Upload
buinhan
View
217
Download
0
Embed Size (px)
Citation preview
SUPPLEMENTAL ONLINE MATERIALS
Supplemental Materials and Methods
Strain and plasmid construction. Strain KLY2962 was generated as described
previously (7). Strains KLY6228 and KLY6231 were generated by crossing KLY2466
and KLY2470, respectively, with KLY2849 {MATα cdc23-1 ade2; a derivative of
RJD1179 (a gift of Ray Deshaies, Caltech, Pasadena, CA)}. To generate strains
expressing NUD1-HA, SLK19-HA, STU2-HA, ASE-HA3, SPC97-HA3, SPC98-HA3, or
TUB4-HA3 under the respective endogenous promoter control, the corresponding loci
were C-terminally tagged with a PCR fragment containing the HA3 epitope using the
method described by Longtine et al. (6). To generate strain KLY6223, strain KLY5401
was mated with strain KLY2675 {MATα URA3::CDC5-HA3; a derivative of YDF13 (a
gift of Leland Johnston, National Institute for Medical Research, London, UK)}. Strain
KLY3098 was generated by crossing strain KLY2962 with strain KLY1548 (α-type
isogenic wild-type). Strain KLY3279 was constructed by integrating a dominant
CDC14TAB6-1 allele (10) at the HIS3 locus.
To generate pKL3266 or pKL3267, a XhoI-StuI DNA fragment bearing either the
NUD1 ORF or the STU2 ORF was cloned into pKL1969 (2) digested with the
corresponding enzymes. Similarly, to construct pKL3205, a StuI fragment bearing SLK19
ORF was inserted into pKL1969 digested with MscI and StuI.
Purification of recombinant proteins from Sf9 cells and kinase assays. To
prepare recombinant proteins, Sf9 cells infected with baculoviruses expressing T7-HA-
Nud1-FLAG, T7-HA-Slk19-FLAG, or T7-HA-Stu2-FLAG were lysed, and then
subjected to pull-down with anti-FLAG M2-agarose (Sigma, St. Louis, MO). Purification
2
of T7-HA-Cdc5-FLAG, T7-HA-cdc5(N209A)-FLAG, GST-Cdc28/His6-Cks1/MBP-
Clb2/GST-Cak1 complex, and GST-cdc28(D145N)/His6-Cks1/MBP-Clb2/GST-Cak1
complex and in vitro kinase assays were carried out as described previously (1). The
resulting samples were then separated by SDS-PAGE as indicated, stained with
Coomassie, and the incorporated 32P was detected by autoradiography.
PBD-binding assays. To prepare mitotic lysates from strain KLY5837 (cdc28-
as1 STU2-HA3), cells were first cultured overnight and treated with nocodazole for 3 h
before harvest. Cells were then lysed with an equal volume of glass beads (Sigma, St.
Louis, MO) in TED buffer {40 mM Tris-Cl (pH 7.5), 0.25 mM EDTA, 1 mM DTT}
supplemented with protease inhibitors. For binding experiment, total cellular lysates were
first diluted with TBSN buffer {20 mM Tris-Cl (pH8.0), 150 mM NaCl, 0.5% NP-40, 5
mM EGTA, 1.5 mM EDTA, 0.5 mM Na3VO4, and 20 mM p-nitrophenyl phosphate
(PNPP)} supplemented with protease inhibitors, and then centrifuged at 15,000 x g for 30
min to clarify heavy cellular materials. The resulting lysates were then incubated with
either bead-bound recombinant GST-fused Plk1 PBD or the corresponding phospho-
pincer H538A K540M (PBD/AM) mutant (3) for 2 h. The bead-associated precipitates
were washed several times with the binding buffer, and then subjected to immunoblotting
analysis.
3
Supplemental References
1. Asano, S., J. E. Park, K. Sakchaisri, L. R. Yu, S. Song, P. Supavilai, T. D.
Veenstra, and K. S. Lee. 2005. Concerted mechanism of Swe1/Wee1 regulation
by multiple kinases in budding yeast. EMBO J. 24:2194-2204.
