Download Changes in the Nuclear Envelope Environment Affect

Copyright Ó 2010 by the Genetics Society of America
DOI: 10.1534/genetics.110.119149
Changes in the Nuclear Envelope Environment Affect Spindle Pole Body
Duplication in Saccharomyces cerevisiae
Keren L. Witkin,* Jennifer M. Friederichs,†,‡ Orna Cohen-Fix*,1 and Sue L. Jaspersen†,‡,1
*
Laboratory of Molecular and Cell Biology, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892, †Stowers Institute for Medical Research,
Kansas City, Missouri 64110 and ‡Department of Molecular and Integrative Physiology,
University of Kansas Medical Center, Kansas City, Kansas 66160
Manuscript received May 21, 2010
Accepted for publication August 12, 2010
ABSTRACT
The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also
containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear
pore complexes (NPCs) and the spindle pole body. To study how properties of the nuclear membrane
affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found
that spo7D cells were sickened by the mutation of genes coding for spindle pole body components and
that spo7D was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for
spindle pole body duplication and for a variety of other nuclear envelope processes. In spo7D cells, the
spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane
composition affects spindle pole body function. The synthetic lethality between spo7D and mps3 mutants
was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the
requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication
can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between
nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes
either compete with the spindle pole body for insertion into the nuclear membrane or affect spindle pole
body duplication by altering the nuclear envelope environment.
T
HE nuclear envelope is composed of distinct outer
and inner nuclear membranes. The outer nuclear
membrane is continuous with the endoplasmic reticulum. The inner nuclear membrane is associated
with a unique set of proteins, some of which mediate
interactions between the nuclear envelope and chromatin (reviewed in Zhao et al. 2009). Nuclear pore
complexes traverse both membranes and allow transport of proteins and solutes between the cytoplasm and
the nucleus. The inner and outer nuclear membranes
fuse in the region surrounding each nuclear pore
complex.
In animal cells, the nuclear envelope disassembles as
cells enter mitosis and reassembles upon mitotic exit.
Nuclear envelope breakdown allows the association of
chromosomes with spindle microtubules, which are
nucleated from centrosomes that reside in the cytoplasm. In contrast, certain types of fungi, such as the
Supporting information is available online at http://www.genetics.org/
cgi/content/full/genetics.110.119149/DC1.
1
Corresponding authors: Laboratory of Molecular and Cell Biology,
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, 8 Center Dr., Bldg. 8, Room 319, Bethesda,
MD 20892. E-mail: [email protected]; and Stowers Institute for
Medical Research, Kansas City, MO 64110 and Department of Molecular
and Integrative Physiology, University of Kansas Medical Center, Kansas
City, KS 66160. E-mail: [email protected]
Genetics 186: 867–883 (November 2010)
budding yeast Saccharomyces cerevisiae, undergo closed
mitosis, where the nuclear envelope remains intact
throughout the entire cell cycle. Closed mitosis is
possible because the yeast centrosome-equivalent, the
spindle pole body (SPB), is embedded in the nuclear
envelope, allowing the SPB to nucleate both cytoplasmic
and nuclear microtubules.
SPB duplication requires a mechanism for inserting
the new SPB into the nuclear envelope (reviewed in
Jaspersen and Winey 2004). The new SPB begins to
form in late G1/early S phase as satellite material
deposited on the cytoplasmic face of an electron-dense
region of the nuclear envelope, called the half-bridge.
The satellite material matures into a duplication plaque,
which is then inserted into the nuclear membrane and
becomes the daughter SPB. Many genes are known to be
required for SPB duplication, and this process has been
carefully examined cytologically (Rose and Fink 1987;
Winey et al. 1991, 1993; Spang et al. 1995; Bullitt et al.
1997; Adams and Kilmartin 1999; Elliott et al. 1999;
Schramm et al. 2000; Jaspersen et al. 2002; Nishikawa
et al. 2003; Araki et al. 2006). However, the exact
mechanisms by which SPB duplication and insertion
occur remain a mystery.
Equally unclear is how nuclear pore complexes are
inserted into an intact nuclear envelope (reviewed in
868
K. L. Witkin et al.
Hetzer and Wente 2009). For both the SPB and
nuclear pore complexes, the inner and outer nuclear
membranes must fuse to allow insertion into the nuclear
envelope. Yeast and vertebrate nuclear pore complexes
each have four pore membrane (POM) nucleoporins
containing transmembrane domains. Other nucleoporins have motifs with potential for bending membranes
or sensing membrane curvature. Thus, certain nuclear
pore complex components may have the ability to alter
the nuclear membrane or stabilize particular membrane conformations (Devos et al. 2004, 2006; Alber
et al. 2007; Drin et al. 2007). It is interesting to note that,
in S. cerevisiae, nuclear pore complexes are enriched in
the vicinity of the SPB (Heath et al. 1995; Winey et al.
1997; Adams and Kilmartin 1999), but the significance
of this phenomenon is not known. The SPB and nuclear
pore complexes share at least two common components, the integral membrane protein Ndc1p and the
small calcium-binding protein Cdc31p (Chial et al.
1998; Fischer et al. 2004). Ndc1p is thought to play a
role in insertion of both SPBs and nuclear pore
complexes into the nuclear membrane.
SUN domain proteins are a conserved family of inner
nuclear membrane proteins that interact with specific
outer nuclear membrane proteins to form a physical
bridge across the nuclear envelope (reviewed in Hiraoka
and Dernburg 2009; Razafsky and Hodzic 2009).
One of the components of the S. cerevisiae SPB is the
SUN domain protein Mps3p. The N terminus of Mps3p
is in the nucleoplasm, while the C terminus, containing
the SUN domain, is found in the space between the
inner and outer nuclear membranes. In addition to the
SPB, Mps3p localizes to multiple foci at the nuclear
periphery, and these two pools of Mps3p have distinct
functions ( Jaspersen et al. 2002, 2006; Nishikawa et al.
2003). At the SPB, Mps3p is required for half-bridge
formation and early steps of SPB duplication, and cells
compromised for Mps3p function accumulate in mitosis
with a single SPB and a monopolar spindle (Jaspersen
et al. 2002; Nishikawa et al. 2003). At the nuclear
periphery, Mps3p is involved in tethering telomeres to
the nuclear envelope in mitosis and meiosis, sequestering DNA double-strand breaks away from recombination factors, and associating with soluble chromatin
proteins (Antoniacci et al. 2004, 2007; Bupp et al. 2007;
Conrad et al. 2007, 2008; Oza et al. 2009; Schober et al.
2009).
While many structural features of the yeast nucleus
have been identified, little is known about how the
physical properties of the nuclear membrane contribute to processes that occur at the nuclear envelope. As
noted above, resident proteins of the nuclear envelope
may affect nuclear membrane properties. In addition,
the nuclear membrane is affected by altering lipid
biosynthesis, for example, by inactivating the phosphatidic acid (PA) phosphohydrolase Pah1p or by inactivating the phosphates complex, made of Spo7p and
Nem1p, which activates Pah1p. In the absence of Spo7p,
Nem1p, or Pah1p, cells exhibit nuclear envelope extensions and extensive ER membrane sheets, and they
also have altered membrane lipid composition, including a decrease in phosphatidylcholine and an increase
in PA, phosphatidylethanolamine, and phosphatidylinositol (Siniossoglou et al. 1998; Santos-Rosa et al.
2005; Campbell et al. 2006; Han et al. 2006). These three
proteins are unique among phospholipid biosynthesis
proteins in their ability to affect nuclear morphology
upon gene disruption (Han et al. 2008). A similar
phenotype was seen upon overexpression of DGK1,
which counteracts the activity of Pah1p by converting
diacylglycerol to PA, leading to an increase in PA levels
at the nuclear envelope (Han et al. 2008). Consistent
with a conserved role for Pah1p in regulating nuclear
envelope processes, deletion of either NEM1 or SPO7 is
synthetically lethal with deletions of certain nucleoporin genes (Siniossoglou et al. 1998), and inactivation of the PAH1 homolog in Caenorhabditis elegans,
LPIN-1, results in defects in nuclear envelope disassembly and reassembly (Golden et al. 2009; Gorjanacz and
Mattaj 2009).
To identify processes that are affected by altered
nuclear membrane properties, we screened for pathways that are compromised in spo7D cells. We found that
SPO7 inactivation strongly influences the SPB. By
screening for proteins that could alleviate spo7Dinduced SPB defects, we uncovered an unexpected
inhibitory role for nucleoporins in SPB function, revealing that nuclear pore complexes, or components
thereof, act antagonistically to the SPB in the nuclear
envelope. Taken together, our findings indicate that the
nuclear envelope environment is important for the
function of protein complexes and biological processes
occurring at the nuclear periphery.
MATERIALS AND METHODS
Yeast strains: All yeast strains are derivatives of W303. The
full genotype of each strain is described in Table 1. Strains
used in supporting information are described in Table
S1. The pURA3-MPS3 plasmid has been described previously
( Jaspersen et al. 2006).
Where indicated, mps3 mutant strains were maintained with
pURA3-MPS3 until immediately prior to the experiment. To
select for cells that spontaneously lost the plasmid, strains were
struck on plates containing 5-fluoroorotic acid (5-FOA). Gene
disruptions were performed according to Longtine et al.
(1998) and Goldstein and McCusker (1999). Yeast transformation, sporulation, and tetrad dissection were done using
standard protocols. For strain requests, contact O.C.-F. (for
strains starting with KW, OCF, or JCY) or S.L.J. (for strains
starting with SLJ).
