Phosphorylation of the chromosomal passenger protein Bir1 is required for localization of Ndc10 to the spindle during anaphase and full spindle elongation

ArticleinMolecular Biology of the Cell 17(3):1065-74 · April 2006with25 Reads
Impact Factor: 4.47 · DOI: 10.1091/mbc.E05-07-0640 · Source: PubMed
Abstract

The Saccharomyces cerevisiae inhibitor of apoptosis (IAP) repeat protein Bir1 localizes as a chromosomal passenger. A deletion analysis of Bir1 identified two regions important for function. The C-terminal region is essential for growth, binds Sli15, and is necessary and sufficient for the localization of Bir1 as a chromosomal passenger. The middle region is not essential but is required to localize the inner kinetochore protein Ndc10 to the spindle during anaphase and to the midzone at telophase. In contrast, precise deletion of the highly conserved IAP repeats conferred no phenotype and did not alter the cell cycle delay caused by loss of cohesin. Bir1 is phosphorylated in a cell cycle-dependent manner. Mutation of all nine CDK consensus sites in the middle region of Bir1 significantly decreased the level of phosphorylation and blocked localization of Ndc10 to the spindle at anaphase. Moreover, immunoprecipitation of Ndc10 with Bir1 was dependent on phosphorylation. The loss of Ndc10 from the anaphase spindle prevented elongation of the spindle beyond 7 microm. We conclude that phosphorylation of the middle region of Bir1 is required to bring Ndc10 to the spindle at anaphase, which is required for full spindle elongation.

Full-text

Available from: Per Widlund
Molecular Biology of the Cell
Vol. 17, 1065–1074, March 2006
Phosphorylation of the Chromosomal Passenger Protein
Bir1 Is Required for Localization of Ndc10 to the Spindle
during Anaphase and Full Spindle Elongation
Per O. Widlund,* John S. Lyssand,* Scott Anderson,
Sherry Niessen,
John R. Yates, III,
and Trisha N. Davis*
*Department of Biochemistry, University of Washington, Seattle, WA 98195-7350; and
Department of Cell
Biology, The Scripps Research Institute, La Jolla, CA 92037
Submitted July 18, 2005; Revised December 16, 2005; Accepted December 20, 2005
Monitoring Editor: Tim Stearns
The Saccharomyces cerevisiae inhibitor of apoptosis (IAP) repeat protein Bir1 localizes as a chromosomal passenger. A
deletion analysis of Bir1 identified two regions important for function. The C-terminal region is essential for growth,
binds Sli15, and is necessary and sufficient for the localization of Bir1 as a chromosomal passenger. The middle region is
not essential but is required to localize the inner kinetochore protein Ndc10 to the spindle during anaphase and to the
midzone at telophase. In contrast, precise deletion of the highly conserved IAP repeats conferred no phenotype and did
not alter the cell cycle delay caused by loss of cohesin. Bir1 is phosphorylated in a cell cycle-dependent manner. Mutation
of all nine CDK consensus sites in the middle region of Bir1 significantly decreased the level of phosphorylation and
blocked localization of Ndc10 to the spindle at anaphase. Moreover, immunoprecipitation of Ndc10 with Bir1 was
dependent on phosphorylation. The loss of Ndc10 from the anaphase spindle prevented elongation of the spindle beyond
7
m. We conclude that phosphorylation of the middle region of Bir1 is required to bring Ndc10 to the spindle at
anaphase, which is required for full spindle elongation.
INTRODUCTION
A successful cell cycle is completed with a single round of
DNA replication, followed by sister chromatid separation,
spindle elongation, and cytokinesis. These events must be
coordinated such that each cell receives its exact comple-
ment of genetic material. Chromosomal passenger proteins
are involved in coordinating cell cycle events that are re-
quired for proper chromosome segregation (reviewed in
Lens and Medema, 2003; Vagnarelli and Earnshaw, 2004).
They have a dynamic localization throughout the cell cycle
as they move from kinetochores to the spindle at anaphase
and concentrate at the spindle midzone before cytokinesis.
Aurora kinase Ipl1 and its binding partner Sli15 are chro-
mosomal passengers that are involved in the spindle check-
point. Ipl1 phosphorylates targets at the kinetochore and is
required to sense the lack of tension caused by mono-ori-
ented or detached chromosomes (Biggins and Murray, 2001;
Pinsky et al., 2003). Sli15 mutants have phenotypes very
similar to Ipl1 mutants, and Sli15 is required for the move-
ment of Ipl1 to the spindle at anaphase (Kim et al., 1999;
Pereira and Schiebel, 2003).
The inhibitor of apoptosis (IAP) repeat protein Bir1 has
been implicated in chromosome segregation (Li et al., 1998;
Uren et al., 1999; Yoon and Carbon, 1999). It copurifies with
Sli15 (Cheeseman et al., 2002). Bir1 shows two-hybrid and
genetic interactions with the inner kinetochore protein
Ndc10 (Yoon and Carbon, 1999). Depletion of Bir1 prevents
localization of Ndc10 to the anaphase spindle (Bouck and
Bloom, 2005). Mutants of Bir1 in Schizosaccharomyces pombe
have defects in chromosome condensation, spindle elonga-
tion, and DNA repair (Morishita et al., 2001; Rajagopalan
and Balasubramanian, 2002).
Here, we show that Bir1 is essential. We mapped the
essential region to a 10-kDa region at the C terminus. This
region is necessary and sufficient for survival and localiza-
tion of Bir1 as a chromosomal passenger. This region is also
sufficient to interact with Sli15. The middle region of Bir1 is
not essential, but it is required to localize Ndc10 to the
spindle at anaphase and to the midzone at telophase. The
middle region is not required for Ndc10’s kinetochore local-
ization. The interaction between Ndc10 and Bir1 is depen-
dent on phosphorylation of Bir1 on CDK consensus sites and
is required for full spindle elongation.
MATERIALS AND METHODS
Media
YPD medium and SD medium were made as described previously (Sherman
et al., 1986). YPD cycloheximide medium is YPD supplemented with 2
g/ml
cycloheximide. SD complete is SD medium supplemented with 50
g/ml
adenine, 25
g/ml uracil, 100
g/ml tryptophan, and 0.1% casamino acids.
SD-uracil low adenine is SD complete lacking uracil and contains 5
g/ml
adenine. NP-40 buffer was described previously (Hazbun et al., 2003).