2. Asano, S., J. E. Park, L. R. Yu, M. Zhou, K. Sakchaisri, C. J. Park, Y. H.
Kang, J. Thorner, T. D. Veenstra, and K. S. Lee. 2006 Direct phosphorylation
and activation of a Nim1-related kinase Gin4 by Elm1 in budding yeast. J. Biol.
Chem. 281:27090-27098.
3. Elia, A. E., P. Rellos, L. F. Haire, J. W. Chao, F. J. Ivins, K. Hoepker, D.
Mohammad, L. C. Cantley, S. J. Smerdon, and M. B. Yaffe. 2003. The
molecular basis for phospho-dependent substrate targeting and regulation of Plks
by the polo-box domain. Cell 115:83-95.
4. Gietz, R. D., and A. Sugnino. 1988. New yeast-Escherichia coli shuttle vectors
constructed with in vitro mutagenized yeast genes lacking six-base pair restriction
sites. Gene 74:527-534.
5. Hill, J. E., A. M. Myers, T. J. Koerner, and A. Tzagoloff. 1993. Yeast/E. coli
shuttle vectors with multiple unique restriction sites. Yeast 2:163-167.
6. Longtine, M. S., A. McKenzie, D. J. Demarini, N. G. Shah, A. Wach, A.
Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for
versatile and economical PCR-based gene deletion and modification in
Saccharomyces cerevisiae. Yeast 14:953-961.
4
7. Park, C. J., S. Song, P. R. Lee, W. Shou, R. J. Deshaies, and K. S. Lee. 2003.
Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p
regulation and Bfa1p/Bub2p-independent cytokinesis. Genetics 163:21-33.
8. Park, J.-E., C. J. Park, K. Sakchaisri, T. Karpova, S. Asano, J. McNally, Y.
Sunwoo, S.-H. Leem, and K. S. Lee. 2004. Novel functional dissection of the
localization-specific roles of budding yeast polo kinase Cdc5p. Mol. Cell. Biol.
24:9873-9886.
9. Sakchaisri, K., S. Asano, L. R. Yu, M. J. Shulewitz, C. J. Park, J. E. Park, Y.
W. Cho, T. D. Veenstra, J. Thorner, and K. S. Lee. 2004. Coupling
morphogenesis to mitotic entry. Proc. Natl. Acad. Sci. USA. 101:4124-4129.
10. Shou, W., J. H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, Z. W. Chen,
J. Jang, A. Shevchenko, H. Charbonneau, and R. J. Deshaies. 1999. Exit from
mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14
from nucleolar RENT complex. Cell 97:233-244.
11. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics 122:19-27.
12. Song, S., T. Z. Grenfell, S. Garfield, R. L. Erikson, and K. S. Lee. 2000.
Essential function of the polo box of Cdc5 in subcellular localization and
induction of cytokinetic structures. Mol. Cell. Biol. 20:286-298.
5
Supplemental Figure Legends
Figure S1. A delayed mitotic exit by the cdc23-1 mutation does not suppress the cdc5-
11-associated growth defect. Strains KLY2470 (CDC5), KLY6231 (CDC5 cdc23-1),
KLY2466 (cdc5-11), and KLY6228 (cdc5-11 cdc23-1) were cultured overnight, serially
diluted, spotted on YEP-glucose, and then incubated at the indicated temperature.
Figure S2. Overexpression of TUB4 rescues the microtubule nucleation defect associated
with the cdc5-11 mutation. Strain KLY2466 (cdc5-11) was transformed with the
indicated plasmids. The resulting transformants were treated with nocodazole and then
released into fresh medium. At the indicated time points, cells were fixed and then
subjected to confocal microscopy as described in the Materials and Methods.