Media and growth conditions: Cells were grown in either
rich media (YPD: 1% yeast extract, 2% peptone, 2% glucose)
or synthetic media (SC: 0.17% yeast nitrogen base, 0.5%
ammonium sulfate, 2% glucose, and amino acid mix lacking
the appropriate amino acid or uracil), as indicated. 5-FOA was
The Nuclear Membrane Affects SPB Duplication
869
TABLE 1
Yeast strains
Strain name
Genotype
JCY565
KW228
KW548
KW576
KW658
KW660
KW661
KW945
KW948
KW949
KW951
KW966
KW967
KW969
KW970
KW971
KW972
KW973
KW983
KW990
KW991
KW992
KW993
KW994
KW995
KW1010
KW1012
KW1013
OCF1533-1A
OCF1533-2C
OCF1533-4B
SLJ001
SLJ715
SLJ717
SLJ751
SLJ839
SLJ843
SLJ910
SLJ2388
SLJ2752
MATa ura3-1 trp1-1 leu2-3,112 spo7DTNAT can1-100
MATa ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 spo7DTNAT mps3-1 can1-100 pURA3-MPS3
MATa leu2-3,112 trp1-1 ura3-1 spo7DTNAT mps3-1 can1-100 pURA3-MPS3
MATa ura3-1 trp1-1 mps3DTHIS3MX6 leu2Tmps3-F592S-LEU2 spo7DTNAT can1-100 pURA3-MPS3
MATa ura3-1 trp1-1 mps3DTHIS3MX6 leu2Tmps3-F592S-LEU2 can1-100 pURA3-MPS3
MATa leu2-3,112 trp1-1 ura3-1 spo7DTNAT can1-100
MATa leu2-3,112 trp1-1 ura3-1 mps3-1 can1-100 pURA3-MPS3
MATa ura3-1 leu2-3,112 trp1-1 mps3-1 nup157DTKAN can1-100 pURA3-MPS3
MATa ura3-1 leu2-3,112 trp1-1 nup157DTKAN can1-100 pURA3-MPS3
MATa ura3-1 leu2-3,112 trp1-1 mps3-1 can1-100 pURA3-MPS3
MATa ura3-1 leu2-3,112 trp1-1 can1-100 pURA3-MPS3
MATa ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 pom152TNAT can1-100
MATa ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 nup157DTNAT can1-100
MATa spc42-11 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 nup157DTKAN bar1ThisG can1-100
MATa mps2-1 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 nup157DTKAN bar1ThisG can1-100
MATa mps2-1 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 pom152TNAT bar1ThisG can1-100
MATa spc29-3 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 nup157DTKAN bar1ThisG can1-100
MATa spc98-2 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 nup157DTKAN bar1ThisG can1-100
MATa spc42-11 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 pom152TNAT bar1ThisG can1-100
MATa ura3-1 leu2-3,112 his3-11,15 mps3DTHIS3MX pom152DTNAT can1-100
MATa ura3-1 leu2-3,112 his3-11,15 pom152DTNAT can1-100
MATa ura3-1 leu2-3,112 his3-11,15 can1-100
MATa ura3-1 leu2-3,112 his3-11,15 lys2D mps3DTHIS3MX nup157DTNAT can1-100
MATa ura3-1 leu2-3,112 his3-11,15 lys2D nup157DTNAT can1-100
MATa ura3-1 leu2-3,112 his3-11,15 lys2D can1-100
MATa kar1D17 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 bar1ThisG nup157DTNAT can1-100 pURA3-KAR1
MATa ura3-1 ade2-1 leu2-3,112 his3-11,15 ndc1DTKAN ndc1-39-TRP1 nup157DTNAT can1-100
MATa ura3-1 ade2-1 leu2-3,112 his3-11,15 ndc1DTKAN ndc1-39-TRP1 can1-100
MATa ura3-1 trp1-1 leu2-3,112 can1-100
MATa leu2-3,112 trp1-1 ura3-1 can1-100
MATa ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 can1-100
MATa bar1ThisG ura3-1 leu2-3,112 trp1-1 his3-11,15 ade2-1 can1-100
MATa spc42-11 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 bar1ThisG can1-100
MATa mps2-1 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 bar1ThisG can1-100
MATa spc29-3 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 bar1ThisG can1-100
MATa spc98-2 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 bar1ThisG can1-100
MATa kar1D17 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 bar1ThisG can1-100 pURA3-KAR1
MATa mps3-1 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 mps3-1 can1-100 pURA3-MPS3
MATa nup60DTHYGMX ura3-1 trp1-1 leu2-3,112 his3-11,15 ade2-1 ade3D can1-100
MATa nup42DTKANMX mps3DTNATMX ura3TMPS3-GFP-URA3 htb2THTB2-mCherry-HYGMX
trp1-1 leu2-3,112 his3-11,15 ade2-1 can1-100
MATa nup188DTKANMX ura3-1 trp1-1 leu2-3,112 his3-11,15 ade2-1 can1-100
MATa mps3-1 pom34DTKANMX ura3-1 trp1-1 leu2-3,112 his3-11,15 ade2-1 can1-100
MATa mps3-1 pom152DTHYGMX ura3-1 trp1-1 leu2-3,112 his3-11,15 ade2-1 can1-100 lys2D
MATa pom34DTKANMX ura3-1 trp1-1 leu2-3,112 his3-11,15 ade2-1 can1-100
MATa pom152DTHYGMX ura3-1 trp1-1 leu2-3,112 his3-11,15 ade2-1 can1-100 lys2D
MATa mps3-1 nup60DTHYGMX ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100
MATa mps3-1 nup42DTKANMX ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100 lys2D
MATa mps3-1 nup188DTKANMX ura3-1 leu2-3,112 his3-11,15 can1-10
MATa mlp2DTHIS5MX mlp1DTKANMX MPS3-HA3-NATMX bar1ThisG ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100
MATa mlp2DTHIS5MX mlp1DTKANMX mps3-1 bar1ThisG ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100 lys2D
MATa nup84DTKANMX MPS3-GFP-NATMX bar1ThisG ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100 lys2D
MATa nup84DTKANMX mps3-1 bar1ThisG ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100 lysD
MATa nup133DTNATMX MPS3-GFP-KANMX bar1ThisG ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100
MATa nup133DTNATMX mps3-1 bar1ThisG ura3-1 trp1-1 leu2-3,112 his3-11,15 can1-100
MATa pom152DTHYGB mps3DTNATMX ade2-1 ade3D100 his3-11,15 leu2-3,112 trp1-1 ura3-1 lys2 can1-100
MATa pom152DTHYGB ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 lys2 can1-100
MATa ade2-1 ade3D100 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100
MATa pom152DTHYGB ade2-1 ade3D100 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100
MATa pom152DTHYGB mps3DTNATMX ade2-1 ade3D100 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100
SLJ3654
SLJ4215
SLJ4216
SLJ4217
SLJ4218
SLJ4221
SLJ4222
SLJ4223
SLJ4233
SLJ4234
SLJ4236
SLJ4237
SLJ4239
SLJ4240
SLJ4259
SLJ4260
SLJ4336
SLJ4338
SLJ4340
870
K. L. Witkin et al.
purchased from US Biologicals (Swampscott, MA). All serial
dilutions were 10-fold. To determine cell cycle distribution of
asynchronous cells, cell cultures in mid-log phase were temperature shifted as indicated and then fixed with 70% ethanol
for 1 hr at room temperature. After sonication and DAPI
staining (Vector Laboratories, Burlingame, CA), cells were
examined using a Nikon Eclipse E800 fluorescent microscope.
All samples were blinded prior to scoring. Cytological methods
used for supporting information material are in File S1.
High-copy suppressor screen: mps3-1 spo7D strains maintained with pURA3-MPS3 were transformed with a LEU2marked 2m library (American Type Culture Collection, Manassas,
VA) and replica-plated to 5-FOA-containing plates. Surviving
cells were retested by streaking on 5-FOA-containing plates.
The suppressing 2m plasmids were isolated and retransformed
into new strains to confirm suppression. The gene responsible
for suppression was then identified by transposon mutagenesis
using the GPS-M mutagenesis system (New England Biolabs,
Ipswich, MA). Briefly, a Tn7-based transposon was integrated
randomly into the plasmid in vitro to disrupt function of
individual genes. For NUP157 and NIC96, suppression by
truncated forms of each gene was then confirmed by PCRamplifying full-length and truncated genes with primers
containing BamHI and SalI restriction sites, followed by
cloning into expression vectors by standard methods of
cloning and ligation. The absence of PCR errors was confirmed by DNA sequencing. Expression vectors were purchased from the American Type Culture Collection.
Flow cytometry: Cells used for flow cytometry in Figure 4
were grown to mid-log phase, fixed with 70% ethanol for 1 hr
at room temperature, treated with RNAse (Roche, Basel,
Switzerland) and proteinase K (Roche), and then sonicated
and resuspended in sytox buffer (180 mm Tris, 180 mm NaCl,
70 mm MgCl2, pH 7.5) containing 1:2500 Sytox Green (Invitrogen, Carlsbad, California). Flow cytometry was then
performed on a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ), using CellQuest Pro software
(BD Biosciences). Cells used for flow cytometry in Figures 5
and 7 were treated as above, except they were grown to late log
phase. After sonication, these cells were resuspended in a
1:1000 dilution of propidium iodide (Sigma-Aldrich, St. Louis),
run on a CyAn ADP Analyzer (Beckman Coulter, Brea, CA) and
analyzed using FlowJo software (Tree Star, Ashland, OR).
Immunofluorescence: Cells were fixed for 45 min to 1 hr in
4% paraformaldehyde and then processed for indirect immunofluorescence microscopy as previously described (Rose et al.
1990 ). A 1:500 dilution of anti-Tub4p ( Jaspersen et al. 2002)
and a 1:50 or 1:500 dilution of the YOL1/34 anti-a-tubulin
(Abcam, Cambridge, MA) antibody were used along with the
appropriate secondary antibodies. DAPI was used to visualize
the DNA.
Transmission electron microscopy: Yeast cells were enriched in G1 using 1 mg/ml a-factor mating pheromone or
left untreated and then frozen on the Leica EM-Pact (Wetzlar,
Germany) at 2050 bar, transferred under liquid nitrogen
into 2% osmium tetroxide/0.1% uranyl acetate/acetone, and
transferred to the Leica AFS. The freeze substitution protocol
was as follows: 90° for 16 hr, raised 4°/hr for 7 hr, 60° for
19 hr, raised 4°/hr for 10 hr, and 20° for 20 hr. Samples were
then removed from the AFS, placed in the refrigerator for 4 hr,
and then allowed to incubate at room temperature for 1 hr.
Samples went through three changes of acetone over 1 hr and
were removed from the planchettes. They were embedded in
acetone/Epon mixtures to final 100% Epon over several days
in a stepwise procedure as described (McDonald 1999). Sixtynanometer serial thin sections were cut on a Leica UC6,
stained with uranyl acetate and Sato’s lead, and imaged on a
FEI Technai Spirit (Hillsboro, OR).
RESULTS
Deletion of SPO7 affects SPB function: Inactivation
of Pah1p, or its activators Nem1p and Spo7p, leads to
alterations in the nuclear and endoplasmic reticulum
membrane (Siniossoglou et al. 1998; Santos-Rosa
et al. 2005; Han et al. 2006). To search for processes that
are affected by changing properties of the nuclear
membrane, we screened for genetic interactions between spo7D and mutations in genes encoding nuclear
envelope proteins. This screen was based on the idea
that if the nuclear envelope environment affects certain
nuclear periphery processes, then altering this environment by inactivating Spo7p might exacerbate the
phenotype of mutants that cause mild defects in these
processes.
A gene of interest was MPS3, needed for SPB
duplication and other processes at the nuclear periphery ( Jaspersen et al. 2002, 2006; Nishikawa et al. 2003;
Bupp et al. 2007; Oza et al. 2009; Schober et al. 2009). To
test whether altering the nuclear membrane affects
Mps3p-related processes, we combined spo7D with mps3-1,
an allele that carries a point mutation in the SUN
domain and is defective in SPB duplication at the
nonpermissive temperature ( Jaspersen et al. 2002).