Plasmids and Strains
The plasmids used in this study are listed in Table 1. The BIR1 open reading
frame (ORF) 560 base pairs 5 and 548 base pairs 3 was cloned into pRS316
to create plasmid pPW02. The plasmid used for the plasmid shuffle is plasmid
This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–07–0640)
on December 28, 2005.
Address correspondence to: Trisha N. Davis (tdavis@uwashington.
edu).
Abbreviations used: CDK, cyclin-dependent kinase; IAP, inhibitor
of apoptosis; ORF, open reading frame.
© 2006 by The American Society for Cell Biology 1065
Page 1
pPW06, which contains ADE3 (from pLI831) and BIR1 in plasmid pTD29
(Geiser et al., 1993; Muller, 1996). The plasmids containing the deletions of
BIR1 were derived from plasmid pPW02 as follows. The QuikChange site-
directed mutagenesis kit (Stratagene, La Jolla, CA) was used to delete codons
15–239 of the BIR1 ORF in pPW02 to create pPW08. Plasmid pPW09 was
made by digesting plasmid pPW02 with NheI, filling in the ends using the
Klenow fragment of DNA polymerase, and ligating. To create plasmids
pPW11 and pPW13-16, pPW02 was digested with the enzymes indicated in
Table 1. The ends were filled in using the Klenow fragment of DNA poly-
merase and ligated. To create plasmid pPW17, pPW02 was digested with the
enzymes indicated in Table 1, treated with mung bean nuclease, and ligated.
Venus and 13xMyc tags were introduced into BIR1 deletion constructs by
ligating fragments from pBS7 (gift from Yeast Resource Center, University of
Washington, Seattle, WA) and pFA6a-13Myc-kanMX6 (Longtine et al., 1998)
containing these tags at the 3 end of the BIR1 ORF in frame. Plasmid pPW129
was created by inserting a synthetic duplex oligonucleotide containing two
copies of the SV40 nuclear localization sequence (NLS) in frame between the
BIR1 ORF and the Venus tag. All point mutations in BIR1 were generated
using the QuikChange or QuikChange Multi site-directed mutagenesis kits
(Stratagene).
The yeast strains used in this study are listed in Table 2. All strains are
derived from W303. C-terminal cyan fluorescent protein (CFP), yellow fluo-
rescent protein (YFP), Venus, 3xHA, 13xMyc, and TAP fusions were created
by amplifying the CFP-hphMX3, CFP-kanMX6, YFP-kanMX6, and Venus-
kanMX6 cassettes from plasmids pBS4, pDH3, pDH6, and pBS7 (all gifts from
Yeast Resource Center) and plasmids pFA6a-13Myc-kanMX6 and pFA6a-
3HA-kanMX6 (Longtine et al., 1998) and plasmid pFA6a-CTAP-MX6-2XPA
(Tasto et al., 2001). Cassettes were integrated in frame at the 3 end of the
target ORF. The BIR1 plasmid shuffle strain, PWY16-4D was constructed by
replacing the BIR1 ORF with the hph gene in a diploid using a PCR cassette
Table 1. Plasmids used in this study
Plasmid Relevant markers Source or reference
pPW02 BIR1 in pRS316 This study
pPW06 BIR1 and ADE3 in 2
m vector This study
pPW08 bir1-IAP in pRS316 This study
pPW09 bir1-876stop in pRS316 This study
pPW11 bir1-BS
a
in pRS316
This study
pPW13 bir1-BclB
a
in pRS316
This study
pPW14 bir1-SN
a
in pRS316
This study
pPW15 bir1-MB
a
in pRS316
This study
pPW16 bir1-MS
a
in pRS316
This study
pPW17 bir1-MN
a
in pRS316
This study
pPW25 bir1 in pRS306 This study
pPW33 bir1-Venus in pRS306 This study
pPW34 bir1-IAP-Venus in pRS306 This study
pPW35 bir1-876stop-Venus in pRS306 This study
pPW36 bir1-BclB
a
-Venus in pRS306
This study
pPW37 bir1-SN
a
-Venus in pRS306
This study
pPW38 bir1-MB
a
-Venus in pRS306
This study
pPW39 bir1-MS
a
-Venus in pRS306
This study
pPW40 bir1-MN
a
-Venus in pRS306
This study
pPW46 bir1-BS
a
-Venus in pRS306
This study
pPW75 bir1 T735A, T747A in pRS316 (bir1-2xA) This study
pPW118 BIR1-13xmyc in pRS306 This study
pPW122 bir1 T735A, T747A-13xmyc in pRS306 (bir1-2xA-13xmyc) This study
pPW124 bir1 S383A, S395A, S552A, S587A, S667A, T684A, S688A, T735A,
T747A in pRS316 (bir1-9xA)
This study
pPW126 bir1 S383A, S395A, S552A, S587A, S667A, T684A, S688A, T735A,
T747A-13xmyc in pRS306 (bir1-9xA-13xmyc)
This study
pPW129 bir1-MN
a
-Venus 2NLS in pRS316
This study
pPW131 bir1-IAP-13xmyc in pRS306 This study
pPW132 bir1-BclB
a
-13xmyc in pRS306
This study
pPW133 bir1-SN
a
-13xmyc in pRS306
This study
pPW134 bir1-MB
a
-13xmyc in pRS306
This study
pPW135 bir1-MS
a
-13xmyc in pRS306
This study
pPW136 bir1-BS
a
-13xmyc in pRS306
This study
pJL13 DBD-BIR1 This study
pJL16 AD-SLI15 This study
pJL19 DBD-bir1C80 This study
pJL20 DBD-bir1C296 This study
pACTII Gal4 DBD LEU2 Clontech (Mountain View, CA)
pGBKT7 Gal4 AD TRP1 Clontech
pRS306 URA3 f1 origin Sikorski and Hieter (1989)
pRS316 CEN6 ARSH4 URA3 f1 origin Sikorski and Hieter (1989)
pTD29 SPC110 LYS2 ADE3 2
m origin Geiser et al. (1993)
pLI831 ADE3 Muller (1996)
pBS4 CFP hphMX3 Yeast Resource Center
pBS7 Venus kanMX6 Yeast Resource Center
pFA6a-13Myc-kanMX6 13xmyc kanMX6 Longtine et al. (1998)
pDH6 YFP kanMX6 Yeast Resource Center
pDH3 CFP kanMX6 Yeast Resource Center
pFA6a-CTAP-MX6–2xPA TAP kanMX6 Tasto et al. (2001)
a
Plasmids were digested with the plasmids indicated (M, MscI; Bcl, BclI; B, BglII; S, SphI; N, NheI), treated with Klenow fragment of DNA
polymerase or mung bean nuclease, and ligated in frame.