Figure S3. Depletion of Cdc5 greatly diminishes proper modification of Nud1, Slk19,
and Stu2. (A-G), To examine whether Cdc5 is required for the modification of indicated
proteins, strain KLY4438 (cdc5∆ + pGAL1-cdc5-1) was C-terminally tagged with a PCR
fragment containing three HA epitopes (HA3) at the respective genomic loci. The
resulting strains KLY5826 (NUD1-HA3) (A), KLY5696 (SLK19-HA3) (B), KLY5828
(STU2-HA3) (C), KLY5707 (ASE1-HA3) (D), KLY5784 (SPC97-HA3) (E), KLY5787
(SPC98-HA3) (F), and KLY5785 (TUB4-HA3) (G) were transformed with either control
YCplac22 vector or YCplac22-CDC5. Cells were cultured overnight in YEP-galactose
medium, arrested with α-factor, and then released to fresh YEP-glucose medium
containing nocodazole. Samples were harvested at the indicated time points and subjected
to immunoblot analyses. Because of the instability of phosphorylated Slk19 in (B), the
6
same membrane was stained with Coomassie (CBB) for loading controls. (H), To
monitor cell cycle progression, an aliquot of the samples harvested was used to determine
the percentages of budded cells by counting >200 cells for each time point after release
from the α-factor block.
Figure S4. Cdc5 is required for proper modification of Nud1, Slk19, and Stu2 in vivo.
(A-C), Strains KLY3098 (cdc5-11) and KLY3279 (cdc5-11 CDC14TAB6-1) were C-
terminally tagged with a three HA-containing DNA fragment at the corresponding
genomic loci. The resulting strains {KLY5855 and KLY5857 (A), KLY5813 and
KLY5816 (B), and KLY5859 and KLY5861 (C)} were arrested in G1 by α-factor
treatment, and then released into fresh YEP-glucose medium at either 23oC or 37oC.
Samples were harvested at the indicated time points for immunoblotting analyses. The
same membranes were then stained with Coomassie (CBB) for loading controls. (D-E),
Aliquots of the cdc5-11 (D) and the cdc5-11 CDC14 TAB6-1 (E) cells were also harvested to
monitor cell cycle progression as in Fig. S2H.
Figure S5. Requirement of Cdc28 activity for proper modification of Nud1, Slk19, and
Stu2 in vivo. (A-C), Strain KLY5401 (cdc28-as1) was C-terminally tagged with a three
HA-containing DNA fragment at the respective genomic loci. The resulting strains
{KLY5849 (A), KLY5820 (B), and KLY5837 (C)} were arrested in G1 by α-factor
treatment, and then released into fresh medium at 30oC. To inhibit Cdc28 activity after
proceeding through G1, 1NM-PP1 (or control DMSO) was added to the cultures 20 min
7
after α-factor release. Total cellular proteins were separated by SDS-PAGE for
immunoblotting analysis with anti-HA antibody. Subsequently, the same membranes
were stained with Coomassie (CBB) for loading controls. Samples harvested at the 120
min time point were counted for budding indices to check cell cycle progression.
Figure S6. The cdc5-11 mutant and several other cdc5 mutants exhibit benomyl-
resistance for growth. (A), Strains KLY2946 (cdc5-1 CDC14 TAB6-1), KLY2950 (cdc5-3
CDC14 TAB6-1), KLY2954 (cdc5-4 CDC14 TAB6-1), KLY2958 (cdc5-7 CDC14 TAB6-1),
KLY2962 (cdc5-11 CDC14 TAB6-1), and KLY2970 (CDC5 CDC14 TAB6-1) were serially
diluted, spotted on YEP-glucose, and incubated at the indicated temperature. The CDC14
TAB6-1 allele was provided to bypass the mitotic exit defect associated with various cdc5
mutations. To test benomyl sensitivity without significantly influencing the cdc5 growth
defect, the benomyl-containing plate was incubated at 30oC. (B), Strains KLY5208
(CDC5), KLY5209 (CDC5∆C-CNM67), KLY5246 (bfa1∆ CDC5∆C-CDC12), and
KLY5210 (CDC5∆C-CNM67 CDC5∆C-CDC12) were spotted as in (A) and then
incubated at 30oC.
Figure S7. Both Cdc5 and Clb2-Cdc28 phosphorylate Nud1, Slk19, and Stu2 in vitro.