Even at the permissive temperature (23°), haploid
mps3-1 cells rapidly diploidize ( Jaspersen et al. 2002),
suggesting that the protein expressed from this allele
has reduced activity. Strains carrying mps3-1, spo7D, or
both mutations were generated in the presence of a
centromeric plasmid coding for MPS3 and URA3
(pURA3-MPS3). In this and subsequent experiments,
growth in the absence of the plasmid was tested by
selecting for cells that have lost the plasmid using 5FOA, which is toxic to cells expressing URA3. While
single mutants were viable on synthetic complete
control media (SC) or 5-FOA, double mutants containing both the mps3-1 and spo7D alleles were unable to
grow on 5-FOA at any temperature tested (Figure 1A;
data not shown). These results suggest that the weakened activity provided by the mps3-1 allele is further
compromised in the absence of Spo7p.
While the mutation in mps3-1 is known to affect SPB
duplication, formally it could also disrupt other functions of Mps3p at the nuclear periphery. To determine
which of Mps3p’s roles is compromised by spo7D, we
tested existing mps3 alleles known to differentially
influence Mps3p function. Alleles tested included the
SUN domain mutant mps3-F592S and two N-terminal
deletions, mps3D2-64 and mps3D75-150, which were
previously shown to be proficient in the Mps3p SPB
function but defective in its other roles at the nuclear
envelope ( Jaspersen et al. 2006; Bupp et al. 2007;
Conrad et al. 2007). mps3-F592S spo7D double mutants
could be recovered in the absence of pURA3-MPS3,
indicating that these alleles are not synthetically lethal
under standard growth conditions (Figure 1B). How-
The Nuclear Membrane Affects SPB Duplication
871
Figure 1.—spo7D has genetic interactions with
mps3 alleles specifically defective at SPB duplication.
(A) Wild-type (OCF15332C), spo7D (KW660), mps3-1
cells containing pURA3MPS3 (KW661), and mps3-1
spo7D
cells
containing
pURA3-MPS3 (KW548) were
plated by serial dilution on
control media (SC) or
plates containing 5-FOA
at 23°. The presence of
5-FOA allows only cells
lacking the URA3 gene to
survive. (B) Wild-type
(OCF1533-1A),
spo7D
( JCY565), mps3-F592S cells
containing pURA3-MPS3
(KW658), and mps3-F592S
spo7D cells containing
pURA3-MPS3 (KW576)
were plated by serial dilution on control media
(SC) or plates containing
5-FOA at the indicated
temperatures. (C) Summary of mps3 mutant alleles, their effects on SPB
duplication, and their effects on cell growth in the
absence of Spo7p. The
Mps3p transmembrane domain (TM) and SUN domain are indicated. The
‘‘x’’ marks the approximate
location of point mutations within the SUN domain. 11, 1, and denote a ‘‘strong defect,’’ ‘‘moderate defect,’’ and ‘‘no defect,’’
respectively. Data for SPB defects are from Jaspersen et al.(2002, 2006) and Bupp et al. (2007). Data for genetic interactions with
spo7D are from Figure 1, A and B, and Figure S1.
ever, the combination of these two mutations led to
lethality at elevated temperatures, as the double mutant
failed to grow at 32°, a temperature at which spo7D
cells grow well and mps3-F592S cells are sick but viable
(Figure 1B). In contrast, spo7D showed no genetic
interactions with either mps3D2-64 or mps3D75-150 at
any temperature tested (Figure S1; data not shown).
Thus, the extent of genetic interaction between the
various mps3 alleles and spo7D correlated with the
severity of the SPB defect of that mps3 allele (Figure
1C; Jaspersen et al. 2002, 2006; Bupp et al. 2007),
suggesting that altering nuclear membrane properties
by deleting the SPO7 gene further compromises mps3
mutants by exacerbating their SPB defect.
If the absence of Spo7p leads to a defect that
compromises SPB function, one prediction is that the
spo7D allele would have genetic interactions with mutations in other SPB components. To examine this, we
tested a panel of SPB mutants coding for proteins from
different parts of the SPB structure or required for different stages of SPB duplication (reviewed in Jaspersen
and Winey 2004). Ndc1p and Mps2p are integral
membrane proteins that localize to the periphery of the
SPB, Kar1p is a transmembrane component of the halfbridge, and Spc42p and Spc29p are nonmembrane SPB
proteins that form the central plaque. All five proteins are
encoded by essential genes, and thus genetic interactions
with spo7D were tested using conditional mutants. Temperature-dependent synthetic sickness was observed between spo7D and ndc1-39, kar1D17, spc29-3, and spc42-11
(Table 2; Figure S2). In addition, we observed genetic
interactions between spo7D and mps2-381, which is
thought to disrupt the physical interaction between
Mps2p and Mps3p ( Jaspersen et al. 2006), but not
between spo7D and mps2-1, which instead affects a separate
function of Mps2p in SPB insertion (Winey et al. 1991)
(Table 2; Figure S2; data not shown). Taken together, the
genetic interactions between spo7D and select mutations
in SPB subunits indicate that, under certain conditions,
Spo7p activity is needed for SPB-associated processes. This
suggests that SPB function may be affected by properties
of the nuclear membrane, such as lipid composition.
Deletion of SPO7 exacerbates the SPB defect of
mps3 mutants: The genetic interactions presented
872
K. L. Witkin et al.
TABLE 2
Genetic interactions between spo7D and SPB mutant alleles
SPB mutant allele
mps2-381
mps2-1
kar1D17
ndc1-39
spc29-3
spc42-11
Genetic interaction with spo7Da
1
1
1
1
1
a
Double mutants were generated and scored for growth:
‘‘’’ indicates no growth defect in the double mutant compared to the single mutants at any temperature tested; ‘‘1’’
indicates synthetic sickness evident by a growth defect in
the double mutant compared to the single mutants.
above suggest that deletion of SPO7 adversely affects
the activity of Mps3p in promoting SPB duplication. It
was formally possible that spo7D cells had a SPB
duplication defect of their own that was additive with
the defect of mps3 alleles. However, using several
methodologies at a range of temperatures, we did not
detect any spindle or SPB abnormalities in spo7D cells
(Figure S3; data not shown). Rather, it was likely that
spo7D created a condition that further weakened the
ability of mutated mps3 gene products to promote SPB
duplication.
Failure in SPB duplication results in the accumulation of cells prior to anaphase (large-budded cells with
a single nucleus) because of their inability to assemble a
bipolar spindle. Indeed, at the nonpermissive temperature (37°), 90% of mps3-F592S cells accumulated as
large-budded cells with a single nucleus, and nearly 60%
of these cells had a monopolar spindle ( Jaspersen et al.
2006). To examine the effect of spo7D on an mps3
mutant strain, a semipermissive temperature was necessary at which only a partial defect in SPB duplication
would occur. To this end, we utilized the mps3-F592S
allele because at semipermissive temperatures the
growth defect of the single mps3-F592S mutant was
notably less severe than that of the mps3-F592S spo7D
double mutant (Figure 1B). Wild-type, spo7D, mps3F592S, and mps3-F592S spo7D double-mutant cells were
grown to mid-log phase at 23° and then shifted to 34° for
6 hr. Fixed cells were stained with DAPI and examined
by microscopy to determine the cell cycle distribution
before and after the temperature shift. At 23°, all four
cultures had 30% large-budded cells with a single
nucleus (Figure 2A, light gray bars). The temperature
shift to 34° had no effect on the cell cycle distribution of
wild-type or spo7D cells, but it doubled the percentage of
large-budded, single-nucleated mps3-F592S cells. mps3F592S spo7D double mutants had a small but reproducible and statistically significant increase over mps3-F592S
cells in the fraction of cells that were large budded with
a single nucleus (P , 0.0001) (Figure 2A, dark gray
bars).
This result is consistent with an exacerbation of the
mps3-F592S SPB duplication defect upon SPO7 deletion.
To test this directly, we determined the fraction of
monopolar spindles in large-budded, single-nucleated
mps3-F592S and mps3-F592S spo7D cells. If deleting SPO7
indeed exacerbated the SPB defect of mps3-F592S, then
the fraction of mitotic cells accumulating with monopolar spindles should be greater in the double mutant
than in the mps3-F592S mutant alone. The SPB and
spindle were visualized using antibodies against Tub4p
(the yeast g-tubulin homolog) and a-tubulin, respectively. After a 6 hr temperature shift to the semipermissive temperature, monopolar spindles were evident
in .45% of the large-budded cells in the double
mutant, but in only 15% of the large-budded cells in
the single mps3-F592S mutant (Figure 2, B and C). Thus,
spo7D exacerbated the SPB duplication defect of mps3F592S.
Interestingly, at the semipermissive temperature, the
mps3-F592S mutants with two SPBs fell into three classes.
While many contained a conventional either short or
long bipolar spindle, a sizable population (51%) had
duplicated and separated SPBs, each with independent
microtubule arrays, but only a single, unsegregated
DNA mass associated with only one of the SPBs (Figure
2D). This phenotype was also observed in mps3-1 cells
grown at the nonpermissive temperature (data not
shown). In the majority of these cells, nuclear microtubules were limited to the SPB associated with the DNA
mass. The second microtubule array was often smaller
and was found outside the nuclear envelope upon
staining with mAB414 antibodies that recognize nuclear
pore complex antigens (Aris and Blobel 1989; data not
shown). While this phenotype has never been reported
for mps3 mutants, it is common among SPB mutants that
fail to properly insert the newly made SPB into the
nuclear envelope (Winey et al. 1991, 1993; Schramm
et al. 2000; Araki et al. 2006). This suggests that Mps3p
may also be involved in late stages of SPB duplication,
such as SPB insertion.
A high-copy suppressor screen reveals a link between
nucleoporins and Mps3p function: To uncover the
biological basis for the genetic interaction between
spo7D and mps3 mutants, we carried out a high-copy
plasmid suppressor screen, aiming to unveil proteins
that upon overexpression could suppress the lethality of
spo7D mps3 double mutants. As expected, the screen
recovered high-copy plasmids containing SPO7, MPS3,
and PAH1 as strong suppressors (data not shown). We
also recovered two additional suppressor plasmids,
called clones 62 and 146, which encoded truncated
forms of the large nucleoporins Nup157p and Nic96p,
respectively. Both proteins are thought to play structural
roles in the nuclear pore complex (Aitchison et al.