P. O. Widlund et al.
Molecular Biology of the Cell1066
Page 2
Table 2. Strains used in this study
Strain Genotype Source or reference
AH109 MATa, trp1-901, leu2-3 112, ura3-52, his3-200, gal4, gal80,
LYS2::GAL1
UAS
-GAL1
TATA-HIS3,
GAL2
UAS
-GAL2
TATA-ADE2,
Clontech
URA3:: MEL1
UAS
-MEL1
TATA-lacZ
BESY49-6C MATa cyh2
r
NDC10-YFP::kanMX6
Yeast Resource Center
BESY46-1D MAT
cyh2
r
NDC10-CFP::hphMX3
Yeast Resource Center
BSY9 MATa/MAT
ade2-1oc/ade2-1oc ADE3/ade3 can1-100/can1-100 CYH2
s
/cyh2
r
;
his3-11,15/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1
Hazun et al. (2003)
PWY8 MATa/MAT
ade3/ade3 BIR1-Venus::kanMX6/BIR1-Venus::kanMX6
CYH2
s
/cyh2
r
NDC10-CFP:kanMX6/NDC10-CFP:kanMX6
This study
PWY16-4D MATa ade3 bir1::hphMX3 lys2::HIS3 with autonomous plasmid pPW06 This study
PWY19 MATa/MAT
ade3/ade3 BIR1-Venus::kanMX6/BIR1-Venus::kanMX6
CYH2
s
/cyh2
r
SLI15-CFP::kanMX6/SLI15-CFP::kanMX6
This study
PWY56 MATa BIR1::bir1-876stop-Venus::kanMX6::URA3 This study
PWY57 MATa BIR1::bir1-MN-Venus::kanMX6::URA3 This study
PWY60 MATa BIR1::BIR1-Venus::kanMX6::URA3 This study
PWY61-4D MATa ade3 BIR1-TAP::kanMX6 cyh2
r
This study
PWY75-6C MATa ade3 BIR1-Venus::kanMX6 NDC10-CFP::hphMX3 This study
PWY76-1C MATa ade3 bir1-IAP-Venus::kanMX6 NDC10-CFP::hphMX3 This study
PWY77-2B MATa ade3 bir1-BclB-Venus::kanMX6 NDC10-CFP::hphMX3 This study
PWY78-6C MATa ade3 bir1-SN-Venus::kanMX6 NDC10-CFP::hphMX3 This study
PWY79-5A MATa ade3 bir1-MB-Venus::kanMX6 NDC10-CFP::hphMX3 This study
PWY80-2C MATa ade3 bir1-MS-Venus::kanMX6 NDC10-CFP::hphMX3 This study
PWY81-1B MATa ade3 bir1-BS-Venus::kanMX6 lys2::HIS3 NDC10-CFP::hphMX3 This study
PWY82-2B MATa ade3 bir1-Venus::kanMX6 SLI15-CFP::hphMX3 This study
PWY83-2A MATa ade3 bir1-IAP-Venus::kanMX6 SLI15-CFP::hphMX3 This study
PWY84-4D MATa ade3 bir1-BclB-Venus::kanMX6 SLI15-CFP::hphMX3 This study
PWY85-4D MATa ade3 bir1-SN-Venus::kanMX6 SLI15-CFP::hphMX3 This study
PWY86-1D MAT
ade3 bir1-MB-Venus::kanMX6 SLI15-CFP::hphMX3 This study
PWY87-5C MATa ade3 bir1-MS-Venus::kanMX6 cyh2
r
SLI15-CFP::hphMX3
This study
PWY88-10A MATa ade3 bir1BS-Venus::kanMX6 cyh2
r
lys2::HIS3 SLI15-CFP::hphMX3
This study
PWY91-10C MATa ade3 bir1::hphMX3 lys2::HIS3 NDC10-YFP::kanMX6 with
autonomous plasmid pPW06
This study
PWY93-3B MATa ade3 BIR1–13xMyc::kanMX6 This study
PWY160-1B MAT
ade3 NDC10-3xHA::kanMX6 This study
PWY192 MATa ade3 bir1::hphMX3::bir1-2xA-13xMyc::kanMX6::URA3 lys2::HIS3
NDC10-YFP::kanMX6
This study
PWY194 MATa ade3 bir1::hphMX3:: bir1-9xA-13xMyc::kanMX6::URA3 lys2::HIS3
NDC10-YFP::kanMX6
This study
PWY199-5B MATa ade3 BIR1-13xMyc::kanMX6 cdc15-2 SLI15-3xHA::kanMX6 This study
PWY200-14C MATa bir1-IAP::hphMX3 his3-11::PCUP1-GFP12-lacI12::HIS3 mcd1-1
PDS1-3xHA::URA3 trp1-1::lacO::TRP1
This study
PWY200-17A MATa his3-11::PCUP1-GFP12-lacI12::HIS3 mcd1-1 PDS1-3xHA::URA3
trp1-1::lacO::TRP1
This study
PWY202-11B MATa BIR1-13xMyc::kanMX6 cdc15-2 NDC10-3xHA::kanMX6 This study
PWY204 MATa ade3 bir1::hphMX3::bir1-IAP-13xMyc::kanMX6:URA3 lys2:HIS3 This study
PWY205 MATa ade3 bir1:hphMX3:bir1-BclB-13xMyc:kanMX6:URA3 lys2:HIS3 This study
PWY206 MATa ade3 bir1:hphMX3:bir1-SN-13xMyc:kanMX6:URA3 lys2:HIS3 This study
PWY207 MATa ade3 bir1:hphMX3:bir1-MB-13xMyc:kanMX6:URA3 lys2:HIS3 This study
PWY208 MATa ade3 bir1:hphMX3:bir1-MS-13xMyc:kanMX6:URA3 lys2:HIS3 This study
PWY209 MATa ade3 bir1:hphMX3:bir1-BS-13xMyc:kanMX6:URA3 lys2:HIS3 This study
JLY15 MATa/MAT
ADE3/ade3 BIR1-Venus:kanMX6/BIR1-Venus:kanMX6 cyh2
r
/
cyh2
r
; his3-11/HIS3 HIS4/his4:CYH2
s
This study
JLY16 MATa/MAT
ADE3/ade3 bir1-IAP-Venus:kanMX6/bir1-IAP-
Venus:kanMX6 cyh2
r
/cyh2
r
; his3-11/HIS3 HIS4/his4:CYH2
s
This study
JLY17 MATa/MAT
ADE3/ade3 bir1-BclB-Venus:kanMX6/bir1-BclB-
Venus:kanMX6 cyh2
r
/cyh2
r
HIS3/HIS3 HIS4/his4:CYH2
s
lys2:HIS3/LYS2
This study
JLY18 MATa/MAT
; ADE3/ADE3 bir1-SN-Venus:kanMX6/bir1-SN-
Venus:kanMX6 cyh2
r
/cyh2
r
HIS3/his3-11,15 his4:CYH2
s
/HIS4
This study
JLY19 MATa/MAT
ADE3/ADE3 bir1-MB-Venus:kanMX6/bir1-MB-
Venus:kanMX6 cyh2
r
/cyh2
r
his3-11,15/HIS3 HIS4/his4:CYH2
s
This study
JLY20 MATa/MAT
ade3/ADE3 bir1-MS-Venus:kanMX6/bir1-MS-
Venus:kanMX6 cyh2
r
/cyh2
r
HIS3/his3-11,15 his4:CYH2
s
/HIS4
This study
JLY21 MATa/MAT
ade3/ADE3 bir1-BS-Venus:kanMX6/bir1-BS-Venus:kanMX6
cyh2
r
/cyh2
r
HIS3/HIS3 his4:CYH2
s
/HIS4 LYS2/lys2:HIS3
This study
TDY93 MATa/MAT
ADE3/ade3 cyh2
r
/cyh2
r
his3-11,15/HIS3 HIS4/his4:CYH2
r
Davis (1992)
W303 MATa ade2–1oc can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1
All strains, except strain AH109, have the same markers as W303 except as shown.