(A-C) GST-Cdc28/His6-Cks1/MBP-Clb2/GST-Cak1 and T7-HA-Cdc5-Flag were
reacted with Nud1, Slk19, or Stu2 in the presence of [γ-32P]-ATP at 30oC. Reactions were
terminated by the addition of SDS sample buffer and then separated by SDS-PAGE.
After staining with Coomassie (CBB), the gel was dried and subjected to
autoradiography. Cdc5 W, T7-HA-Cdc5-FLAG; cdc5 N, T7-HA-cdc5(N209A)-FLAG;
8
Cdc28 W, GST-Cdc28/His6-Cks1/MBP-Clb2/GST-Cak1 complex; cdc28 D, GST-
cdc28(D145N)/His6-Cks1/MBP-Clb2/GST-Cak1 complex; Nud1, T7-HA-Nud1-FLAG;
Slk19, T7-HA-Slk19-FLAG; Stu2, T7-HA-Stu2-FLAG. Brackets indicate
phosphorylated signals for Nud1, Slk19, or Stu2, respectively, whereas arrows indicate
autophosphorylated Cdc5. Both wild-type GST-Cdc28 and the kinase-inactive GST-
cdc28(D145N) mutant were phosphorylated by GST-Cak1.
Figure S8. Inhibition of Cdc28 activity results in downregulation of Cdc5. (A-B), Strain
KLY6223 (cdc28-as1 CDC5-HA3) was cultured overnight, arrested in G1 by α-factor
treatment, then released into YEP-glucose containing nocodazole to arrest the cells in
prometaphase. In order to inhibit the mitotic Cdc28 activity at two distinct stages of the
cell cycle, cells were treated with 0.5 µM 1NM-PP1 either 30 min (A) or 70 min (B) after
release from the α-factor block. Total cellular proteins prepared at each time point were
subjected to immunoblotting analyses.
Figure S9. Cdc5-dependent modification of Stu2 requires both Cdc28 activity and the
intact PBD. (A), Mitotic lysates prepared from strain KLY5837 (cdc28-as1 STU2-HA3)
were subjected to pull-down assays with either bead-bound GST-PBD or the
corresponding GST-PBD (H538A K540M) mutant (AM). The precipitates were
immunoblotted with anti-HA antibody. The same membrane was stained with Coomassie
(CBB) for loading controls. Asterisk, a non-specific protein that cross-reacts with anti-
HA antibody. (B), Strain KLY5837 (cdc28-as1 STU2-HA3) was transformed with
pGAL1-CDC5 plasmid (pKL882). The resulting strain was cultured overnight in YEP-
9
raffinose, arrested with α-factor, and then released into YEP-galactose medium
containing nocodazole to express the GAL1 promoter-controlled CDC5. To acutely
inhibit Cdc28 activity without interfering with G1 progression, 1NM-PP1 (or control
DMSO) was added to the cultures 30 min after α-factor release. Total cellular proteins
were prepared at the indicated time points for immunoblot analyses. The same membrane
was stained for loading control. (C), Strain KLY5837 was transformed with either
pGAL1-CDC5 (pKL882), pGAL1-CDC5((W517F V518A L530A) (pKL883), or pGAL1-
CDC5 (N209A) (pKL884). The resulting cells were cultured in YEP-raffinose overnight,
and then shifted to YEP-galactose containing nocodazole for the indicated lengths of
time. Samples were harvested at 120 min after induction and subjected to
immunoblotting analyses.
cdc5-11
cdc5-11
cdc5-11
CDC5
CDC5 cdc23-1
CDC5 cdc23-1
CDC5 cdc23-1
cdc5-11 cdc23-1
23o C
27o C
30o C
CDC5
cdc5-11 cdc23-1
CDC5
cdc5-11 cdc23-1
Park et al., Fig. S1
Park et al., Fig. S2cdc5-11
0 min 9 min 15min
+ pCDC5
+ V
ecto
r+
pTUB4,
2µµµµ
Tub4-HA3
60 min 120 minαααα noc.