1995b; Zabel et al. 1996; Kosova et al. 2000). Interestingly, both suppressor clones were missing the 39 ends of
their respective nucleoporin genes. Clone 62 coded for
The Nuclear Membrane Affects SPB Duplication
873
Figure 2.—The SPB defect of
mps3 mutants is exacerbated in
the absence of Spo7p. (A) Wildtype (OCF1533-1A), spo7D
( JCY565), mps3-F592S (KW658),
and mps3-F592S spo7D cells
(KW576) were grown to mid-log
phase at 23° and then shifted to
the indicated temperature for
6 hr. The percentage of cells just
prior to anaphase (large-budded
cells with a single nucleus) is
graphed. Strains containing the
mps3-F592S allele were maintained with the pURA3-MPS3
plasmid, which was removed using 5-FOA immediately prior to
the experiment (see materials
and methods). A total of 300
cells (wild type and spo7D) or
500 cells (mps3-F592S and
mps3-F592S spo7D) were counted
over three or five independent
experiments. Error bars indicate
standard deviation. (B) mps3F592S (KW658; n ¼ 183 over
three independent experiments)
and mps3-F592S spo7D cells
(KW576; n ¼ 218 over three independent experiments) were treated as in A and then subjected to indirect immunofluorescence
using antibodies against Tub4p to visualize SPBs and against a-tubulin to visualize microtubules. The percentage of large-budded
cells containing monopolar spindles is graphed. Error bars indicate standard deviation. The asterisk denotes statistical significance by Pearson’s x2 test (P-value , 0.0001). (C and D) Representative examples of large-budded cells stained with antibodies
to Tub4p (SPB) or a-tubulin (spindle) and treated with DAPI. In the merged image, the SPB is shown in green and the microtubules in red. (C) Example of a large-budded cell with a monopolar spindle. (D) Example of a large-budded cell with duplicated,
microtubule-associated SPBs in both mother and daughter cells, but only a single DAPI mass.
the first 1204 amino acids of Nup157p (of 1391 amino
acids), whereas clone 146 coded for the first 670 amino
acids of Nic96p (of 839 amino acids). We named these
suppressor alleles NUP157-T and NIC96-T, respectively.
To compare full-length and truncated forms of each
gene without contribution from overlapping open
reading frames on the original plasmids, full-length
and truncated forms of each gene were subcloned into a
centromere-based plasmid behind the TEF promoter.
In this context, NUP157-T suppressed as well as the
original suppressor plasmid (clone 62), and NIC96-T
suppressed better than clone 146 (Figure 3). In contrast, full-length forms of either gene had no suppressor
activity (Figure 3). Further experimentation revealed
that each truncated gene rescued the temperature
sensitivity of the mps3-1 mutant but not the phenotypes
associated with SPO7 deletion (Figure S4 and Figure
S5). These results suggest that both NUP157-T and
NIC96-T suppress synthetic lethality between mps3-1
and spo7D by fixing a defect associated with mps3-1.
Deletion of NUP157 rescues the SPB defect of the
mps3-1 allele: Because only truncated forms of NUP157
and NIC96 suppressed the temperature sensitivity of
mps3-1, we speculated that the truncated genes might
act as antimorphic alleles. For example, a nonfunctional
truncated nucleoporin still might replace the full-
length protein in the nuclear pore complex, effectively
causing a loss-of-function phenotype. If this were the
case, then suppressor activity might be phenocopied by
the complete deletion of these genes. Because NIC96 is
an essential gene (and thus cannot be deleted), but
NUP157 is not, we focused on the deletion of NUP157.
mps3-1 and mps3-1 nup157D strains, each containing an
MPS3 plasmid, were assayed for the ability to grow
without the plasmid at 37°. While strains containing the
mps3-1 mutation alone were unable to grow without the
plasmid at 37°, deletion of NUP157 restored viability
under these conditions (Figure 4A). Thus, NUP157-T,
and most likely NIC96-T, act as suppressors by causing
loss of Nup157p and Nic96p function, respectively.
Because the essential function of Mps3p is its role in
SPB duplication, we investigated whether NUP157 deletion improved SPB function in mps3-1 cells. As a
consequence of SPB malfunction, haploid mps3-1 cells
become diploid even at the permissive temperature
immediately following loss of an MPS3-containing
plasmid or immediately upon germination after meiosis
( Jaspersen et al. 2002). To determine whether nup157D
suppresses the diploidization phenotype of mps3-1 mutants, we analyzed the ploidy of wild-type, mps3-1, nup157D,
and mps3-1 nup157D strains. Each strain was initially
haploid due to the presence of a wild-type copy of MPS3
874
K. L. Witkin et al.
Figure 3.—Identification of high-copy suppressors of synthetic lethality in spo7D mps3 mutants. spo7D mps3-1 cells maintained with the
pURA3-MPS3 plasmid (KW228) were transformed with the indicated suppressor plasmids
(clones 62 and 146), an empty vector, and constructs containing full-length (-FL) or truncated
forms (-T ) of NUP157 and NIC96 cloned behind
the TEF promoter. Each construct was tested by
serial dilution for the ability to restore growth
at 23° on plates containing 5-FOA or control
(SC) media.
carried on the pURA3-MPS3 plasmid. After selection for
cells that have lost the plasmid, independent cultures
were grown to mid-log phase and examined by flow
cytometry to determine their DNA content. As expected,
mps3-1 mutant strains always had double the DNA
content compared to a wild-type strain (n ¼ 20; for a
representative example, see Figure 4B). Strikingly, we
found that the DNA content of mps3-1 nup157D double
mutants (n ¼ 20) was similar to wild-type cells, containing
peaks representing 1N and 2N DNA content (Figure 4B).
Thus, deleting NUP157 suppressed the diploidization
phenotype of mps3-1 mutants, consistent with improved
SPB function.
We also found that the mitotic delay of mps3-1 cells,
caused by the inability to duplicate SPBs, was completely
eliminated by deletion of NUP157 (Figure 4C). To
confirm that this was due to restoration of SPB duplication, we used immunofluorescence to examine SPB and
spindle morphology in large-budded cells of each
genotype, as described in Figure 2B. While wild-type
and nup157D cells had only a very small proportion of
large-budded cells with monopolar spindles, mps3-1
mutants exhibited 45% monopolar spindles (Figure
4D). Deletion of NUP157 in mps3-1 cells reduced this
number to wild-type levels (Figure 4D). Taken together,
these results suggest that the SPB defects of mps3-1 cells
are rescued by NUP157 deletion.
Specific nucleoporin deletions suppress the growth
defects of mps3 mutants: It was previously reported that
deletion of genes coding for the POM nucleoporins
Pom152p or Pom34p can rescue defects seen in certain
mutants that disrupt SPB insertion, including ndc1,
bbp1, and mps2 mutants (Chial et al. 1998; Sezen et al.
2009). To test whether POM gene deletions could also
relieve defects associated with MPS3 mutation, POM152
and POM34 were individually deleted in haploid strains
containing either wild-type MPS3 or mps3-1 alleles.
Strains were then assayed for growth on rich media at
23° and 37°. Deletion of either POM gene restored
growth at 37° to mps3-1 mutants and also rescued the
genome diploidization phenotype (Figure 5). Thus, loss
of POM nucleoporins is at least as effective as NUP157
deletion in alleviating the SPB defect of mps3 mutants
(also see below).
To expand on the relationship between MPS3 and
nucleoporin genes, we tested a panel of additional
nucleoporin deletions to determine their effectiveness
at rescuing mps3-1 phenotypes. Double mutants containing mps3-1 and individual deletion alleles of several
nonessential nucleoporin genes were created and analyzed for ploidy and temperature sensitivity. Of the
additional genes tested, only nup42D both rescued the
diploidization phenotype and restored growth at elevated temperatures to the mps3-1 mutant (Table 3;
Figure 5). This was somewhat surprising as Nup42p is
a peripheral nucleoporin, rather than a structural
nucleoporin like the other suppressors identified to
this point (Strahm et al. 1999). nup188D acted as a
partial suppressor, rescuing temperature sensitivity but
not the diploidization phenotype, as did simultaneous
deletion of MLP1 and MLP2 (Table 3; Figure 5). All
other nucleoporin deletions tested, including nup84D,
nup60D, nup133D, and mlp1D, and mlp2D individually,
failed to suppress either temperature sensitivity or
diploidization of mps3-1 (Table 3). Thus, while suppression of the SPB defect of mps3-1 cells is not an exclusive
The Nuclear Membrane Affects SPB Duplication
875
Figure
4.—Deletion
of
NUP157 rescues the SPB defect
of mps3-1 cells. (A) Wild-type
(KW951), nup157D (KW948),
mps3-1 (KW949), and nup157
mps3-1 cells (KW945), all initially
maintained with the pURA3MPS3 plasmid, were assayed for
growth at 30° and 37° by serial
dilution on control (SC) or 5FOA-containing
media.
(B)
DNA analysis by flow cytometry
of cells described above and treated with 5-FOA to select for cells
that have lost the pURA3-MPS3
plasmid and then grown to midlog phase at 30° and fixed with
ethanol. In each case, 20 individual cultures of cells with the indicated genotypes were tested, and
representative examples are
shown. (C) Cells described above
were treated with 5-FOA to select
for cells that had lost the pURA3MPS3 plasmid, were grown to
mid-log phase, and were then
shifted to the indicated temperature for 3 hr. The percentage of
total cells arrested just prior to
anaphase as large-budded cells
with a single nucleus is graphed.
Light gray: growth at 30°. Dark
gray: following a 3-hr shift to
37°. For each sample, 400 cells
were counted over four independent experiments. Error bars indicate standard deviation. (D) Cells treated as in C were stained with antibodies to the SPB and
microtubules as described in Figure 2. The graph illustrates the percentage of large-budded cells that contain monopolar spindles.
For each sample, .100 cells were counted in each of two independent experiments. Error bars indicate standard deviation. The
asterisk denotes statistical significance by Pierson’s x2 test (P-value , 0.0001).
property of nup157D, it is specific to particular nucleoporin deletions. Moreover, while there is considerable
overlap between the nucleoporin deletions that suppress mps2D and mps3-1, the spectrum of suppressing
mutants is not identical: nup188D could partially suppress mps3-1 but not mps2D (Sezen et al. 2009),
and nup157D suppressed mps3-1 but not mps2-1 (see
below).
nup157D and pom152D rescue distinct SPB mutants:
The results above establish a genetic relationship
between MPS3 and genes encoding subunits of the
nuclear pore complex. To extend this analysis, we
examined the spectrum of SPB mutants that could be
suppressed by nucleoporin deletions, initially focusing
on nup157D. NUP157 was deleted in cells containing
one of several conditional mutant alleles that affect
different structural parts of the SPB or different stages
of SPB duplication (mps2-1, spc42-11, spc98-2, kar1D17,
ndc1-39, and spc29-3). Growth of single SPB mutants and
double mutants with nup157D was compared at permissive and restrictive temperatures. The temperature
sensitivity of the spc42-11 allele was partially suppressed
by the NUP157 deletion, which was evident by slight
growth at 37° (Figure 6A). In contrast, nup157D did not
rescue the temperature sensitivity of spc29-3, spc98-2,
kar1D17, ndc1-39, or mps2-1 mutants (Figure 6A). This
suggests that, while suppression by nup157D is not specific
to mps3 mutants, it is limited to a small subset of SPB
genes that likely collaborate in a common function.
Since nup157D, pom152D, and pom34D equivalently
suppress the temperature sensitivity of mps3-1 (Figure
5), we asked whether deletion of POM genes would also
rescue temperature sensitivity of the spc42-11 allele. To
test this, POM152 and POM34 were individually deleted
in haploid strains containing spc42-11 or mps2-1 and
assayed for growth on rich media at 23° and 37°. The
mps2-1 allele was used as a control because POM deletions are known to rescue this mutant (Sezen et al.