Ndc10 Spindle Localization Requires Bir1
Vol. 17, March 2006 1067
Page 3
generated from plasmid pBS4, followed by transformation of plasmid pPW06
and subsequent sporulation. PWY91-10C is a modified BIR1 plasmid shuffle
strain containing Ndc10-CFP. All functional BIR1 deletions, truncations, and
point mutants were introduced as the sole copy at the BIR1 locus in
PWY16-4D or PWY91-10C using the gene replacement method as described
previously (Widlund and Davis, 2005).
Fluorescence Microscopy
Live cell imaging was performed with a DeltaVision microscopy system from
Applied Precision (Issaquah, WA). The system incorporates an Olympus
IL-70 microscope, a u-plan-apo 100 oil objective (1.35 numerical aperture), a
CoolSnap HQ digital camera from Roper Scientific (Tucson, AZ), and optical
filter sets from Omega Optical (Battleboro, VT). Cells were mounted on a 1%
agarose pad containing SD-complete medium (Muller et al., 2005). Single
sections were taken. The images were analyzed using DeltaVision softWoRx
software. Images were converted to TIFF format in 24-bit RGB.
Chromosome Loss Assays
The rates of loss of a tagged chromosome III was assayed as described
previously except that cycloheximide was used at a final concentration of 2
g/ml (Runge et al., 1991; Davis, 1992). Nineteen small colonies were picked
after2dofgrowth at room temperature on YPD media and suspended in 1
ml of distilled H
2
O. Suspensions were then sonicated, and 100
l of each
suspension was plated onto each of 19 YPD-cycloheximide plates. Plates were
incubated at 30°C for 4 d. To determine the average number of colony-
forming units per colony, aliquots (50
l) of each suspension were pooled and
titered on YPD medium. Calculation of the rate of chromosome loss was
performed by the method of the median (Lea and Coulson, 1949). The number
of chromosome loss events per colony was determined from the median
number of cycloheximide resistant cells per colony. This number was then
divided by the average number of cell divisions per colony to give the rates
of chromosome loss per cell division.
Protein Purification
Tandem-affinity purifications of Bir1 and mass spectrometry analysis were
performed as described previously (Hazbun et al., 2003) except in the case of
the low-stringency purification, where NaCl concentration was kept at 150
mM (Hazbun et al., 2003). Sites of phosphorylation were identified by mass
spectrometry as described previously (MacCoss et al., 2002). All mass spec-
trometry data generated for this study are available at www.yeastrc.org/pdr.
Immunological Techniques
Lysate was prepared from strains carrying Myc-tagged Bir1 or hemagglutinin
(HA)-tagged Ndc10 according to the tandem-affinity purification protocol
except that the clarification step was at 15,000 rpm for 10 min. Aliquots (1 ml)
were prepared from the 50 ml of total cleared lysate and frozen at 80°C.
Bir1-Myc was immunoprecipitated from 1 ml of lysate with 25
l of Sepharose
beads conjugated with 9E11 monoclonal
-Myc antibodies (Covance, Prince-
ton, NJ). Beads were washed with NP-40 buffer and then buffer 3 (New
England Biolabs, Ipswich, MA). The beads were incubated at 37°C for1hin
buffer 3 in the presence or absence of 100 U/ml calf intestinal phosphatase
(CIP). Lysate from cells carrying Ndc10-3xHA was precleared by incubating
with 100-
l bed volume of Sepharose 6B (GE Healthcare, Buckinghamshire,
United Kingdom) for 30 min. The precleared lysate was added to
-Myc-
conjugated Sepharose beads (control) and to the Bir1-bound beads and incu-
bated for 2 h. Beads were then washed three times in NP-40 buffer, and
proteins were eluted by boiling in denaturing SDS-PAGE gel loading buffer.
All samples were subjected to electrophoresis on SDS-polyacrylamide gels
and transferred to nitrocellulose membranes. Membranes were blocked with
Odyssey block (Li-Cor, Lincoln, NE). The anti-HA antibody was mouse
monoclonal 12CA5 (Roche Diagnostics, Indianapolis, IN). The anti-Myc anti-
body used was mouse monoclonal 9E10 (Santa Cruz Biotechnology, Santa
Cruz, CA). The secondary antibody was goat
-mouse Alexa Fluor 680
(Molecular Probes, Eugene, OR). Membranes were scanned and quantified
using the Odyssey system (Li-Cor).
RESULTS
S. cerevisiae Bir1 Localizes as a Chromosomal Passenger
Localization of full-length Bir1 has proven difficult, although
it seems to associate with microtubules (Uren et al., 1999;
Yoon and Carbon, 1999; Huh et al., 2003). We successfully
tagged and localized endogenous full-length Bir1 with the
YFP variant Venus. Bir1-Venus shows a dynamic localiza-
tion through the cell cycle that is similar to chromosomal
passenger proteins in vertebrates (Figure 1). Bir1 colocalizes
with Sli15 at all stages of the cell cycle. Bir1 seems to move
from kinetochores to the spindle at anaphase and concen-
trates at the midzone late in the cell cycle.