+ pC
DC
5
+ ve
cto
r
+ ve
cto
r
+ p C
DC
5
Ase1-HA3
60 min 120 minαααα noc.
+ pC
DC
5
+ ve
cto
r
+ ve
cto
r
+ p C
DC
5
BA60 min 120 min
αααα noc.
+ pC
DC
5
+ ve
cto
r
+ ve
cto
r
+ p C
DC
5
Nud1-HA3
C
D
Stu2-HA3
60 min 120 minαααα noc.
+ pC
DC
5
+ ve
cto
r
+ ve
cto
r
+ p C
DC
5
Spc97-HA3
60 min 120 minαααα noc.
+ pC
DC
5
+ ve
cto
r
+ ve
cto
r
+ p C
DC
5
Spc98-HA3
60 min 120 minαααα noc.
+ pC
DC
5
+ ve
cto
r
+ ve
cto
r
+ p C
DC
5
Slk19-HA3
60 min 120 minαααα noc.
+ pC
DC
5
+ ve
cto
r
+ ve
cto
r
+ p C
DC
5
FE
G Hcdc5∆∆∆∆ + pGAL1-cdc5-1
0
20
40
60
100
80
60 90300 120after G1 release (min)
% la
rge-
bu
dd
ed c
ells
Park et al., Fig. S3
CBB
Bn
o t
ag
Cdc5
Nud1-HA3
CB
B
60 min 120 minαααα noc.
37o C
23o C
37o C
23o C
cdc5-10 cdc5-11 TAB6-1
60 min 120 minαααα noc.
37o C
23o C
37o C
23o C
no
tag
60 min 120 minαααα noc.
37o C
23o C
37o C
23o C
Cdc5
cdc5-10 cdc5-11 TAB6-1
60 min 120 minαααα noc.
37o C
23o C
37o C
23o C
Stu2-HA3
CB
B
C
A
Park et al., Fig. S4
cdc5-11 TAB6-1
60 min23oC 37oC
120 min23oC 37oC23oC
0 min
0
20
40
60
80
100
% c
ells
60 min23oC 37oC
120 min23oC 37oC23oC
0 min
cdc5-11
0
20
40
60
80
100
% c
ells
no
tag
60 min 120 minαααα noc.
37o C
23o C
37o C
23o C
Cdc5
cdc5-10 cdc5-11 TAB6-1
60 min 120 minαααα noc.
37o C
23o C
37o C
23o C
CB
B
D
E
Slk19-HA3
B
A
Park et al., Fig. S5
Slk19-HA3
CB
B
90 min 120 min
DM
SO
1NM
30 min 60 minαααα noc.
0 m
in
DM
SO
1NM
DM
SO
1NM
DM
SO
1NM
cdc28-as1
DM
SO
1NM
DM
SO
1NM
DM
SO
1NM
0
20
40
60
80
100
% c
ells
90 min 120 min
DM
SO
1NM
30 min 60 minαααα noc.
0 m
in
DM
SO
1NM
DM
SO
1NM
DM
SO
1NM
cdc28-as1
Nud1-HA3
CB
B
DM
SO
1NM
DM
SO
1NM
DM
SO
1NM
0
20
40
60
80
100
% c
ells
C
Stu2-HA3
CB
B
90 min 120 min
DM
SO
1NM
30 min 60 minαααα noc.