2009). Neither POM gene deletion rescued the temperature sensitivity of the spc42-11 allele (Figure 6B; data
not shown). Furthermore, while deletion of either
POM152 or POM34 rescued the mps2-1 mutation (Figure
6B; data not shown), the NUP157 deletion did not (Figure
6A). Taken together, these data indicate that nucleoporin
gene deletions have differential effects on the requirement for individual SPB subunits.
876
K. L. Witkin et al.
Figure 5.—Specific nucleoporin gene deletions rescue mps3-1 phenotypes. Strains of the indicated genotypes were plated by serial dilution
at 23° and 37°. Samples of each strain were grown
at 23°, fixed with ethanol, and analyzed by flow
cytometry (right); 1N, 2N, and 4N DNA peaks
are indicated below. The strains used were SLJ001,
SLJ910, SLJ4217, SLJ4215, SLJ4218, SLJ4216,
SLJ4239, SLJ4240, SLJ4236, SLJ4237, SLJ2752,
SLJ4222, SLJ2388, SLJ4221, SLJ3654, SLJ4223,
SLJ4233, and SLJ4234.
Deletion of NUP157 or POM152 renders MPS3
nonessential: Loss of Nup157p or POM nucleoporins
could affect Mps3p directly, for example, by changing
the localization, stability, or conformation of the mps3-1
protein product; or it could act more generally to
modulate SPB function such that Mps3p function is
no longer required. To distinguish between these two
possibilities, we made diploid strains heterozygous for
mps3D and for either nup157D or pom152D. After meiosis
and sporulation, progeny were inspected for segregation of the mps3D allele. As expected, no mps3D cells
were recovered in otherwise wild-type backgrounds.
Strikingly, we found that mps3D nup157D double mutants were viable, although compromised for growth
compared to wild type and nup157D controls (Figure
7A).Furthermore, mps3D pom152D progeny were not
only viable, but also exhibited robust growth similar to
wild type and pom152D controls (Table 4; Figure 7B).
Asynchronous mps3D pom152D cells had a cell cycle
distribution similar to wild type (Figure 7C) and a
haploid DNA profile by flow cytometry (Figure 7D).
Thus, in the absence of either Nup157p or Pom152p,
MPS3 is no longer an essential gene.
If deletion of other nucleoporin genes suppressed
mps3-1 by a common mechanism shared with nup157D
and pom152D, then they also might be expected to
suppress the mps3D allele. Interestingly, despite their
ability to rescue the mps3-1 allele (Figure 5), neither
nup42D nor nup188D were able to restore growth to
mps3D (data not shown). Thus, while different nucleoporin deletions can rescue SPB mutations, our results
suggest that they do so in distinct ways. Those that
rescue the mps3D allele alleviate the need for any Mps3p
protein product, suggesting that under these conditions
the process of SPB duplication occurs by a noncanonical
pathway, completely independently of Mps3p. In contrast, those that can suppress mps3-1 but not mps3D may
rescue the SPB defect by affecting the compromised
Mps3p protein so that SPB duplication can still occur via
the canonical pathway (i.e., Mps3-dependent), or they
may activate the noncanonical pathway but to insufficient levels.
mps3D pom152D cells have intact spindles and SPBs:
How do the nucleoporin deletion mutations bypass the
requirement for Mps3p? One possibility is that these
bypass suppressors act by alleviating any need for the
TABLE 3
Suppression of mps3-1 phenotypes by nucleoporin
gene deletions
Full suppressiona
nup157D
pom152D
pom34D
nup42D
Incomplete
suppressionb
nup188D
mlp1D mlp2D
No suppressionc
nup84D
nup60D
nup133D
mlp1D
mlp2D
a
Suppression of both temperature sensitivity and ploidy
phenotypes.
b
Suppression of temperature sensitivity but not ploidy phenotypes.
c
No suppression of either temperature sensitivity or ploidy
phenotypes.
The Nuclear Membrane Affects SPB Duplication
Figure 6.—The effects of nup157D and pom152D on various
SPB mutant alleles. Strains of the indicated genotypes were
plated on rich media (YPD) by serial dilution at 23° and
37°. The following strains were used: (A) OCF1533-4B,
KW967, SLJ715, KW969, SLJ839, KW973, SLJ751, KW972,
SLJ717, KW970, SLJ843, KW1010, KW1013, and KW1012.
For SLJ843 and KW1010, 5-FOA was used immediately prior
to the experiment to select for cells that have lost the
pURA3-KAR1 plasmid. (B) The following strains were used:
KW966, KW983, and KW971.
SPB, perhaps by allowing formation of acentrosomal
spindles. While acentrosomal spindles have never been
reported in S. cerevisiae, they are found in oocytes,
plants, and some mutant animals lacking centrosomes
(Walczak and Heald 2008). If this were the mechanism of suppression, then nup157D and pom152D would
be expected to also restore viability to deletion alleles of
other essential SPB genes. To test this, double mutants
were made containing both pom152D and individual
SPB deletions covered by a wild-type copy of the SPB
877
gene on a URA3-marked plasmid and then tested for
growth on plates containing 5-FOA. While POM152
deletion suppressed the mps2D allele, as reported previously (Sezen et al. 2009), it did not restore growth to
kar1D, spc98D, nbp1D, spc42D, nud1D, mps1D, or ndc1D
(Table 4; Figure S6; data not shown). This suggests that
pom152D cells still require an intact SPB for spindle
formation; however, this SPB requires neither Mps3p
nor Mps2p.
To examine the SPB and spindle in mps3D pom152D
cells directly, we performed immunofluoresence analysis of asynchronous cells using antibodies against Tub4p
and a-tubulin to visualize SPBs and microtubules,
respectively. This analysis revealed that mps3D pom152D
mutants form bipolar spindles (Figure 7E). Quantitative
measurement of spindle length revealed that average
length and range of spindle lengths in mps3D pom152D
cells were similar to those of wild-type and pom152D cells
(Figure S7). However, compared to the controls, mps3D
pom152D cells had a paucity of cells containing intermediate length spindles (Figure S7). This suggests
that, while mps3D pom152D cells are capable of SPB
duplication, bipolar spindle formation, and spindle
elongation, their spindles are not identical to those of
wild-type cells.
To examine the structure of the SPB in cells lacking
Mps3p, pom152D and pom152D mps3D cells were examined by electron microscopy (EM). Complete serial
sections through the nuclei of 10 pom152D and 25
pom152D mps3D mutants demonstrated that the overall
architecture of the SPB, including its size, laminar
structure, microtubule-organizing ability, and location
in the nuclear envelope, was very similar between the
two strains, with two notable exceptions (Figure 8).
First, the density of the bridge or half-bridge was
decreased in pom152D mps3D compared to pom152D.
This was particularly notable on the nuclear side of the
half-bridge (Figure 8, E–G); in some cases, the cytoplasmic region lost its association with the nuclear envelope
(Figure 8D). Despite this difference, pom152D mps3D
mutants still appear to undergo SPB duplication in
much the same way as wild-type cells. We were able
to observe all of the known intermediates in the canonical SPB duplication pathway in cells lacking Mps3,
including a satellite (Figure 8E) and duplication-plaque
structure (Figure 8F) at the distal tip of the half-bridge
as well as duplicated side-by-side SPBs connected by
a complete bridge (Figure 8G). Thus, the Mps3pindependent mechanism of SPB duplication is largely
similar to the conventional Mps3p-dependent pathway
at least at the cytological level. The second major
difference that we frequently observed was that
pom152D mps3D SPBs, but not pom152D SPBs, were
often present on nuclear invaginations (8 of 25 SPBs;
Figure 8, B and C) even though the overall shape of
nuclei was round. Given that microtubule nucleation
from the inner and outer plaque and the half-bridge
878
K. L. Witkin et al.
Figure 7.—mps3D strains are viable in the absence of Nup157p or Pom152p. (A and B)
Strains of the indicated genotypes were plated
by serial dilution at 30°. The following strains
were included: (A) KW995 (wild type), KW994
(nup157D), and KW993 (mps3D nup157D). (B)
KW992 (wild type), KW991 (pom152D), and
KW990 (mps3D pom152D). (C) Asynchronous
cells of the strains described in B were grown
to mid-log phase, fixed with ethanol, and then
examined for cell cycle distribution. Differences
between wild type and the mps3D pom152D double
mutant are not statistically significant (P . 0.05;
for each sample, n ¼ 300 over three independent
experiments). Error bars indicate standard deviation. (D) Wild-type cells (SLJ4336), pom152D cells
(SLJ4338), and the mps3D pom152D double mutants (SLJ4340) were grown to mid-log phase,
fixed with ethanol, and analyzed by flow cytometry. (E) The same strains were grown to mid-log
phase and processed for indirect immunofluorescence as described in Figure 2. Images show microtubules (green: anti-a-tubulin), SPBs (red:
anti-Tub4p), and DNA (blue: DAPI). Arrows indicate short bipolar spindles, and arrowheads mark
long bipolar spindles.
appears normal in pom152D mps3D mutants and there
are no obvious changes in nuclear morphology, we
assume that this invagination does not interfere with
SPB function. We suspect that it may be due to a
nonessential role of Mps3p in chromosome segregation
or perhaps in affecting the rigidity of the nuclear
membrane surrounding the SPB. Whatever the defect,
pom152D mps3D mutants are able to form bipolar
spindles (Figure 8H), indicating that under certain
conditions, Mps3p is not essential for SPB duplication
and spindle formation.
DISCUSSION
The function of Mps3p in SPB duplication is
sensitive to the nuclear envelope environment: In this
study, we aimed to identify nuclear structures or processes that are affected by alteration of nuclear membrane properties. We found that the function of Mps3p
was greatly affected by inactivation of Spo7p (Figure 1, A
and B). Mps3p plays dual roles at the nuclear envelope,
acting in both SPB duplication and as a molecular tether
that physically links nucleoplasmic factors (such as
telomeres) to the nuclear periphery. Three lines of
evidence suggest that the synthetic lethality between
mps3-1 and spo7D occurs because Spo7p affects the
function of Mps3p at the SPB, rather than at the nuclear
periphery. First, the genetic interactions between spo7D
and mps3 are seen with mps3 alleles that are defective in
SPB duplication, and not with mps3 alleles that are
defective in telomere tethering (Figure 1). Second,
spo7D has genetic interactions with mutant alleles of
other SPB genes (Table 2; Figure S2). Finally, we saw an
exacerbation of the SPB duplication defect in mps3F592S mutants upon SPO7 deletion (Figure 2). Work
from the Rose lab has identified genetic interactions
between kar1D17 and deletion of the gene coding for
Nem1p, Spo7p’s partner in the phosphatase complex
(Khalfan 2001). Like Mps3p, Kar1p is a transmembrane component of the SPB, and both have been
implicated in Cdc31p recruitment early in the SPB
duplication pathway. Expression of the dominant
CDC31-16 allele rescued both temperature sensitivity
of kar1D17 and its synthetic lethality with nem1D,
suggesting that the genetic interaction between kar1D17
and nem1D stems from an SPB defect (Khalfan 2001),
consistent with our findings here. Taken together, these
data suggest that the Spo7p/Nem1p complex, possibly
through its role in determining membrane composition, affects SPB processes in general and Mps3p
function in particular.