We compared the localization of Bir1-Venus and Ndc10-
CFP. Bir1 and Ndc10 colocalize at kinetochores before an-
aphase. During anaphase, Bir1 leaves the kinetochore,
whereas the majority of Ndc10 remains. Bir1 and a pool of
Ndc10 colocalize to the spindle during anaphase and at the
midzone during telophase.
BIR1 Is Essential
There has been some dispute as to whether BIR1 is essential
for viability. Two groups have reported BIR1 is not essential
Figure 1. Bir1 localizes as a chromosomal passenger. (A–C) Strain
PWY19, expressing Bir1 with a C-terminal Venus tag and Sli15 with
a C-terminal CFP tag expressed under control of their endogenous
promoters, was imaged as described in Materials and Methods. (D–F)
Strain PWY8, expressing Bir1 with a C-terminal Venus tag and
Ndc10 with a C-terminal CFP tag, expressed under control of their
endogenous promoters, was imaged as described in Materials and
Methods. (A) Bir1-Venus and Sli15-CFP colocalize on kinetochores
before anaphase. (B) At anaphase, Bir1 and Sli15 colocalize on the
mitotic spindle. (C) Bir1-Venus and Sli15-CFP colocalize on the
spindle in late anaphase and begin to concentrate at the spindle
midzone as cells approach cytokinesis. (D) Bir1-Venus colocalizes
with Ndc10-CFP before anaphase. (E) Bir1-Venus and Ndc10-CFP
localization overlaps on the spindle but not on kinetochores during
anaphase. (F) Bir1-Venus and Ndc10-CFP localization overlaps on
the spindle and at the midzone as cells approach cytokinesis.
P. O. Widlund et al.
Molecular Biology of the Cell1068
Page 4
(Uren et al., 1999; Yoon and Carbon, 1999), and one group
reported that BIR1 is essential (Li et al., 2000). We found that
BIR1 is essential. We replaced one copy of the BIR1 ORF
with the hygromycin B resistance gene, hph, in a diploid.
Colony PCR using primers flanking the ORF confirmed
the presence of a deleted copy and a wild-type copy in the
diploid. Sporulation and tetrad dissection of the diploid
isolates gave two live and two dead spores. The two live
spores in each of 24 tetrads were hygromycin B sensitive.
Li and coworkers proposed that any viable bir1 haploids
isolated from a heterozygous diploid contain a wild-type
copy of BIR1. We tested this possibility directly. Our
BIR1/bir1::hph heterozygous diploid is also heterozygous
for the recessive cyh
r
allele. We sporulated this strain and
selected for haploids on medium containing cyclohexi-
mide. Forty-eight of 276 cycloheximide-resistant spores
isolated were resistant to hygromycin B indicating the
presence of a disrupted copy of BIR1. Colony PCR of six
of these viable cells containing bir1::hph confirmed the
presence of both a wild-type and a disrupted copy of
BIR1.
Finally, we constructed a plasmid shuffle strain for BIR1,
where a haploid has the endogenous copy of BIR1 disrupted
and carries a multicopy plasmid carrying BIR1 and ADE3.If
BIR1 is not essential, then the strain should form colonies
with white sectors, the white sectors being cells that have
lost the only copy of BIR1, which is on the ADE3 plasmid.
However, this strain forms solid red colonies, indicating that
BIR1 is essential for viability.
The C Terminus of Bir1 Is Essential
Because BIR1 is essential, we performed a deletion analysis
to define the essential regions of BIR1. Deletions and trun-
cations of Bir1 were expressed from the BIR1 promoter in an
ARS/CEN vector (Figure 2A). These constructs were trans-
formed into the plasmid shuffle strain PWY16-4D to assess
their ability to support the growth of yeast cells. Only the
C-terminal truncation (bir1-876stop) and the largest deletion
(bir1-MN) were unable to support growth (Figure 2A).
We localized the two inactive deletions. Bir1-876stop is
present in the nucleus but does not localize to the kinet-
ochore or spindle (Figure 3A). Bir1-MN seems uniformly
spread throughout the cell and is not found in the nucleus
(Figure 3A). Addition of a nuclear localization sequence to
Bir1-MN partially restored proper localization (Figure
3A). The NLS-Bir1-MN is also able to support growth,
indicating that Bir1-MN lacks an NLS but is otherwise
functional. Therefore, the C terminus is the essential re-
gion of Bir1 and is required for kinetochore and spindle
localization.
The Essential C Terminus of Bir1 Interacts with Sli15
We purified TAP-tagged Bir1 and identified the copurifying
proteins by mass spectrometry. In six of six purifications,
Sli15 copurified with Bir1. Ipl1 was not detected even when
Bir1 was purified under low stringency. These results are
consistent with a model in which Bir1 forms a complex with
Sli15 that does not contain Ipl1.
The region of Bir1 responsible for interaction with Sli15
was mapped by directed two-hybrid experiments. Full-
length Bir1 showed a strong interaction with full-length
Sli15 (Figure 4A). The C-terminal 297 amino acids (C termi-
nus of bir1-MB) and the C-terminal 80 amino acids (C
terminus of bir1-MN) also showed interaction with full-
length Sli15 (Figure 4B).
The IAP Repeats of Bir1 Are Not Required for the Spindle
Checkpoint
The function of the IAP repeats in Bir1 is not known. Dele-
tion of the repeats did not affect Bir1’s localization or ex-
Figure 2. Bir1 deletions and truncations.
(A) Internally deleted and truncated ver-
sions of BIR1 were constructed as de-
scribed in Materials and Methods. Sectoring
is shown as the percentage of colonies that
formed white sectors in red colonies when
transformed with the version of BIR1
shown, expressed from an autonomously
replicating plasmid (plasmids are given in
Table 1). For all other analyses, the dele-
tions and truncations were integrated as
the only copy of bir1 at the endogenous
locus as described previously (Widlund
and Davis, 2005). Strains are listed in Ta-
ble 2. Bir1, Sli15, and Ndc10 localizations
are scored as absent (), present at a re-
duced level in some cells (/), present
at a reduced level in all cells (), or
present at a normal level in all cells (⫹⫹).