0 m
in
DM
SO
1NM
DM
SO
1NM
DM
SO
1NM
cdc28-as1
DM
SO
1NM
DM
SO
1NM
DM
SO
1NM
0
20
40
60
80
100
% c
ells
30oC 30oC + 15 µµµµg/ml benomyl
cdc5-1 TAB6-1
cdc5-3 TAB6-1
cdc5-4 TAB6-1
cdc5-7 TAB6-1
cdc5-11 TAB6-1
CDC5 TAB6-1
23oC
Park et al., Fig. S6
B
A
control 5 µµµµg/ml Benomyl 10 µµµµg/ml Benomyl
+ pCDC5
+ pCDC5∆∆∆∆C-CNM67
bfa1∆∆∆∆
+ pCDC5∆∆∆∆C-CNM67+ pCDC5∆∆∆∆C-CDC12
+ pCDC5∆∆∆∆C-CDC12
30oC
cdc5∆∆∆∆
Cdc5
Stu2
: Cdc5W W
+-
NW W D- -- - -
--
+ + + +- -: Cdc28-Clb2: Stu2
W W
+-
NW W D- -- - -
--
+ + + +- -
Autorad CBB
Cdc5
Slk19
: Cdc5W W
+-
NW W D- -- - -
--
+ + + +- -: Cdc28-Clb2: Slk19
W W
+-
NW W D- -- - -
--
+ + + +- -
Autorad CBB
B
A
Park et al., Fig. S7
C
Cdc5
Nud1
Autorad CBB
: Cdc5W W
+-
NW W D- -- - -
--
+ + + +- -: Cdc28-Clb2: Nud1
W W
+-
NW W D- -- - -
--
+ + + +- -
MBP-Clb2
GST-Cak1GST-Cdc28
MBP-Clb2
GST-Cak1GST-Cdc28
MBP-Clb2
GST-Cak1GST-Cdc28
0 20 40 60 80 100
120
140
αααα noc.
DMSO 1NM-PP10 20 40 60 80 10
0
120
140
αααα noc.
0 20 40 60 80 100
120
140
αααα noc.
DMSO 1NM-PP1
0 20 40 60 80 100
120
140
αααα noc.
Park et al., Fig. S8
cdc2
8-as
1 C
DC
5-H
A3
Cdc5-HA3
Cdc28
Cdc5-HA3
Cdc28
A
cdc2
8-as
1 C
DC
5-H
A3
B
A
Park et al., Fig. S9
: pGAL1-CDC5WT
0 min
FA
A
NA
WT
120 min
FA
A
NA
Stu2-HA3
EGFP-HA-Cdc5
0' 120'
YPR
YPG + Noc
αααα-H
Aαααα
-GF
P0
min
40 min
DM
SO
1NM
-PP
1
70 min
DM
SO
1NM
-PP
1
100 minD
MS
O
1NM
-PP
1130 min
DM
SO
1NM
-PP
1
160 min
DM
SO
1NM
-PP
1
160 min
DM
SO
1NM
-PP
1
αααα-H
AC
BB
Stu2-HA3
YPG YPD
cdc28-as1 STU2-HA3 + pGAL1-CDC5
0' 40' 70' 100' 130' 160'
YPR + ααααF
YPG + Noc
1NM-PP1
B
: lysates+ + - -Inp
ut
(4%
)
: GST-PBDWT AM WT AM
Stu2-HA3
Inp
ut
(12%
)
*
GST-PBDαααα
-HA
CB
B
C
Table S1. Strains used in this study
Strain a Genotype Source
KLY1546 a MATa his3-11,15 leu2-3,112 trp1-1 ura3-1 Lab stock
KLY2470 KLY1546 LEU2::TUB1-GFP cdc5∆::KanMX6 (7)
TRP1::CDC5-HA3
KLY2466 KLY1546 LEU2::TUB1-GFP cdc5∆::KanMX6 This study
TRP1::cdc5-11-HA3
KLY6231 KLY2470 cdc23-1 This study
KLY6228 KLY2466 cdc23-1 This study
KLY2946 cdc5∆::KanMX6 LEU2::TUB1-GFP (7)
HIS3::CDC14TAB6-1 TRP1::cdc5-1-HA3
KLY2950 cdc5∆::KanMX6 LEU2::TUB1-GFP (7)
HIS3::CDC14TAB6-1 TRP1::cdc5-3-HA3
KLY2954 cdc5∆::KanMX6 LEU2::TUB1-GFP (7)
HIS3::CDC14TAB6-1 TRP1::cdc5-4-HA3
