GLC7 encodes the catalytic subunit of type 1 protein serine/threonine phosphatase (PP1) in the yeast Saccharomyces cerevisiae. Here we have characterized the temperature-sensitive glc7-10 allele, which displays aberrant bud morphology and an abnormal actin cytoskeleton at the restrictive temperature. At 37 degrees C glc7-10 strains accumulated a high proportion of budded cells with an unmigrated nucleus, duplicated spindle pole bodies, a short spindle, delocalized cortical actin and 2C DNA content, indicating a cell cycle block prior to the metaphase to anaphase transition. glc7-10 was suppressed by growth on high osmolarity medium and exhibited temperature-sensitive cell lysis upon hypo-osmotic stress. Pkc1p, the yeast protein kinase C homolog which is thought to regulate the Mpk1p MAP kinase pathway involved in cell wall remodelling and polarized cell growth, was found to act as a dosage suppressor of glc7-10. Although neither activation of BCK1 (MEKK) by the dominant BCK1-20 mutation nor increased dosage of MKK1 (MEK) or MPK1 (MAP kinase) mimicked PKC1 as a glc7-10 dosage suppressor, extra copies of genes encoding upstream components of the Pkc1p pathway such as ROM2, RHO2, HCS77/WSC1/SLG1 and MID2 also suppressed glc7-10 effectively. Conversely, mpk1delta glc7-10 and bck1delta glc7-10 double mutants displayed a synthetic cell lysis defect compared with each single mutant and glc7-10 was hypersensitive to reduced PKC1 function, displaying highly aberrant morphologies and inviability even at the normally permissive temperature of 26 degrees C. Dephosphorylation by PP1 therefore functions positively to promote cell integrity, bud morphology and polarization of the actin cytoskeleton and glc7-10 cells require higher levels of Pkc1p activity to sustain these functions.

REFERENCES

Alberts
A. S.
,
Bouquin
N.
,
Johnston
L. H.
,
Treisman
R.
(
1998
).
Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7.
J. Biol. Chem
273
,
8616
8622
Axton
J. M.
,
Dombradi
V.
,
Cohen
P.
,
Glover
D. M.
(
1990
).
One of the protein phosphatase 1 isozymes in Drosophila is essential for mitosis.
Cell
63
,
33
46
Baker
S. H.
,
Frederick
D. L.
,
Bloecher
A.
,
Tatchell
K.
(
1997
).
Alanine-scanning mutagenesis of protein phosphatase type I in the yeast Saccharomyces cerevisiae.
Genetics
145
,
615
626
Bi
E.
,
Maddox
P.
,
Lew
D. J.
,
Salmon
E. D.
,
McMillan
J. N.
,
Yeh
E.
,
Pringle
J. R.
(
1998
).
Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis.
J. Cell Biol
142
,
1301
1312
Black
S.
,
Andrews
P. D.
,
Sneddon
A. A.
,
Stark
M. J. R.
(
1995
).
A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1.
Yeast
11
,
747
759
Bloecher
A.
,
Tatchell
K.
(
1999
).
Defects in S. cerevisiae protein phosphatase type 1 activate the spindle/kinetochore checkpoint.
Genes Dev
13
,
517
522
Borgia
P. T.
(
1992
).
Roles of the orlA, tsE, and bimG genes of Aspergillus nidulans in chitin synthesis.
J. Bacteriol
174
,
384
389
Brown
J. L.
,
Bussey
H.
,
Stewart
R. C.
(
1994
).
Yeast Skn7p functions in a eukaryotic two-component regulatory pathway.
EMBO J
13
,
5186
5194
Butler
A. R.
,
White
J. H.
,
Stark
M. J. R.
(
1991
).
Analysis of the response of Saccharomyces cerevisiae cells to Kluyveromyces lactis toxin.
J. Gen. Microbiol
137
,
1749
1757
Cali
B. M.
,
Doyle
T. C.
,
Botstein
D.
,
Fink
G. R.
(
1998
).
Multiple functions for actin during filamentous growth of Saccharomyces cerevisiae.
Mol. Biol. Cell
9
,
1873
1889
Cohen
P.
(
1989
).
The structure and regulation of protein phosphatases.
Annu. Rev. Biochem
58
,
453
508
Costigan
C.
,
Gehrung
S.
,
Snyder
M.
(
1992
).
A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.
Mol. Cell. Biol
12
,
1162
1178
Costigan
C.
,
Kolodrubetz
D.
,
Snyder
M.
(
1994
).
NHP6A and NHP6B, which encode HMG1-Like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway.
Mol. Cell. Biol
14
,
2391
2403
Di Como
C. J.
,
Chang
H.