Expression levels are given relative to
wild type as quantified from the Western
blot shown in B. Chromosome loss rate
was measured as described in Materials
and Methods. The rate of loss of chromo-
some III in a wild-type strain was 1.05
10
5
per cell division. NA, not applicable;
ND, not determined. (B) Western blot
analysis of the protein levels of Bir1 wild-
type and Bir1 deletions. 13xMyc tagged
versions of all deletions under control of the BIR1 promoter were used to replace BIR1 at the endogenous locus. Strains are listed in Table
2. Strains were grown to mid-log phase, and extracts were prepared by precipitation with trichloroacetic acid as described previously (Wright
et al., 1989). Equal amounts of total protein were loaded in each lane as determined from a Coomassie blue-stained gel. Protein was detected
by antibodies against the Myc epitope (Santa Cruz Biotechnology).
Ndc10 Spindle Localization Requires Bir1
Vol. 17, March 2006 1069
Page 5
pression (Figures 2 and 3B). The IAP repeats were not re-
quired for Sli15 localization or Ndc10 localization (Figure 3,
B and C). Deletion of the IAP repeats did not alter the rate of
chromosome loss as measured by a fluctuation analysis
(Figure 2A). Because Bir1 has been linked to Ipl1 function
and Ipl1 homologues can phosphorylate IAP repeats in vitro
(Leverson et al., 2002; Wheatley et al., 2004), we examined
whether the IAP repeats were required for the tension
checkpoint. Cells carrying a temperature-sensitive mutation
in cohesin, mcd1-1, delay in the cell cycle, and this delay is
Figure 3. Localization of Bir1, Sli15, and Ndc10 in bir1 deletion mutants. (A) Two bir1 mutants that could not independently support growth,
Bir1-876stop and Bir1-MN, were tagged with Venus and integrated into a wild-type haploid and imaged as described in Materials and Methods.
Bir1-876stop-Venus shows diffuse nuclear localization and is absent from kinetochores and the spindle. Bir1-MN-Venus is spread throughout the
cell and is unable to efficiently localize to the nucleus. Bir1-MN NLS on an ARS/CEN vector was transformed into PWY16]hyphen]4D. Addition
of an SV40 NLS partially restores localization of Bir1-MN to the spindle. (B) Functional deletion mutants of BIR1 were tagged with Venus and
were used to replace wild-type BIR1 as the only copy as described previously (Widlund and Davis, 2005). The localizations of the tagged mutant
proteins were compared with the localization of Sli15-CFP. First column, the deletion mutants of Bir1 localize normally except the bir1-SN and
bir1-MB mutants, which display fainter spindle localizations. Second column, Sli15 localizes in all the mutants with reduced levels in bir1-SN
and bir1-MB. Third column, merged image with Bir1-Venus in green and Sli15-CFP in red. Fourth column, differential interference contrast (DIC)
image. (C) Localization of Ndc10-CFP in the bir1 deletion mutants. First column, deletion mutants of Bir1. Second column, Ndc10 is mislocalized
in all but the bir1-IAP mutant strain. Third column, merged image with Bir1-Venus in green and Ndc10-CFP in red. Fourth column, DIC image.
P. O. Widlund et al.
Molecular Biology of the Cell1070
Page 6
dependent on Ipl1 (Biggins and Murray, 2001). We found
that deletion of the IAP repeats did not alter the cell cycle
delay caused by the mcd1-1 mutation (Figure 5).
Deletions of the Middle Region of Bir1 Alter Ndc10
Localization and Increase the Rate of Chromosome Loss
Deletions in the middle region of Bir1 could support growth.
These deletions localized properly except the bir1-SN and
the bir1-MB mutants showed weaker spindle localization
(Figure 3). All deletions also expressed well (Figure 2). The
deletions of the middle region did not alter localization of
Sli15 (Figure 3B). Localization of Ndc10 to the kinetochore
was not altered. However, localization of Ndc10 to the an-
aphase spindle and the midzone was prevented (Figure 3C).
Moreover, the middle region deletions caused a moderate-
to-severe increase in the rate of chromosome loss as ana-
lyzed by a fluctuation analysis (Figure 2A) (Runge et al.,
1991; Davis, 1992).
Bir1 Is Cell Cycle Regulated
Bir1 expression levels fluctuate through the cell cycle (Figure
6A). Bir1 is barely expressed in cells arrested with
-factor.
Protein levels increase after release from the arrest. Bir1 is
also phosphorylated in a cell cycle-dependent manner. Phos-
phorylation occurs as the cells enter S phase and is removed
late in the cell cycle (Figure 6A). Immunoprecipitated Bir1
migrates on SDS-PAGE as multiple bands, which can be
condensed to one band by treatment with phosphatase,
demonstrating that the mobility shift is because of phos-
phorylation (Figure 6B). Bir1 is heavily modified at a cdc15
cell cycle arrest, indicating phosphorylation is maintained
into telophase (Figure 6C).
Phosphorylation of Bir1 Is Required for Interaction with
Ndc10 and Efficient Spindle Elongation
We identified which sites were phosphorylated in vivo by
analyzing purified Bir1 by mass spectrometry (Figure 6D).
The two sites, T735 and T747, were minimal consensus sites
for Cdc28 phosphorylation.
Mutation of the two identified sites diminished but did
not abolish phosphorylation (Figure 6E, bir1-2A). Mutation
of all nine cdc28 consensus sites in the central region (S383A,
Figure 4. The C terminus of Bir1 interacts with Sli15. Full-length
Sli15 was fused to the Gal4 DNA binding domain (DBD). Full-
length and fragments of Bir1 were fused to the Gal4 activation
domain. Combinations of Sli15 and Bir1 fragments were cotrans-
formed into strain AH-109 and plated on SD-Leu-Trp medium to
select for both plasmids. Transformants were then tested for growth
on SD-Leu-Trp-His plates containing 3 mM 3-aminotriazole (3AT).
(A) Strain AH109 containing Bir1 tagged with the Gal4 DNA bind-
ing domain and Sli15 tagged with the Gal4 activation domain was
assayed for growth on SD-Leu-Trp-His plates containing 3 mM
3AT. AH109 cotransformed with AD-Sli15 and the DBD alone or
DBD-Bir1 and the AD alone were used as controls. (B) Strain AH109
containing AD-SLI15 and one of two DBD tagged C-terminal frag-
ments of Bir1 were assayed for growth as described in A. AH109
cotransformed with the AD alone and either DBD tagged C-termi-
nal fragment of Bir1 were used as controls.
