI site at base 112 to the Sacl site at 896, and pph22-81 extends from the NsiI site at base 25 to the EcoNI site at 772. The GAL1 :PPH22 allele, used for regulated expression of PP2A, has a deletion of the PPH22 promoter from the XbaI site at -279 to the BstBI site at -10. The deleted DNA was replaced by the GALI-TPK2 hybrid promoter from pJN92, using the pop-in/pop-out method (4). Other methods. The methods used for yeast genetics and molecular cloning have been described previously (37, 38). Yeast cells were grown in YPD or YPGal (1% yeast extract, 2% peptone, 2% glucose or galactose). PPH3 was cloned from the pHR81 library (37) by low-stringency hybridization (32), using a PPH22 probe corresponding to positions - 162 to 649 (see Fig. 2C). The tree in Fig. 3 was computed from distance data, using the Fitch-Margoliash procedure (18) as previously described (32). Only positions for which all
Corresponding author. 4876
PROTEIN PHOSPHATASE 2A IN S. CEREVISIAE
VOL. 11, 1991 TABLE 1. Yeast strainsa Strain
H310 .
TABLE 2. Tetrad data
Relevant genotype a pph2l-81:: URA3 a pph22-81::HIS3 a pph2l-81::HIS3 GAL1:PPH22 a pph2-b81::HIS3 GALI :PPH22
H314 . H328 . pph3: :LEU2 H336 . H340 ..a pph21-81::LEU2 GALI:PPH22 a pph2l-81::HIS3 H341 . H346 ..a pph2l::HIS3 pph22::LEU2 a All strains also have the ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-I ura3-1 markers of W303-1A (48).
Ascus type
Interval
pph2l-pph22 pph2l-cdc9 pph22-cdc9 pph2l-trpl pph3-trpl pph3-rad55 a
sequences could be aligned were used, which correspond to residues 20 to 294 in human PP2A (1). 4',6'-Diamidino-2phenylindole (DAPI) staining and photography were carried out as previously described (32). Nucleotide sequence accession numbers. The EMBL accession numbers of the sequences are X58856 (PPH21), X58857 (PPH22), and X58858 (PPH3). RESULTS Cloning of three yeast genes encoding PP2A-related phosphatases. In a search for high-copy-number suppressors of the GAL] promoter (37, 38), we cloned a plasmid containing the 5' half of the TFPI gene (43). The suppressor activity was mapped to a GAL4 site in TFPI, which inhibits GAL] expression by promoter competition (37). During the characterization of this plasmid, we found that a nearby gene encodes a protein highly similar to mammalian PP2A. Since results for Xenopus cells suggest that PP2A plays an important role in cell cycle control (17, 28), we decided to investigate its function in yeast cells. Initial studies showed that disruption of the PP2A gene had no effect. This is usually the case when proteins are encoded by duplicated genes. Indeed, Southern analysis revealed a second, closely related gene, which we cloned by colony hybridization. Disruption of the second PP2A gene was also without effect. In contrast, disruption of both genes caused a very severe growth defect, but some doubledisrupted cells were still alive. This prompted us to search for more genes. By low-stringency hybridization, we cloned a third gene which is only distantly related to the other two. Disruption of the third gene did not affect growth, but spores disrupted for all three genes failed to grow. We conclude that PP2A performs an important function in yeast cells. The first two of our cloned genes can both provide this function and sustain normal growth, but the third gene is only marginally capable of doing so. One Saccharomyces gene encoding a PP2A-related phosphatase has already been described. This is SIT4, which was renamed PPHI (for protein phosphatase) when its sequence became known (2). The first two of our cloned genes are highly similar to each other and encode true homologs of mammalian PP2A (see below). We will refer to them as PPH21 and PPH22. These two genes were also recently cloned by Sneddon et al. (44), and one of them, PPH22, was cloned by Sutton et al. (47). The third gene, which is only distantly related to the mammalian PP2A genes, we will call PPH3. Mapping of the PPH genes. All three PPH genes were located to chromosome 4 in Southern blots of yeast DNA separated on a contour-clamped homogeneous electric field gel (7). We proceeded to map the genes by tetrad analysis.
4877
Parental P Parentl
Nonparental
ditype 80
ditype 5
139 109 37 64 126
1 3 17 15 0
Tetratype Tertp 92 35 63 123 288 0
Map distance
(cM), 35.5 11.5 23.1 79.2 57.0 0
Calculated as described by Ma and Mortimer (33).
PPH21 and PPH22 are both on the left arm of chromosome 4. Thus, PPH22 is 23 centimorgans (cM) telomeric to cdc9, while PPH21 is 12 cM centromeric to cdc9, near RPBI and ARF2 (Table 2). In fact, the 3' end of the ARF2 gene (45) is present in our cloned DNA (Fig. 1). Interestingly, the closest neighbor of PPH22 on the genetic map is ARFJ (35). The fact that PPH21 and PPH22 map near the duplicated ARF genes suggests that a duplication of several genes may have occurred, like that which created the CYCI and CYC7 regions (34). However, the restriction maps of PPH22 and ARFI do not extend far enough to establish a possible physical linkage of these two genes. The PPH3 gene was mapped to the right arm of chromosome 4, 57 cM from trpl. This is close to the rad5S locus, and further crosses revealed that PPH3 is tightly linked to radS5 (Table 2). A comparison of the restriction maps (31) shows that the 3' end of PPH3 is adjacent to the 5' end of RAD55 (Fig. 1). Interestingly, the PP2A-related PPHIISIT4 gene is also located on chromosome 4 (2). Sequences of the PPH genes. The sequences of the three genes are shown in Fig. 2. PPH21 and PPH22 are highly similar within the coding regions. In particular, the 5' parts of the genes are almost identical in sequence, whereas the 3' parts are more divergent. There are also a few limited similarities between the upstream regions, but none of these correspond to known regulatory sites (50). Between PPH22 and TFPI there is a small open reading frame (Fig. 1), which would encode a 32-kDa protein with no significant similarity to known sequences. Disruption of the open reading frame produced no obvious phenotype, and Northern (RNA) analysis failed to reveal a transcript. PPH3 differs considerably in sequence from PPH21 and PPH22. The upstream region of PPH3 has one possible CXC IB O S E
X
Ikb IIh11UL1IKL~ 1Ik PPH21
ARF2
CECCBE S V KXHOB VCE H
EH
ll11111 11111 lI I 11 1 1 PPH22 K
H
TFP1
EL H S VCHLE
IINII 11111111
