Temperature sensitive (ts) nimA mutants of Aspergillus nidulans arrest at a unique point in G2 which is post activation of CDC2. Here we show that this G2 arrest is due to loss of nimA function and that it is dependent on BIMAAPC3, a component of the anaphase promoting complex/cyclosome (APC/C). Whereas nimA single mutants arrested in G2 with decondensed chromatin and interphase microtubule arrays, nimA, bimAAPC3 double mutants arrested growth with condensed chromatin and aster-like microtubule arrays. nimA, bimAAPC3 double mutants entered mitosis with kinetics similar to bimAAPC3 single mutants and wild-type cells, indicating a checkpoint-like role for BIMAAPC3 in G2. Even cells which had been depleted for NIMA protein and which contained insignificant levels of NIMA kinase activity entered mitosis on inactivation of bimAAPC3. BIMAAPC3 was present in a >25S complex containing BIMEAPC1, and bimAAPC3 mutants were sensitive to elevated CYCLIN B expression, consistent with BIMAAPC3 being a component of the APC/C. Inactivation of bimAAPC3 had little affect on the steady state levels of the B-type cyclin, NIMECyclin B. Our results indicate that BIMAAPC3, and most likely the APC/C itself, is activated in G2 in nimA mutants. We propose that APC/C activation is part of a novel, late G2 checkpoint, which responds to a defective process or structure in nimA mutants, and which prevents inappropriate entry into mitosis.

REFERENCES

Cohen-Fix
O.
,
Peters
J. M.
,
Kirschner
M. W.
,
Koshland
D.
(
1996
).
Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p.
Genes Dev
10
,
3081
3093
Engle
D. B.
,
Osmani
S. A.
,
Osmani
A. H.
,
Rosborough
S.
,
Xiang
X.
,
Morris
N. R.
(
1990
).
A negative regulator of mitosis in Aspergillus is a putative membrane-spanning protein.
J. Biol. Chem
265
,
16132
16137
Fry
A. M.
,
Nigg
E. A.
(
1995
).
Cell cycle. The NIMA kinase joins forces with Cdc2.
Curr. Biol
5
,
1122
1125
Funabiki
H.
,
Yamano
H.
,
Nagao
K.
,
Tanaka
H.
,
Yasuda
H.
,
Hunt
T.
,
Yanagida
M.
(
1997
).
Fission yeast Cut2 required for anaphase has two destruction boxes.
EMBO J
16
,
5977
5987
Gwynne
D. I.
,
Buxton
F. P.
,
Sibley
S.
,
Davies
R. W.
,
Lockington
R. A.
,
Scazzocchio
C.
,
Sealy-Lewis
H. M.
(
1987
).
Comparison of the cis-acting control regions of two coordinately controlled genes involved in ethanol utilization in Aspergillus nidulans.
Gene
51
,
205
216
Harris
S. D.
,
Hamer
J. E.
(
1995
).
sepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis.
EMBO J
14
,
5244
5257
Hershko
A.
,
Ganoth
D.
,
Sudakin
V.
,
Dahan
A.
,
Cohen
L. H.
,
Luca
F. C.
,
Ruderman
J. V.
,
Eytan
E.
(
1994
).
Components of a system that ligates cyclin to ubiquitin and their regulation by the protein kinase cdc2.
J. Biol. Chem
269
,
4940
4946
Hirano
T.
,
Hiraoka
Y.
,
Yanagida
M.
(
1988
).
A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2 +that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.
J. Cell Biol
106
,
1171
1183
Irniger
S.
,
Piatti
S.
,
Michaelis
C.
,
Nasmyth
K.
(
1995
).
Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast.
Cell
81
,
269
277
Irniger
S.
,
Nasmyth
K.
(
1997
).
The anaphase-promoting complex is required in G1arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S-phase.
J. Cell Sci
110
,
1523
1531
James
S. W.
,
Mirabito
P. M.
,
Scacheri
P. C.
,
Morris
N. R.
(
1995
).
The Aspergillus nidulans bimE (blocked-in-mitosis) gene encodes multiple cell cycle functions involved in mitotic checkpoint control and mitosis.
J. Cell Sci
108
,
3485
3499
Juang
Y. L.
,
Huang
J.
,
Peters
J. M.
,
McLaughlin
M. E.
,
Tai
C. Y.
,
Pellman
D.
(
1997
).
APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle.
Science
275
,
1311
1314
Kafer
E.
(
1977
).
Meiotic and mitotic recombination in Aspergillus nidulans and its chromosomal aberrations.
Advan. Genet
19
,
33
131
King
R. W.
,
Peters
J. M.
,
Tugendreich
S.