KLY2958 cdc5∆::KanMX6 LEU2::TUB1-GFP (7)
HIS3::CDC14TAB6-1 TRP1::cdc5-7-HA3
KLY2962 cdc5∆::KanMX6 LEU2::TUB1-GFP This study
HIS3::CDC14TAB6-1 TRP1::cdc5-11-HA3
KLY2970 cdc5∆::KanMX6 LEU2::TUB1-GFP (7)
HIS3::CDC14TAB6-1 TRP1::CDC5-HA3
KLY5208 MATa cdc5∆::HphMX4 SCCI-HA3::KanMX6 + (8)
YCplac111-EGFP-CDC5
2
KLY5209 MATa cdc5∆::HphMX4 SCCI-HA3::KanMX6 + (8)
pRS314-CDC5∆C-CNM67 + YCplac33-GAL1-PLK1
KLY5246 MATa cdc5∆::HphMX4 bfa1∆::his5+ SCC1-HA3::KanMX6 (8)
+ pRS315-CDC5∆C-CDC12 + YCplac33-GAL1-PLK1
KLY5210 MATa cdc5∆::HphMX4 SCCI-HA3::KanMX6 (8)
+ pRS314-CDC5C∆-CNM67 + pRS315-GFP-
CDC5∆C-CDC12 + YCplac33-GAL1-PLK1
KLY4438 MATa cdc5∆::HphMX4 + (9)
YCplac33-GAL1-HA-EGFP-cdc5-1
KLY5826 KLY 4438 HphMX4∆::KanMX6 NUD1-HA3::HphMX4 This study
KLY5696 KLY4438 SLK19-HA3::KanMX6 This study
KLY5828 KLY4438 STU2-HA3::KanMX6 This study
KLY5707 KLY4438 ASE1-HA3::KanMX6 This study
KLY5784 KLY4438 SPC97-HA3::KanMX6 This study
KLY5787 KLY4438 HphMX4∆::KanMX6 SPC98-HA3::HphMX4 This study
KLY5785 KLY4438 TUB4-HA3::KanMX6 This study
KLY3098 MATa cdc5∆::KanMX6 TRP1::cdc5-11 This study
KLY3279 KLY3098 HIS3::CDC14TAB6-1 This study
KLY5855 KLY3098 NUD1-HA3::HphMX4 This study
KLY5857 KLY3279 NUD1-HA3:: HphMX4 This study
KLY5813 KLY3098 SLK19-HA3::HphMX4 This study
KLY5816 KLY3279 SLK19-HA3::HphMX4 This study
KLY5859 KLY3098 STU2-HA3::HphMX4 This study
3
KLY5861 KLY3279 STU2-HA3::HphMX4 This study
KLY5401 MATa cdc28-as1 bar1 (1)
KLY5849 KLY5401 NUD1-HA3::HphMX4 This study
KLY5820 KLY5401 SLK19-HA3::KanMX6 This study
KLY5837 KLY5401 STU2-HA3::KanMX6 This study
KLY6223 MATa cdc28-as1 URA3::CDC5-HA3 This study
a KLY1546 is in W303-1A genetic background.
4
Table S2. Plasmids used in this study.
Name Description Source
YCplac111 CEN, LEU2 (4)
pRS314 CEN, TRP1 (11)
pRS315 CEN, LEU2 (11)
YCplac22 CEN, TRP1 (5)
pSK754 YCplac111, EGFP -CDC5 (8)
pKL 2563 pRS314, CDC5∆-CNM67-GFP (8)
pKL2438 pRS315, EGFP-CDC5∆-CDC12 (8)
pKL321 YCplac22, CDC5 (8)
pKL3266 pBlueBacHis2B, HA-NUD1-Flag This study
pKL3205 pBlueBacHis2B, HA-SLK19-Flag This study
pKL3267 pBlueBacHis2B, HA-STU2-Flag This study
pKL882 YCplac22, GAL1-EGFP2-CDC5∆DB (12)
pKL883 YCplac22, GAL1-EGFP2- (12)
cdc5(W517F V518A L530A)∆DB
pKL884 YCplac22, GAL1-EGFP2-cdc5(N209A)∆DB (12)