,
Arndt
K. T.
(
1995
).
Activation of CLN1 and CLN2 G1 cyclin gene expression by BCK2.
Mol. Cell. Biol
15
,
1835
1846
Doonan
J. H.
,
Morris
N. R.
(
1989
).
The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1.
Cell
57
,
987
996
Drgonova
J.
,
Drgon
T.
,
Tanaka
K.
,
Kollar
R.
,
Chen
G. C.
,
Ford
R. A.
,
Chan
C. S. M.
,
Takai
Y.
,
Cabib
E.
(
1996
).
Rho1p, a yeast protein at the interface between cell polarization and morphogenesis.
Science
272
,
277
279
Egloff
M. P.
,
Cohen
P. T. W.
,
Reinemer
P.
,
Barford
D.
(
1995
).
Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate.
J. Mol. Biol
254
,
942
959
Epstein
C. B.
,
Cross
F. R.
(
1994
).
Genes that can bypass the CLN requirement for Saccharomyces cerevisiae cell cycle start.
Mol. Cell. Biol
14
,
2041
2047
Fernandez
A.
,
Brautigan
D. L.
,
Lamb
N. J. C.
(
1992
).
Protein phosphatase type 1 in mammalian cell mitosis—chromosomal localization and Involvement in mitotic exit.
J. Cell Biol
116
,
1421
1430
Gietz
R. D.
,
Sugino
A.
(
1988
).
New yeast- Escherichia coli shuttle vectors constructed with in vitro mutagenised yeast genes lacking six-base pair restriction sites.
Gene
74
,
527
534
Gietz
R. D.
,
St. Jean
A.
,
Woods
R. A.
,
Schiestl
R. H.
(
1992
).
Improved method for high efficiency transformation of intact yeast cells.
Nucl. Acids Res
20
,
1425
–.
Gray
J. V.
,
Ogas
J. P.
,
Kamada
Y.
,
Stone
M.
,
Levin
D. E.
,
Herskowitz
I.
(
1997
).
A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator.
EMBO J
16
,
4924
4937
Helliwell
S. B.
,
Howald
I.
,
Barbet
N.
,
Hall
M. N.
(
1998
).
TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae.
Genetics
148
,
99
112
Helliwell
S. B.
,
Schmidt
A.
,
Ohya
Y.
,
Hall
M. N.
(
1998
).
The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton.
Curr. Biol
8
,
1211
1214
Hill
J. E.
,
Myers
A. M.
,
Koerner
T. J.
,
Tzagoloff
A.
(
1986
).
Yeast/ E. coli shuttle vectors with multiple unique restriction sites.
Yeast
2
,
163
168
Hisamoto
N.
,
Sugimoto
K.
,
Matsumoto
K.
(
1994
).
The GLC7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M.
Mol. Cell. Biol
14
,
3158
3165
Hughes
V.
,
Muller
A.
,
Stark
M. J. R.
,
Cohen
P. T. W.
(
1993
).
Both isoforms of protein phosphatase Z are essential for the maintenance of cell size and integrity in Saccharomyces cerevisiae in response to osmotic stress.
Eur. J. Biochem
216
,
269
279
Igual
J. C.
,
Johnson
A. L.
,
Johnston
L. H.
(
1996
).
Coordinated regulation of gene expression by the cell cycle transcription factor SWI4 and the protein kinase C MAP kinase pathway for yeast cell integrity.
EMBO J
15
,
5001
5013
Irie
K.
,
Takase
M.
,
Lee
K. S.
,
Levin
D. E.
,
Araki
H.
,
Matsumoto
K.
,
Oshima
Y.
(
1993
).
MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase kinase homologs, function in the pathway mediated by protein kinase C.
Mol. Cell. Biol
13
,
3076
3083
Ishii
K.
,
Kumada
K.
,
Toda
T.
,
Yanagida
M.
(
1996
).
Requirement for PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+.
EMBO J
15
,
6629
6640
Jacoby
J. J.
,
Nilius
S. M.
,
Heinisch
J. J.
(
1998
).
A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene.
Mol. Gen. Genet
258
,
148
155
Kahana
J. A.
,
Schnapp
B. J.
,
Silver
P. A.
(
1995
).
Kinetics of spindle pole body separation in budding yeast.
Proc. Nat. Acad. Sci. USA
92
,
9707
9711
Kamada
Y.
,
Qadota
H.
,
Python
C. P.
,
Anraku
Y.
,
Ohya
Y.
,
Levin
D. E.
(
1996
).
Activation of yeast protein kinase C by Rho1 GTPase.