,
Rolfe
M.
,
Hieter
P.
,
Kirschner
M. W.
(
1995
).
A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.
Cell
81
,
279
288
King
R. W.
,
Deshaies
R. J.
,
Peters
J. M.
,
Kirschner
M. W.
(
1996
).
How proteolysis drives the cell cycle.
Science
274
,
1652
1659
Kirk
K. E.
,
Morris
N. R.
(
1993
).
Either-tubulin isogene product is sufficient for microtubule function during all stages of growth and differentiation in Aspergillus nidulans.
Mol. Cell Biol
13
,
4465
4476
Koerner
T. J.
,
Hill
J. E.
,
Myers
A. M.
,
Tzagoloff
A.
(
1991
).
High-Expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors.
Meth. Enzymol
194
,
477
490
Kumada
K.
,
Su
S.
,
Yanagida
M.
,
Toda
T.
(
1995
).
Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation.
J. Cell Sci
108
,
895
905
Lu
K. P.
,
Osmani
S. A.
,
Means
A. R.
(
1993
).
Properties and regulation of the cell cycle-specific NimA protein kinase of Aspergillus nidulans.
J. Biol. Chem
268
,
8769
8776
Lu
K. P.
,
Means
A. R.
(
1994
).
Expression of the noncatalytic domain of the NIMA kinase causes a G2arrest in Aspergillus nidulans.
EMBO J
13
,
2103
2113
Lu
K. P.
,
Hunter
T.
(
1995
).
Evidence for a NIMA-like mitotic pathway in vertebrate cells.
Cell
81
,
413
424
Mirabito
P. M.
,
Morris
N. R.
(
1993
).
BIMA, a TPR-containing protein required for mitosis, localizes to the spindle pole body in Aspergillus nidulans.
J. Cell Biol
120
,
959
968
Morris
N. R.
(
1976
).
Mitotic mutants of Aspergillus nidulans.
Genet. Res
26
,
237
254
O'Connell
M. J.
,
Osmani
A. H.
,
Morris
N. R.
,
Osmani
S. A.
(
1992
).
An extra copy of nimE CyclinBelevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans.
EMBO J
11
,
2139
2149
O'Connell
M. J.
,
Norbury
C.
,
Nurse
P.
(
1994
).
Premature chromatin condensation upon accumulation of NIMA.
EMBO J
13
,
4926
4937
O'Donnell
K. L.
,
Osmani
A. H.
,
Osmani
S. A.
,
Morris
N. R.
(
1991
).
bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans.
J. Cell Sci
99
,
711
719
Osmani
S. A.
,
May
G. S.
,
Morris
N. R.
(
1987
).
Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans.
J. Cell Biol
104
,
1495
1504
Osmani
S. A.
,
Engle
D. B.
,
Doonan
J. H.
,
Morris
N. R.
(
1988
).
Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene.
Cell
52
,
241
251
Osmani
A.
,
McGuire
S. L.
,
Osmani
S. A.
(
1991
).
Parallel activation of the NIMA and p34 cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans.
Cell
67
,
283
291
Osmani
A. H.
,
O'Donnell
K.
,
Pu
R. T.
,
Osmani
S. A.
(
1991
).
Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint.
EMBO J
10
,
2669
2679
Osmani
S. A.
,
Ye
X. S.
(
1996
).
Cell cycle regulation in Aspergillus by two protein kinases.
Biochem. J
317
,
633
641
Pellman
D.
,
Bagget
M.
,
Tu
Y. H.
,
Fink
G. R.
,
Tu
H. T.
(
1995
).
Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae.
J. Cell Biol
130
,
1373
1385
Peters
J.-M.
,
King
R. W.
,
Hoog
C.
,
Kirschner
M. W.
(
1996
).
Identification of BIME as a subunit of the anaphase-promoting complex.
Science
274
,
1199
1201
Pontecorvo
G.
,
Roper
J. A.
,
Hemmons
L. M.
,
MacDonald
K. D.
,
Buffon
A. W. J.
(
1953
).
The genetics of Aspergillus nidulans.
Advan. Genet
5
,
141
238
Pu
R. T.
,
Osmani
S. A.
(
1995
).
Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.
EMBO J
14
,
995
1003
Rio
D. C.
,
Laski
F. A.
,
Rubin
G. M.
(
1986
).
Identification and immunochemical analysis of biologically active Drosophila P element transposase.
Cell
44
,
21
32
Ruther
U.
,
Muller-Hill
B.
(
1983
).
Easy identification of cDNA clones.
EMBO J
2
,
1791
1794
Samejima
I.