How could spo7D affect SPB function? The only
known function of Spo7p is to regulate lipid biosynthe-
The Nuclear Membrane Affects SPB Duplication
TABLE 4
Suppression of SPB deletions with pom152D
SPB deletion
mps3D
mps2D
mps1D
nbp1D
ndc1D
kar1D
nud1D
spc42D
spc98D
Suppression by pom152Da
1
1
a
Double mutants were generated in the presence of a
URA3-marked plasmid containing a wild-type copyof the
SPB gene and scored for growth on 5-FOA: ‘‘1’’ indicates robust growth similar to wild type strains; ‘‘’’ indicates no
growth on 5-FOA.
sis through Pah1p, and inactivation of Pah1 leads to
alteration in membrane composition. Thus, our data
are consistent with the possibility that Mps3p is sensitive
to changes in its membrane environment that occur
upon SPO7 deletion. This is further supported by our
observation that the genetic interaction between spo7D
and mps3-1 can be suppressed by altering another
nuclear envelope component, namely the nuclear pore
complex (Figure 3). Both the SPB and the nuclear pore
complexes are large structures embedded in the inner
and outer nuclear membranes. Data from several
groups suggest that physical properties of the nuclear
membrane can affect the process of inserting nuclear
pore complexes. For example, specific alleles of the
ACC1 gene, affecting the limiting step in very-long-chain
fatty acid synthesis, alter the structure of the nuclear
envelope and inhibit nuclear pore complex assembly
(Schneiter et al. 1996). Nuclear pore complex abnormalities are also seen upon deletion of the APQ12 gene,
and these defects are remedied upon treatment with
benzyl alcohol, which increases membrane fluidity
(Scarcelli et al. 2007). More recently, it was shown
that brr6-1 mutants with an abnormal accumulation of
certain lipids also display defects in nuclear pore
complex assembly (Hodge et al. 2010). Moreover,
affecting membrane fluidity or rigidity by benzyl alcohol or palmitic acid, respectively, interferes with the
normal distribution of the nucleoporin Gle2p (Izawa
et al. 2004; Scarcelli et al. 2007). Finally, reticulon
proteins required for membrane curvature have been
implicated in NPC biogenesis in yeast and vertebrate
cells (Dawson et al. 2009). Interestingly, spo7D cells also
display enhanced sensitivity to mutation of nucleoporin
genes (Siniossoglou et al. 1998). In light of these
results, we interpret the genetic interactions between
spo7D and SPB genes to indicate that changes in the
nuclear membrane composition affect SPB duplication and insertion into the nuclear envelope.
879
SPB duplication defects in mps3 cells are suppressed
by altering nuclear pore complex composition: A highcopy suppressor screen revealed that deletion or mutation of certain nucleoporin genes suppresses the SPB
duplication defect of mps3-1 (Figures 3–5). This raises
the possibility that nuclear pore complexes may cause
an additional type of nuclear envelope alteration that
affects SPB function. Several of the nucleoporin genes
that upon deletion suppress mps3 mutants code for
proteins implicated in nuclear pore complex insertion
or assembly, including Nup157p, Nic96p, Nup188p,
Pom152p, and Pom34p (Nehrbass et al. 1996; Zabel
et al. 1996; Gomez-Ospina et al. 2000; Madrid et al. 2006;
Miao et al. 2006; Dawson et al. 2009; Flemming et al.
2009; Makio et al. 2009; Onischenko et al. 2009). Since
many of these genes are not essential for viability, while
nuclear pore complexes are essential, these gene
deletions are unlikely to suppress the mps3-1 defect by
eliminating nuclear pore complexes or drastically reducing their number. Indeed, many of these genes
interact with other partially redundant nucleoporins
also involved in nuclear pore complex insertion and
biogenesis (e.g., NUP157 and NUP170). Furthermore,
other labs have documented no obvious change in the
steady-state number of nuclear pore complexes in the
absence of either Nup157p or Pom152p (Madrid et al.
2006; Makio et al. 2009).
Thus, we propose two possible models for how
nucleoporin gene deletions alter the nuclear pore
complex to allow compromised SPBs to more easily
duplicate. First, altered nuclear pore complexes lacking
specific nucleoporins may affect the physical properties
of the nuclear membrane in a way that creates a
favorable condition for SPB duplication, allowing a
compromised SPB duplication machinery to function
more effectively. Consistent with this possibility is the
observation that the strongest suppression of SPB
mutants is mediated by deletion of the POM nucleoporins, which physically contact the nuclear membrane and
are thus good candidates for proteins that can affect
membrane properties. However, not all nucleoporins
with predicted membrane-interaction domains are capable of suppression; for example, nup133D has no
suppressor activity, despite encoding a protein with
an ALPS domain (Drin et al. 2007). Conversely, not
all suppressing nucleoporin deletions are membraneassociated proteins. For example, Nup42p has not been
shown to be involved in either NPC assembly or insertion, and yet nup42D is a strong suppressor of mps3-1.
It is, however, possible that deletion of NUP42 may
influence NPC structure such that its interactions with
the surrounding nuclear membrane are altered.
Several groups have reported altered nuclear membrane structure upon deletion or mutation of certain
nuclear pore complex components. For example, mutants compromised for NUP116 (Wente and Blobel
1993); NUP145 (Wente and Blobel 1994), NUP120
880
K. L. Witkin et al.
Figure 8.—Electron microscopy analysis of
SPBs in cells lacking Mps3p. (A) EM image from
pom152D cells (SLJ4260) of duplicated side-byside SPBs connected by a bridge. Asterisk indicates vesicle structures in close proximity to the
SPB. (B–H) EM images from pom152D mps3D mutant cells (SLJ4259). (B and C) Low magnification (B) and high magnification (C) image of
SPB present on a nuclear invagination. An arrowhead points to electron-dense material resembling a satellite, and an arrow marks a
cytoplasmic microtubule emanating from the
half-bridge region of the SPB. (D) An unduplicated SPB with a half-bridge that appears to have
lost association with the nuclear envelope (arrowhead). (E and F) SPBs that contain electron-dense
material resembling a satellite (E, arrowhead) or
duplication plaque (F, arrowhead) at the distal
tip of the half-bridge. (G) Duplicated side-by-side
SPBs connected by a bridge. Asterisk indicates
vesicle structures located in close proximity to
the SPB. (H) Serial section images showing a bipolar spindle. The first SPB is apparent in H (left)
while the second SPB is apparent in H (right).
Bars, 100 nm.
(Aitchison et al. 1995a; Heath et al. 1995), NUP1
(Bogerd et al. 1994; DeHoratius and Silver 1996),
NUP133 (Pemberton et al. 1995), NUP85 (Goldstein
et al. 1996), NUP84 (Siniossoglou et al. 1996), or the
combination of NUP170 and POM152 (Aitchison et al.
1995b) all have nuclear envelope structural abnormalities. It is possible that these previously described
structural changes are related to those that facilitate
SPB duplication in the absence of certain nucleoporins.
An alternative hypothesis for the mechanism of
suppression is that the SPB and nuclear pore complexes
compete for a limiting factor and that the SPB relies
even more heavily on this putative factor when certain
SPB proteins are weakened by mutation. Elimination of
certain nucleoporins may reduce the affinity of the
nuclear pore complex for this putative factor, thus
favoring its association with the SPB instead. Indeed,
the two complexes do have several features in common.
First, several reports indicate that nuclear pore complexes may be preferentially inserted into the nuclear
envelope in the vicinity of the SPB (Heath et al. 1995;
Winey et al. 1997; Adams and Kilmartin 1999). Second,
they share common components: Ndc1p is a functional
part of both complexes, and Cdc31p and Mlp2p have
each been reported to co-purify with both SPB and
nuclear pore complex components (Chial et al. 1998;
Fischer et al. 2004; Niepel et al. 2005). There may also
be other shared components that have yet to be discovered. These shared features between the SPB and
nuclear pore complexes raise the intriguing possibility
that the two complexes may be inserted by common
mechanisms or may rely on common membraneremodeling machinery.
Competition for a limiting insertion factor is also
consistent with the tendency of SPB mutants to diploidize. The propensity of SPB mutants to diploidize
might be due to cell cycle progression in the absence of
SPB duplication. But why do these cells remain as stable
diploids rather than continue to increase in ploidy?
When cells diploidize, not only their genetic content but
also their cell and nuclear volumes increase 2-fold,
possibly reflecting a doubling in protein synthesis
capacity (Galitski et al. 1999; Jorgensen et al. 2007;
Neumann and Nurse 2007). However, the surface area
of the nucleus increases by only 1.6-fold. Thus, the
relative concentration of this putative limiting factor in
the nuclear membrane may be higher in diploids than
in haploids, allowing cells with compromised SPBs to
survive only as diploid cells.
An alternative SPB duplication pathway in cells lacking both SPB components and specific nucleoporins:
Remarkably, pom152D and nup157D were able to suppress the lethality associated with mps3D. Prior to this
study, Mps3p was thought to be essential for SPB
duplication. This observation is reminiscent of the
finding that certain mutations in nuclear pore complex
genes, including pom152D but not nup157D, can suppress the lethality associated with the deletion of several
SPB genes, including mps2D, which are required for
insertion of the daughter SPB into the nuclear mem-
The Nuclear Membrane Affects SPB Duplication
brane following SPB duplication. Our findings suggest
that SPB duplication can occur through a noncanonical
pathway that does not involve Mps3p and that this
pathway resembles Mps3p-dependent SPB duplication
in that the same structural intermediates were observed.
Interestingly, the essential function of the other integral
membrane component of the half-bridge, Kar1p, in SPB
duplication can also be bypassed, not by deletion of the
same nucleoporins that rescue mps3D, but by a dominant allele of CDC31 or by mutation of the small
ubiquitin-like gene DSK2 (Vallen et al. 1994; Biggins
et al. 1996). Although the structure of the SPB in cells
lacking KAR1 was not determined, the fact that the two
structural components of the half-bridge can be eliminated is consistent with the notion that the SPB may be
able to duplicate by more than one mechanism and that
there may be an intrinsic plasticity built into this
organelle. In addition, the requirement for certain
SPB proteins, particularly integral membrane components, might be highly dependent on proteins and
other factors present in the nuclear membrane.
There is not a strict correlation between the SPB
deletions that are rescued by nup157D and those
rescued by POM nucleoporin deletions. The spc42-11
allele is rescued only by nup157D, whereas mps2-1 is
rescued only by POM deletions (Figure 6). Similarly,
only a subset of the nucleoporin deletions that rescue
the mps3-1 allele restore viability to mps3D. Further
studies are needed to determine the exact mechanism
by which the different nucleoporin deletions suppress
each SPB allele and to elucidate the mechanism by
which SPBs can duplicate in the complete absence of
Mps3p. However, regardless of the precise mechanism,
our data show that Mps3p is required only for SPB
duplication in the presence of intact nuclear pore
complexes and that an alternative SPB duplication
pathway exists under conditions where nucleoporin deletions have altered the nuclear envelope environment.