J. Biol. Chem
271
,
9193
9196
Karpova
T. S.
,
Lepetit
M. M.
,
Cooper
J. A.
(
1993
).
Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth.
Genetics
135
,
693
709
Ketela
T.
,
Green
R.
,
Bussey
H.
(
1999
).
Saccharomyces cerevisiae Mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway.
J. Bacteriol
181
,
3330
3340
Lee
K. S.
,
Levin
D. E.
(
1992
).
Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog.
Mol. Cell. Biol
12
,
172
182
Lee
K. S.
,
Hines
L. K.
,
Levin
D. E.
(
1993
).
A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1 -mediated pathway.
Mol. Cell. Biol
13
,
5843
5853
Lee
K. S.
,
Irie
K.
,
Gotoh
Y.
,
Watanabe
Y.
,
Araki
H.
,
Nishida
E.
,
Matsumoto
K.
,
Levin
D. E.
(
1993
).
A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C.
Mol. Cell. Biol
13
,
3067
3075
Levin
D. E.
,
Fields
F. O.
,
Kunisawa
R.
,
Bishop
J. M.
,
Thorner
J.
(
1990
).
A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle.
Cell
62
,
213
224
Levin
D. E.
,
Bartlett-Heubusch
E.
(
1992
).
Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect.
J. Cell Biol
116
,
1221
1229
Levin
D. E.
,
Errede
B.
(
1995
).
The proliferation of MAP kinase signaling pathways in yeast.
Curr. Opin. Cell Biol
7
,
197
202
Lew
D. J.
,
Reed
S. I.
(
1993
).
Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins.
J. Cell Biol
120
,
1305
1320
Longtine
M. S.
,
Demarini
D. J.
,
Valencik
M. L.
,
Alawar
O. S.
,
Fares
H.
,
Devirgilio
C.
,
Pringle
J. R.
(
1996
).
The septins: roles in cytokinesis and other processes.
Curr. Opin. Cell Biol
8
,
106
119
MacKelvie
S. H.
,
Andrews
P. D.
,
Stark
M. J. R.
(
1995
).
The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase.
Mol. Cell. Biol
15
,
3777
3785
Madaule
P.
,
Axel
R.
,
Myers
A. M.
(
1987
).
Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae.
Proc. Nat. Acad. Sci. USA
84
,
779
783
Madden
K.
,
Sheu
Y. J.
,
Baetz
K.
,
Andrews
B.
,
Snyder
M.
(
1997
).
SBF cell cycle regulator as a target of the yeast PKC-MAP kinase pathway.
Science
275
,
1781
1784
Manning
B. D.
,
Padmanabha
R.
,
Snyder
M.
(
1997
).
The Rho-GEF Rom2p localizes to sites of polarized cell growth and participates in cytoskeletal functions in Saccharomyces cerevisiae.
Mol. Biol. Cell
8
,
1829
1844
Mayer
A.
,
Wickner
W.
,
Haas
A.
(
1996
).
Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles.
Cell
85
,
83
94
Mazzoni
C.
,
Zarzov
P.
,
Rambourg
A.
,
Mann
C.
(
1993
).
The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisia e.
J. Cell Biol
123
,
1821
1833
Morgan
B. A.
,
Bouquin
N.
,
Merrill
G. F.
,
Johnston
L. H.
(
1995
).
A yeast transcription factor bypassing the requirement for SBF and DSC1/MBF in budding yeast has homology to bacterial signal transduction proteins.
EMBO J
14
,
5679
5689
Nickas
M. E.
,
Yaffe
M. P.
(
1996
).
BRO1, a novel gene that interacts with components of the Pkc1p-mitogen-activated protein kinase pathway in Saccharomyces cerevisiae.
Mol. Cell. Biol
16
,
2585
2593
Nonaka
H.
,
Tanaka
K.
,
Hirano
H.
,
Fujiwara
T.
,
Kohno
H.
,
Umikawa
M.
,
Mino
A.
,
Takai
Y.
(
1995
).
A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae.
EMBO J
14
,
5931
5938
Ohkura
H.
,
Kinoshita
N.
,
Miyatani
S.
,
Toda
T.
,
Yanagida
M.
(
1989
).
The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases.
Cell
57
,
997
1007
Ozaki
K.
,
Tanaka
K.
,
Imamura
H.
,
Hihara
T.
,
Kameyama
T.
,
Nonaka
H.
,
Hirano
H.
,
Matsuura
Y.
,
Takai
Y.
(
1996
).
Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae.
EMBO J
15
,
2196
2207
Paravicini
G.
,
Cooper
M.
,
Friedli
L.
,
Smith
D. J.
,
Carpentier
J. L.
,
Klig
L. S.
,
Payton
M. A.
(
1992
).