Figure 5. The IAP repeats of Bir1 are not required for the tension
checkpoint. Cells carrying the bir1-IAP allele and a temperature-
sensitive cohesin mutation, mcd1-1, were arrested in G
1
with
-fac
-
tor and released to the restrictive temperature of 37°C as described
previously (Yoder et al., 2005). HA-tagged securin (Pds1) levels were
monitored by Western blot analysis to determine if cells arrested in
anaphase. Pds1 persists at the restrictive temperature in both the
IAP mutant and the controls, indicating that the IAP repeats are
dispensable for the tension checkpoint.
Figure 6. Bir1 is phosphorylated in a cell cycle-dependent manner.
(A) Haploid cells expressing TAP-tagged Bir1 were synchronized by
treatment with
-factor and released as described previously (Yoder
et al., 2005). At each time point, extracts were prepared by precipi-
tation with trichloroacetic acid (Wright et al., 1989). Bir1 protein
levels were detected by probing for the protein A motif in the TAP
tag using a peroxidase
-peroxidase antibody (Sigma, St. Louis,
MO). Adh1 was used as a control for equal protein loading in all
lanes. (B) Cells containing Myc-tagged Bir1 and the temperature-
sensitive cdc15-2 allele were arrested at telophase by shifting to the
restrictive temperature and lysed. Immunoprecipitated Bir1 was
incubated at 37°C for1hintheabsence or presence of CIP. (C) Cells
containing the temperature-sensitive cdc15-2 allele and Myc-tagged
Bir1 were grown at the permissive temperature (25°C) or arrested at
the restrictive temperature (37°C). Extracts were prepared by pre-
cipitation with trichloroacetic acid (Wright et al., 1989). Precipitated
protein was analyzed by Western blot using antibody to the Myc
epitope. (D) Bir1-TAP was purified by tandem affinity purification
in the presence of phosphatase inhibitors from 4 liters of culture
grown to 150 Klett units as described in Materials and Methods.
One-fourth of the purified protein was subjected to electrophoresis
on a 10% polyacrylamide gel. The gel was silver stained (Bio-Rad,
Hercules, CA). The remaining purified Bir1 was analyzed by mass
spectrometry to identify phosphorylated peptides. (E) Strains ex-
pressing Myc-tagged versions of the indicated phospho-mutants of
Bir1 were grown to mid-log phase, and extracts were prepared by
precipitation with trichloroacetic acid and analyzed by Western blot
analysis using antibody against the Myc epitope.
Ndc10 Spindle Localization Requires Bir1
Vol. 17, March 2006 1071
Page 7
S395A, S552A, S587A, S667A, T684A, S688A, T735A, and
T747A) dramatically decreased the shift (Figure 6E, bir1-
9xA). Mutation of these nine sites prevents localization of
Ndc10 to the spindle at anaphase (Figure 7A). Moreover,
treatment of Bir1-13xMyc with phosphatase disrupts the in
vitro interaction between Ndc10 and Bir1 (Figure 7B).
Finally, we examined the consequence of loss of Ndc10
from the anaphase spindle and midzone by characterizing
the bir1-9xA mutant. Cells were synchronized by treatment
with
-factor and then released and progression through the
cell cycle was analyzed. Bud emergence and cytokinesis
occurred at the same time in mutant and wild-type cultures
(Figure 8A). Next, we measured the length of the mutant
and wild-type anaphase spindles in a cycling population of
cells. The mutant anaphase spindles were shorter with an
average length of 4.9
m compared with an average length
of 5.4
m for wild-type spindles. This was a statistically
significant difference to a 99% confidence level with Stu-
dent’s t test. The main difference between wild-type and
mutant was a lack of long spindles greater than 7
min
length in the mutant cells. Less than 1% of the mutant
anaphase spindles were above 7
m in length compared
with 12% of wild-type anaphase spindles (Figure 8B).
DISCUSSION
Bir1 is a chromosomal passenger protein implicated in chro-
mosome segregation. We have shown that the C terminus is
necessary and sufficient for Bir1 kinetochore and spindle
localization and is therefore critical for its functions as a
chromosomal passenger. In fact, when fused to a nuclear
localization sequence, the C-terminal 80 amino acids are
sufficient for survival. The C-terminal 80 amino acids are
also sufficient to interact with Sli15.
Figure 7. Phosphorylation of Bir1 is required for interaction with
Ndc10. (A) The bir1-9xA mutant was transformed into the modified
plasmid shuffle strain PWY91-10c. Ndc10-YFP was localized in the
presence of this bir1 mutant as described in Materials and Methods.
Localization of Ndc10-YFP in the presence of wild-type Bir1 is
shown as a control. B. The interaction between Ndc10 and Bir1
depends on phosphorylation of Bir1. Bir1-13xMyc was immunopre-
cipitated from whole cell extract with Myc-conjugated Sepharose
beads and either dephosphorylated with CIP or left untreated.
Whole cell extract carrying Ndc10-3xHA was then added to both the
CIP-treated and untreated beads. The presence of Bir1 and Ndc10
were assayed by Western blot analysis using antibodies recognizing
Myc and HA, respectively. Lane 1, Ndc10-3xHA extract alone. Lane
2, Ndc10-3xHA extract bound directly to Myc-conjugated Sepharose
beads (control). Lane 3, Ndc10 bound to phosphorylated Bir1. Lane
4, Ndc10 bound to dephosphorylated Bir1.
Figure 8. Full spindle elongation is prevented in the bir1-9xA
mutant. (A) Cultures of strains BESY49-6c and PWY194 were syn-
chronized by treatment with
-factor as described previously (Yo-
der et al., 2005). At the given times, samples were removed and fixed
in 3.7% formaldehyde. Budding and cytokinesis was determined by
phase contrast microscopy. Time is in minutes after release from
-factor. Triangles denote strain BESY49-6c (control) and squares
denote PWY194 (bir1-9xA). At least 100 cells were analyzed at each
time point. (B) Strains BESY49-6c and PWY194 were imaged as
described in Materials and Methods except that 12 0.4-
m sections
were taken. Spindles were considered to be at anaphase when one
spindle pole had entered the bud. Distance between spindles poles
(as detected by Ndc10-Venus) three or fewer sections apart were
measured in three dimensions using softWoRx software; 108 spin-
dles were measured for each strain.