We thank Mark Winey for strains and helpful discussions, Joseph
Campbell for advice and technical support, and Kathryn Wagner for
assistance with flow cytometry. We are grateful to Rhonda Trimble for
assistance with EM and to Neal Freedman for help with statistical
analysis. We thank Mark Rose for sharing unpublished data and for
insightful suggestions on immunofluorescence data. We also thank
Kevin O’Connell, Leslie Barbour, Micah Webster, Daphna JosephStrauss, and William Glassford for discussion and comments on the
manuscript. K.L.W. is funded by a National Institute of Diabetes and
Digestive and Kidney Diseases (NIDDK) intramural Nancy Nossal
fellowship award, O.C.F. is funded by an intramural NIDDK grant, and
S.L.J. is supported by funds from the Stowers Institute for Medical
Research.
LITERATURE CITED
Adams, I. R., and J. V. Kilmartin, 1999 Localization of core spindle
pole body (SPB) components during SPB duplication in Saccharomyces cerevisiae. J. Cell Biol. 145: 809–823.
Aitchison, J. D., G. Blobel and M. P. Rout, 1995a Nup120p: a
yeast nucleoporin required for NPC distribution and mRNA
transport. J. Cell Biol. 131: 1659–1675.
881
Aitchison, J. D., M. P. Rout, M. Marelli, G. Blobel and R. W.
Wozniak, 1995b Two novel related yeast nucleoporins
Nup170p and Nup157p: complementation with the vertebrate
homologue Nup155p and functional interactions with the yeast
nuclear pore-membrane protein Pom152p. J. Cell Biol. 131:
1133–1148.
Alber, F., S. Dokudovskaya, L. M. Veenhoff, W. Zhang, J. Kipper
et al., 2007 The molecular architecture of the nuclear pore
complex. Nature 450: 695–701.
Antoniacci, L. M., M. A. Kenna, P. Uetz, S. Fields and R. V.
Skibbens, 2004 The spindle pole body assembly component
mps3p/nep98p functions in sister chromatid cohesion. J. Biol.
Chem. 279: 49542–49550.
Antoniacci, L. M., M. A. Kenna and R. V. Skibbens, 2007 The nuclear envelope and spindle pole body-associated Mps3 protein
bind telomere regulators and function in telomere clustering.
Cell Cycle 6: 75–79.
Araki, Y., C. K. Lau, H. Maekawa, S. L. Jaspersen, T. H. Giddings,
Jr. et al., 2006 The Saccharomyces cerevisiae spindle pole body
(SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p
and Mps2p. Mol. Biol. Cell 17: 1959–1970.
Aris, J. P., and G. Blobel, 1989 Yeast nuclear envelope proteins
cross react with an antibody against mammalian pore complex
proteins. J. Cell Biol. 108: 2059–2067.
Biggins, S., I. Ivanovska and M. D. Rose, 1996 Yeast ubiquitin-like
genes are involved in duplication of the microtubule organizing
center. J. Cell Biol. 133: 1331–1346.
Bogerd, A. M., J. A. Hoffman, D. C. Amberg, G. R. Fink and L. I.
Davis, 1994 nup1 mutants exhibit pleiotropic defects in nuclear pore complex function. J. Cell Biol. 127: 319–332.
Bullitt, E., M. P. Rout, J. V. Kilmartin and C. W. Akey, 1997 The
yeast spindle pole body is assembled around a central crystal of
Spc42p. Cell 89: 1077–1086.
Bupp, J. M., A. E. Martin, E. S. Stensrud and S. L. Jaspersen,
2007 Telomere anchoring at the nuclear periphery requires
the budding yeast Sad1-UNC-84 domain protein Mps3. J. Cell
Biol. 179: 845–854.
Campbell, J. L., A. Lorenz, K. L. Witkin, T. Hays, J. Loidl et al.,
2006 Yeast nuclear envelope subdomains with distinct abilities to resist membrane expansion. Mol. Biol. Cell. 17: 1768–
1778.
Chial, H. J., M. P. Rout, T. H. Giddings and M. Winey,
1998 Saccharomyces cerevisiae Ndc1p is a shared component
of nuclear pore complexes and spindle pole bodies. J. Cell Biol.
143: 1789–1800.
Conrad, M. N., C. Y. Lee, J. L. Wilkerson and M. E. Dresser,
2007 MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 104: 8863–8868.
Conrad, M. N., C. Y. Lee, G. Chao, M. Shinohara, H. Kosaka et al.,
2008 Rapid telomere movement in meiotic prophase is promoted by NDJ1, MPS3, and CSM4 and is modulated by recombination. Cell 133: 1175–1187.
Dawson, T. R., M. D. Lazarus, M. W. Hetzer and S. R. Wente,
2009 ER membrane-bending proteins are necessary for de novo
nuclear pore formation. J. Cell Biol. 184: 659–675.
DeHoratius, C., and P. A. Silver, 1996 Nuclear transport defects
and nuclear envelope alterations are associated with mutation
of the Saccharomyces cerevisiae NPL4 gene. Mol. Biol. Cell 7:
1835–1855.
Devos, D., S. Dokudovskaya, F. Alber, R. Williams, B. T. Chait
et al., 2004 Components of coated vesicles and nuclear pore
complexes share a common molecular architecture. PLoS Biol.
2: e380.
Devos, D., S. Dokudovskaya, R. Williams, F. Alber, N. Eswar et al.,
2006 Simple fold composition and modular architecture of the
nuclear pore complex. Proc. Natl. Acad. Sci. USA 103: 2172–
2177.
Drin, G., J. F. Casella, R. Gautier, T. Boehmer, T. U. Schwartz
et al., 2007 A general amphipathic alpha-helical motif for sensing membrane curvature. Nat. Struct. Mol. Biol. 14: 138–146.
Elliott, S., M. Knop, G. Schlenstedt and E. Schiebel,
1999 Spc29p is a component of the Spc110p subcomplex
and is essential for spindle pole body duplication. Proc. Natl.
Acad. Sci. USA 96: 6205–6210.
882
K. L. Witkin et al.
Fischer, T., S. Rodriguez-Navarro, G. Pereira, A. Racz, E. Schiebel
et al., 2004 Yeast centrin Cdc31 is linked to the nuclear mRNA
export machinery. Nat. Cell Biol. 6: 840–848.
Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Bottcher et al.,
2009 Two structurally distinct domains of the nucleoporin
Nup170 cooperate to tether a subset of nucleoporins to nuclear
pores. J. Cell Biol. 185: 387–395.
Galitski, T., A. J. Saldanha, C. A. Styles, E. S. Lander and G. R.
Fink, 1999 Ploidy regulation of gene expression. Science
285: 251–254.
Golden, A., J. Liu and O. Cohen-Fix, 2009 Inactivation of the C.
elegans lipin homolog leads to ER disorganization and to defects
in the breakdown and reassembly of the nuclear envelope. J. Cell
Sci. 122: 1970–1978.
Goldstein, A. L., and J. H. McCusker, 1999 Three new dominant
drug resistance cassettes for gene disruption in Saccharomyces
cerevisiae. Yeast 15: 1541–1553.
Goldstein, A. L., C. A. Snay, C. V. Heath and C. N. Cole,
1996 Pleiotropic nuclear defects associated with a conditional
allele of the novel nucleoporin Rat9p/Nup85p. Mol. Biol. Cell
7: 917–934.
Gomez-Ospina, N., G. Morgan, T. H. Giddings, Jr., B. Kosova, E.
Hurt et al., 2000 Yeast nuclear pore complex assembly defects
determined by nuclear envelope reconstruction. J. Struct. Biol.
132: 1–5.
Gorjanacz, M., and I. W. Mattaj, 2009 Lipin is required for efficient breakdown of the nuclear envelope in Caenorhabditis elegans. J. Cell Sci. 122: 1963–1969.
Han, G. S., W. I. Wu and G. M. Carman, 2006 The Saccharomyces
cerevisiae Lipin homolog is a Mg21-dependent phosphatidate
phosphatase enzyme. J. Biol. Chem. 281: 9210–9218.
Han, G. S., L. O’Hara, G. M. Carman and S. Siniossoglou,
2008 An unconventional diacylglycerol kinase that regulates
phospholipid synthesis and nuclear membrane growth. J. Biol.
Chem. 283: 20433–20442.
Heath, C. V., C. S. Copeland, D. C. Amberg, V. Del Priore,
M. Snyder et al., 1995 Nuclear pore complex clustering and nuclear accumulation of poly(A)1 RNA associated with mutation of
the Saccharomyces cerevisiae RAT2/NUP120 gene. J. Cell Biol.
131: 1677–1697.
Hetzer, M. W., and S. R. Wente, 2009 Border control at the nucleus: biogenesis and organization of the nuclear membrane
and pore complexes. Dev. Cell 17: 606–616.
Hiraoka, Y., and A. F. Dernburg, 2009 The SUN rises on meiotic
chromosome dynamics. Dev. Cell 17: 598–605.
Hodge, C. A., V. Choudhary, M. J. Wolyniak, J. J. Scarcelli,
R. Schneiter et al., 2010 Integral membrane proteins Brr6
and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum. J. Cell Sci. 123: 141–151.
Izawa, S., R. Takemura and Y. Inoue, 2004 Gle2p is essential to induce adaptation of the export of bulk poly(A)1 mRNA to heat
shock in Saccharomyces cerevisiae. J. Biol. Chem. 279: 35469–
35478.
Jaspersen, S. L., and M. Winey, 2004 The budding yeast spindle
pole body: structure, duplication, and function. Annu. Rev. Cell
Dev. Biol. 20: 1–28.
Jaspersen, S. L., T. H. Giddings, Jr. and M. Winey, 2002 Mps3p is a
novel component of the yeast spindle pole body that interacts
with the yeast centrin homologue Cdc31p. J. Cell Biol. 159:
945–956.
Jaspersen, S. L., A. E. Martin, G. Glazko, T. H. Giddings, Jr.,
G. Morgan et al., 2006 The Sad1-UNC-84 homology domain
in Mps3 interacts with Mps2 to connect the spindle pole body
with the nuclear envelope. J. Cell Biol. 174: 665–675.
Jorgensen, P., N. P. Edgington, B. L. Schneider, I. Rupes, M. Tyers
et al., 2007 The size of the nucleus increases as yeast cells grow.
Mol. Biol. Cell 18: 3523–3532.
Khalfan, W. A., 2001 Genetic interactions between a spindle pole
body (SPB) gene KAR1, and the PKC1 MAP kinase cascade and
nuclear envelope proteins. Ph.D. Thesis, Princeton University
Press, Princeton, NJ.
Kosova, B., N. Pante, C. Rollenhagen, A. Podtelejnikov, M.
Mann et al., 2000 Mlp2p, a component of nuclear pore attached intranuclear filaments, associates with nic96p. J. Biol.
Chem. 275: 343–350.