,
Yanagida
M.
(
1994
).
Bypassing anaphase by fission yeast cut9 mutation: Requirement of cut9 +to initiate anaphase.
J. Cell Biol
127
,
1655
1670
Starborg
M.
,
Brundell
E.
,
Gell
K.
,
Hoog
C.
(
1994
).
A novel murine gene encoding a 216-kDa protein is related to a mitotic checkpoint regulator previously identified in Aspergillus nidulans.
J. Biol Chem
269
,
24133
24137
Surana
U.
,
Amon
A.
,
Dowzer
C.
,
McGrew
J.
,
Byers
B.
,
Nasmyth
K.
(
1995
).
Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast.
EMBO J
12
,
1969
1978
Tugendreich
S.
,
Tomkiel
J.
,
Earnshaw
W.
,
Hieter
P.
(
1995
).
CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition.
Cell
81
,
261
268
Upshall
A.
,
Mortimore
I. D.
(
1984
).
Isolation of aneuploid-generating mutants of Aspergillus nidulans one of which is defective in interphase of the cell cycle.
Genetics
108
,
107
121
Waring
R. B.
,
May
G. S.
,
Morris
N. R.
(
1989
).
Characterization of an inducible expression system in Aspergillus nidulan s using alcA and tubulin-coding genes.
Gene
79
,
119
130
Yamada
H.
,
Kumada
K.
,
Yanagida
M.
(
1997
).
Distinct subunit functions and cell cycle regulated phosphorylation of the 20S APC/Cyclosome required for anaphase in fission yeast.
J. Cell Sci
110
,
1793
1804
Yamamoto
A.
,
Guacci
V.
,
Koshland
D.
(
1996
).
Pds1p is required for faithful execution of anaphase in yeast, Saccharomyces cerevisiae.
J. Cell Biol
133
,
85
97
Yamamoto
A.
,
Guacci
V.
,
Koshland
D.
(
1996
).
Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathways.
J. Cell Biol
133
,
99
110
Yamashita
Y. M.
,
Nakaseko
Y.
,
Samejima
I.
,
Kumada
K.
,
Yamada
H.
,
Michaelson
D.
,
Yanagida
M.
(
1996
).
20S cyclosome complex formation and proteolytic activity inhibited by the cAMP/PKA pathway.
Nature
384
,
276
279
Ye
X. S.
,
Xu
G.
,
Pu
R. T.
,
Fincher
R. R.
,
McGuire
S. L.
,
Osmani
A. H.
,
Osmani
S. A.
(
1995
).
The NIMA protein kinase is hyperphosphorylated and activated downstream of p34 cdc2 /Cyclin B: Coordination of two mitosis promoting kinases.
EMBO J
14
,
986
994
Ye
X. S.
,
Fincher
R. R.
,
Tang
A.
,
O'Donnell
K.
,
Osmani
S. A.
(
1996
).
Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.
EMBO J
15
,
3599
3610
Ye
X. S.
,
Xu
G.
,
Fincher
R. R.
,
Osmani
S. A.
(
1997
).
Characterization of NIMA protein kinase in Aspergillus nidulans.
Meth. Enzymol
283
,
520
532
Ye
X. S.
,
Fincher
R. R.
,
Tang
A.
,
McNeal
K. K.
,
Gygax
S. E.
,
Wexler
A. N.
,
Ryan
K. B.
,
James
S. W.
,
Osmani
S. A.
(
1997
).
Proteolysis and tyrosine phosphorylation of p34cdc2/Cyclin B: The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2.
J. Biol. Chem
272
,
33384
33393
Yu
H.
,
Peters
J.-M.
,
King
R. W.
,
Page
A. M.
,
Hieter
P.
,
Kirschner
M. W.
(
1998
).
Identification of a Cullin homology in a subunit of the anaphase-promoting complex.
Science
279
,
1219
1222
Zachariae
W.
,
Nasmyth
K.
(
1996
).
TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast.
Mol. Biol. Cell
7
,
791
801
Zachariae
W.
,
Shin
T. H.
,
Galova
M.
,
Obermaier
B.
,
Nasmyth
K.
(
1996
).
Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae.
Science
274
,
1201
1204
Zachariae
W.
,
Shevchenko
A.
,
Andrews
P. D.
,
Ciosk
R.
,
Galova
M.
,
Stark
M. J. R.
,
Mann
M.
,
Nasmyth
K.
(
1998
).
Mass spectrometric analysis of the anaphase-promoting complex from yeast: Identification of a subunit related to Cullins.
Science
279
,
1216
1219
This content is only available via PDF.