P. O. Widlund et al.
Molecular Biology of the Cell1072
Page 8
The IAP repeats of Bir1 seem to be completely dispensable
for normal growth. Precise deletion of these repeats did not
affect localization of Bir1 itself, localization of Sli15 or local-
ization of Ndc10. They were also not important for high-
fidelity chromosome segregation or the spindle checkpoint
because the bir1-IAP mutant behaved normally in re-
sponse to loss of cohesin. This suggests that in Bir1 homo-
logues, such as survivin, the C terminus but not the IAP
repeats are important in their role at the mitotic spindle.
The middle region of Bir1 is required to localize Ndc10 to
the anaphase spindle and to the midzone but is not required
for kinetochore localization of Ndc10. The C-terminal half of
Bir1 is required for a strong two-hybrid interaction with
Ndc10 (Yoon and Carbon, 1999). We find that even small
deletions in this region abolish Ndc10 localization. More-
over, the interaction between Bir1 and Ndc10 is regulated by
phosphorylation. Mutation of the nine CDK minimal con-
sensus sites in the middle region of Bir1 reduced the mobil-
ity shift as seen by Western blot analysis and prevented
Ndc10 localization to the anaphase spindle.
We found that Bir1-TAP copurified with Sli15. Sli15-TAP
copurifies with Bir1 (Cheeseman et al., 2002). Bir1 and Sli15
colocalize throughout the cell cycle. Both Bir1 and Sli15 are
heavily modified in a cdc20-based metaphase arrest (Sulli-
van et al., 2004). However, the modification of Sli15 is re-
moved earlier in the cell cycle than the modification of Bir1.
Sli15 is dephosphorylated at anaphase by the FEAR network
(Pereira and Schiebel, 2003), whereas we find that Bir1 re-
mains modified in a cdc15-2 arrest when the FEAR network
is active but the mitotic exit network is not. Despite their
colocalization throughout the cell cycle, Bir1 and Sli15 are
regulated differently.
We propose that unphosphorylated Sli15 brings phos-
phorylated Bir1 to the spindle at anaphase. Phosphorylated
Bir1, in turn, brings Ndc10 to the spindle and midzone.
Ndc10 remains associated with Bir1 until after spindle de-
polymerization because it has been shown that Ndc10 tracks
plus ends of depolymerizing interpolar microtubules (Bouck
and Bloom, 2005). At or after mitotic exit, Bir1 is dephos-
phorylated, disrupting interaction with Ndc10.
Loss of Ndc10 from the anaphase spindle by mutating the
phosphorylation sites in Bir1 caused both an increase in
chromosome loss and a defect in spindle elongation. How-
ever, we do not see a severe cytokinesis defect as is de-
scribed for the ndc10-1 mutant (Bouck and Bloom, 2005).
Ndc10’s major role in cytokinesis seems to be independent
of its spindle and midzone localization. The inability to fully
elongate the anaphase spindle may increase the possibility
of missegregation of rare lagging chromosomes resulting in
the increase in the rate of chromosome loss we observed in
mutants in the middle region of Bir1.
ACKNOWLEDGMENTS
We are grateful to Eric Muller for critical reading of the manuscript. We thank
Susan Francis and Brian Kennedy for helpful discussions. P.O.W., J.S.L., and
T.N.D. were funded by National Institutes of Health Grant R01 GM-40506.
P.O.W. was funded in part by Public Health Service National Research
Service Award T32 GM-07270 from National Institute of General Medical
Sciences. J.R.Y., S. A., and S. N. were funded by National Institutes of Health
Grant P41 RR-11823
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    • "Interestingly, distinct CPC subcomplexes exist that appear to carry out specific functions. Among the proteins recruited to the midzone in anaphase by CPC components are the kinetochore protein Ndc10 (Cbf2), which binds to Bir1 (Bouck and Bloom 2005; Widlund et al. 2006; Thomas and Kaplan 2007; Rozelle et al. 2011). Ndc10, Bir1, and Sli15 form an alternative CPC that lacks Ipl1 and regulates spindle elongation (Rozelle et al. 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Studies on budding yeast have exposed the highly conserved mechanisms by which duplicated chromosomes are evenly distributed to daughter cells at the metaphase-anaphase transition. The establishment of proteinaceous bridges between sister chromatids, a function provided by a ring-shaped complex known as cohesin, is central to accurate segregation. It is the destruction of this cohesin that triggers the segregation of chromosomes following their proper attachment to microtubules. Since it is irreversible, this process must be tightly controlled and driven to completion. Furthermore, during meiosis, modifications must be put in place to allow the segregation of maternal and paternal chromosomes in the first division for gamete formation. Here, I review the pioneering work from budding yeast that has led to a molecular understanding of the establishment and destruction of cohesion.
    Preview · Article · Jan 2014 · Genetics
    • "The CPC complex is also part of the inner kinetochore, although it is not a core kinetochore complex. Composed of the Ipl1 protein kinase (Aurora B), Sli15 (INCENP), Bir1 (Survivin), and Nbl1 (Borealin) proteins, this complex associates with kinetochores from G1 until anaphase (Widlund et al. 2006; Carmena et al. 2012). The CPC association with the inner kinetochore is mediated via its interaction with CBF3 through Bir1 (Yoon and Carbon 1999; Sandall et al. 2006), and a separate pool may be localized through binding COMA via Sli15 (Knockleby and Vogel 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: The propagation of all organisms depends on the accurate and orderly segregation of chromosomes in mitosis and meiosis. Budding yeast has long served as an outstanding model organism to identify the components and underlying mechanisms that regulate chromosome segregation. This review focuses on the kinetochore, the macromolecular protein complex that assembles on centromeric chromatin and maintains persistent load-bearing attachments to the dynamic tips of spindle microtubules. The kinetochore also serves as a regulatory hub for the spindle checkpoint, ensuring that cell cycle progression is coupled to the achievement of proper microtubule-kinetochore attachments. Progress in understanding the composition and overall architecture of the kinetochore, as well as its properties in making and regulating microtubule attachments and the spindle checkpoint, is discussed.
    Full-text · Article · Aug 2013 · Genetics
    • "Similar to mammalian survivin (also known as Birc5), the budding yeast Bir1 contains a single RING-finger domain. It is cell cycle regulated and localizes to the centromeres until metaphase-anaphase transition but remains in the equatorial zone as the sister chromatids separate (Uren et al., 1998Uren et al., , 2000 Widlund et al., 2006). Anti-apoptotic function has also been reported for survivin and the yeast homolog Bir1. "
    [Show abstract] [Hide abstract] ABSTRACT: Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H(2)O(2) treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.
    Full-text · Article · Aug 2012 · Frontiers in Oncology
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