The osmotic integrity of the yeast cell requires a functional PKC1 gene product.
Mol. Cell. Biol
12
,
4896
4905
Peters
C.
,
Andrews
P. D.
,
Stark
M. J.
,
Cesaro-Tadic
S.
,
Glatz
A.
,
Podtelejnikov
A.
,
Mann
M.
,
Mayer
A.
(
1999
).
Control of the terminal step of intracellular membrane fusion by protein phosphatase 1.
Science
285
,
1084
1087
Pringle
J. R.
,
Adams
A. E. M.
,
Drubin
D. G.
,
Haarer
B. K.
(
1991
).
Immunofluorescence methods for yeast.
Meth. Enzymol
194
,
565
602
Qadota
H.
,
Python
C. P.
,
Inoue
S. B.
,
Arisawa
M.
,
Anraku
Y.
,
Zheng
Y.
,
Watanabe
T.
,
Levin
D. E.
,
Ohya
Y.
(
1996
).
Identification of yeast Rho1p GTPase as a regulatory subunit of 1, 3-beta-glucan synthase.
Science
272
,
279
281
Rajavel
M.
,
Philip
B.
,
Buehrer
B. M.
,
Errede
B.
,
Levin
D. E.
(
1999
).
Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae.
Mol. Cell. Biol
19
,
3969
3976
Ram
A. F. J.
,
Wolters
A.
,
Tenhoopen
R.
,
Klis
F. M.
(
1994
).
A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white.
Yeast
10
,
1019
1030
Richman
T. J.
,
Sawyer
M. M.
,
Johnson
D. I.
(
1999
).
The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating theapical-isotropic switch and nuclear division in yeast.
J. Biol. Chem
274
,
16861
16870
Roemer
T.
,
Bussey
H.
(
1991
).
Yeast beta-glucan synthesis: KRE6 encodes a predicted type-II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro.
Proc. Nat. Acad. Sci. USA
88
,
11295
11299
Roemer
T.
,
Delaney
S.
,
Bussey
H.
(
1993
).
SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis.
Mol. Cell. Biol
13
,
4039
4048
Roemer
T.
,
Paravicini
G.
,
Payton
M. A.
,
Bussey
H.
(
1994
).
Characterisation of the yeast (16)beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly.
J. Cell Biol
127
,
567
579
Rothstein
R.
(
1991
).
Targeting, disruption, replacement, and allele rescue: Integrative DNA transformation in yeast.
Meth. Enzymol
194
,
281
301
Sassoon
I.
,
Severin
F.
,
Andrews
P. D.
,
Taba
M.-R.
,
Kaplan
K. B.
,
Ashford
A. J.
,
Stark
M. J. R.
,
Sorger
P. K.
,
Hyman
A. A.
(
1999
).
Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p.
Genes Dev
13
,
545
555
Schmidt
A.
,
Bickle
M.
,
Beck
T.
,
Hall
M. N.
(
1997
).
The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2.
Cell
88
,
531
542
Schwob
E.
,
Bohm
T.
,
Mendenhall
M. D.
,
Nasmyth
K.
(
1994
).
The B-type cyclin kinase inhibitor p40Sic1controls the G1 to S transition in S. cerevisiae.
Cell
79
,
233
244
Sikorski
R. S.
,
Hieter
P.
(
1989
).
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics
122
,
19
27
Sikorski
R. S.
,
Boeke
J. D.
(
1991
).
In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast.
Meth. Enzymol
194
,
302
318
Stark
M. J. R.
(
1996
).
Yeast protein serine/threonine phosphatases: multiple roles and diverse regulation.
Yeast
12
,
1647
1675
Surana
U.
,
Robitsch
H.
,
Price
C.
,
Schuster
T.
,
Fitch
I.
,
Futcher
A. B.
,
Nasmyth
K.
(
1991
).
The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae.
Cell
65
,
145
161
Verna
J.
,
Lodder
A.
,
Lee
K.
,
Vagts
A.
,
Ballester
R.
(
1997
).
A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae.
Proc. Nat. Acad. Sci. USA
94
,
13804
13809
Watanabe
M.
,
Chen
C. Y.
,
Levin
D. E.
(
1994
).
Saccharomyces cerevisiae PKC1 encodes a protein kinase C (PKC) homolog with a substrate specificity similar to that of mammalian PKC.
J. Biol. Chem
269
,
16829
16836
Yamochi
W.
,
Tanaka
K.
,
Nonaka
H.
,
Maeda
A.
,
Musha
T.
,
Takai
Y.
(
1994
).
Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.
J. Cell Biol
125
,
1077
1093
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