Longtine, M. S., A. McKenzie, III, D. J. Demarini, N. G. Shah,
A. Wach et al., 1998 Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953–961.
Madrid, A. S., J. Mancuso, W. Z. Cande and K. Weis, 2006 The role
of the integral membrane nucleoporins Ndc1p and Pom152p in
nuclear pore complex assembly and function. J. Cell Biol. 173:
361–371.
Makio, T., L. H. Stanton, C. C. Lin, D. S. Goldfarb, K. Weis et al.,
2009 The nucleoporins Nup170p and Nup157p are essential
for nuclear pore complex assembly. J. Cell Biol. 185: 459–473.
McDonald, K., 1999 High-pressure freezing for preservation of
high resolution fine structure and antigenicity for immunolabeling. Methods Mol. Biol. 117: 77–97.
Miao, M., K. J. Ryan and S. R. Wente, 2006 The integral membrane
protein Pom34p functionally links nucleoporin subcomplexes.
Genetics 172: 1441–1457.
Nehrbass, U., M. P. Rout, S. Maguire, G. Blobel and R. W.
Wozniak, 1996 The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear
pore complex. J. Cell Biol. 133: 1153–1162.
Neumann, F. R., and P. Nurse, 2007 Nuclear size control in fission
yeast. J. Cell Biol. 179: 593–600.
Niepel, M., C. Strambio-de-Castillia, J. Fasolo, B. T. Chait and
M. P. Rout, 2005 The nuclear pore complex-associated protein, Mlp2p, binds to the yeast spindle pole body and promotes
its efficient assembly. J. Cell Biol. 170: 225–235.
Nishikawa, S., Y. Terazawa, T. Nakayama, A. Hirata, T. Makio
et al., 2003 Nep98p is a component of the yeast spindle pole
body and essential for nuclear division and fusion. J. Biol. Chem.
278: 9938–9943.
Onischenko, E., L. H. Stanton, A. S. Madrid, T. Kieselbach and
K. Weis, 2009 Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance. J. Cell Biol. 185:
475–491.
Oza, P., S. L. Jaspersen, A. Miele, J. Dekker and C. L. Peterson,
2009 Mechanisms that regulate localization of a DNA doublestrand break to the nuclear periphery. Genes Dev. 23: 912–927.
Pemberton, L. F., M. P. Rout and G. Blobel, 1995 Disruption of
the nucleoporin gene NUP133 results in clustering of nuclear
pore complexes. Proc. Natl. Acad. Sci. USA 92: 1187–1191.
Razafsky, D., and D. Hodzic, 2009 Bringing KASH under the SUN:
the many faces of nucleo-cytoskeletal connections. J. Cell Biol.
186: 461–472.
Rose, M. D., and G. R. Fink, 1987 KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast.
Cell 48: 1047–1060.
Rose, M. D., F. Winston and P. Heither, 1990 Methods in Yeast
Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Santos-Rosa, H., J. Leung, N. Grimsey, S. Peak-Chew and S.
Siniossoglou, 2005 The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth. EMBO J. 24:
1931–1941.
Scarcelli, J. J., C. A. Hodge and C. N. Cole, 2007 The yeast integral
membrane protein Apq12 potentially links membrane dynamics
to assembly of nuclear pore complexes. J. Cell Biol. 178: 799–812.
Schneiter, R., M. Hitomi, A. S. Ivessa, E. V. Fasch, S. D. Kohlwein
et al., 1996 A yeast acetyl coenzyme A carboxylase mutant links
very-long-chain fatty acid synthesis to the structure and function
of the nuclear membrane-pore complex. Mol. Cell. Biol. 16:
7161–7172.
Schober, H., H. Ferreira, V. Kalck, L. R. Gehlen and S. M. Gasser,
2009 Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination. Genes
Dev. 23: 928–938.
Schramm, C., S. Elliott, A. Shevchenko and E. Schiebel,
2000 The Bbp1p-Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication. EMBO J. 19:
421–433.
Sezen, B., M. Seedorf and E. Schiebel, 2009 The SESA network
links duplication of the yeast centrosome with the protein translation machinery. Genes Dev. 23: 1559–1570.
Siniossoglou, S., C. Wimmer, M. Rieger, V. Doye, H. Tekotte et al.,
1996 A novel complex of nucleoporins, which includes Sec13p
The Nuclear Membrane Affects SPB Duplication
and a Sec13p homolog, is essential for normal nuclear pores. Cell
84: 265–275.
Siniossoglou, S., H. Santos-Rosa, J. Rappsilber, M. Mann and
E. Hurt, 1998 A novel complex of membrane proteins required
for formation of a spherical nucleus. EMBO J. 17: 6449–6464.
Spang, A., I. Courtney, K. Grein, M. Matzner and E. Schiebel,
1995 The Cdc31p-binding protein Kar1p is a component of
the half bridge of the yeast spindle pole body. J. Cell Biol. 128:
863–877.
Strahm, Y., B. Fahrenkrog, D. Zenklusen, E. Rychner, J. Kantor
et al., 1999 The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FGnucleoporin Rip1p, the DEAD-box protein Rat8p/Dbp5p and a
new protein Ymr 255p. EMBO J. 18: 5761–5777.
Vallen, E. A., W. Ho, M. Winey and M. D. Rose, 1994 Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces
cerevisiae. Genetics 137: 407–422.
Walczak, C. E., and R. Heald, 2008 Mechanisms of mitotic spindle
assembly and function. Int. Rev. Cytol. 265: 111–158.
Wente, S. R., and G. Blobel, 1993 A temperature-sensitive NUP116
null mutant forms a nuclear envelope seal over the yeast nuclear
pore complex thereby blocking nucleocytoplasmic traffic. J. Cell
Biol. 123: 275–284.
883
Wente, S. R., and G. Blobel, 1994 NUP145 encodes a novel
yeast glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin
required for nuclear envelope structure. J. Cell Biol. 125: 955–969.
Winey, M., L. Goetsch, P. Baum and B. Byers, 1991 MPS1 and
MPS2: novel yeast genes defining distinct steps of spindle pole
body duplication. J. Cell Biol. 114: 745–754.
Winey, M., M. A. Hoyt, C. Chan, L. Goetsch, D. Botstein et al.,
1993 NDC1: a nuclear periphery component required for yeast
spindle pole body duplication. J. Cell Biol. 122: 743–751.
Winey, M., D. Yarar, T. H. Giddings, Jr. and D. N. Mastronarde,
1997 Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional
reconstruction from electron micrographs of nuclear envelopes.
Mol. Biol. Cell 8: 2119–2132.
Zabel, U., V. Doye, H. Tekotte, R. Wepf, P. Grandi et al.,
1996 Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p. J. Cell Biol.
133: 1141–1152.
Zhao, R., M. S. Bodnar and D. L. Spector, 2009 Nuclear neighborhoods and gene expression. Curr. Opin. Genet. Dev. 19: 172–179.
Communicating editor: M. D. Rose
GENETICS
Supporting Information
http://www.genetics.org/cgi/content/full/genetics.110.119149/DC1
Changes in the Nuclear Envelope Environment Affect Spindle Pole Body
Duplication in Saccharomyces cerevisiae
Keren L. Witkin, Jennifer M. Friederichs, Orna Cohen-Fix and Sue L. Jaspersen
Copyright Ó 2010 by the Genetics Society of America
DOI: 10.1534/genetics.110.119149
2 SI
K. L. Witkin et al.
FIGURE S1.—No genetic interactions between spo7 and N-terminal truncation mutants of MPS3. Wild-type cells
(KW340), spo7 cells (KW343), mps32-64 cells (KW332), mps32-64 spo7 cells (KW334), mps375-150 cells
(KW336), and mps375-150 spo7 cells (KW338) were plated on rich media at the indicated temperatures. At 37˚C,
spo7 does not grow in the presence or absence of MPS3 mutations (data not shown).
K. L. Witkin et al.
3 SI
FIGURE S2.—Genetic interactions between spo7 and other SPB mutant alleles. (A) Wild-type cells (KW696), spo7
cells (KW693), mps2-381 cells (KW514), and double mutants (KW515), all initially containing a URA3-marked MPS2
plasmid, were assayed for growth at 23˚C on 5-FOA and control plates (SC). (B) Wild-type cells (KW573), spo7 cells
(KW572), kar117 cells (KW571), and double mutants containing a URA3-marked KAR1 plasmid (KW570), were
assayed for growth at 23˚C on 5-FOA and control plates (SC). (C-E) Cells of the indicated genotypes were tested for
growth on rich media at the indicated temperatures. The strains utilized are: (C) KW562, KW561, KW560, KW559
(D) KW543, KW509, KW542, KW538 (E) KW594, KW595, KW596, KW598.
4 SI
K. L. Witkin et al.
FIGURE S3.—spo7 cells have no apparent SPB defect on their own. While spo7 cells are viable, they exhibit
reduced growth at elevated temperatures (SINIOSSOGLOU et al. 1998). Although spo7 mutants do not accumulate in
mitosis even at the restrictive temperature (data not shown), it was formally possible that their temperature-dependent
growth defect was due to spindle or SPB defects. We therefore assayed cell cycle progression and spindle elongation in
spo7 cells. Wild-type (OCF1533-28B; black) and spo7 (KW459; grey) cells were arrested in G1 with alpha-factor
mating pheromone at 30˚C, and then released into fresh media at 37˚C. At the time points indicated, cells were
removed and either fixed with ethanol and examined microscopically to determine the budding index (A) or fixed with
paraformaldehyde and processed for immunofluorescence with antibodies against a-tubulin to examine the mitotic
spindle (B). spo7 cells delayed in their release from G1 compared to wild-type cells. However, after this initial delay,
spo7 cells progressed through the cell cycle with similar kinetics to wild-type cells. They also built and elongated their
spindle at the appropriate time relative to G1 release.
K. L. Witkin et al.
5 SI
FIGURE S4.—Truncated forms of NUP157 and NIC96 suppress mps3-1 defects. The original suppressor clones
(clones 62 and 146) and subcloned truncated nucleoporin genes were tested for their ability to suppress the mps3-1
temperature-sensitivity phenotype (strain SLJ910) by plating in serial dilutions on SC plates at 30°C or 34°C. NUP157T was subcloned onto a 2μ plasmid behind the GPD promoter (pGPD) and NIC96-T was transferred to a CEN plasmid
behind the TEF promoter (pTEF), because these constructs were found to confer maximal suppression of synthetic
lethality (data not shown). We found that both clone 62 and pGPD-NUP157-T partially suppressed the temperature
sensitivity of the mps3-1 allele by allowing growth at 34˚C. Although the original clone 146 did not suppress the
temperature-sensitivity of mps3-1, pTEF-NIC96-T did restore growth at 34˚C. An empty vector and the MPS3 gene on a
2μ plasmid were used as controls. At 37˚C, only cells containing plasmids expressing MPS3 were viable (not shown).
Prior to the experiment, all strains were maintained with pURA3-MPS3; plates containing 5-FOA were used to select
for cells lacking the plasmid prior to plating on SC plates as described above.
6 SI
K. L. Witkin et al.
FIGURE S5.—The nucleoporin suppressor clones do not rescue phenotypes of spo7 cells. (A) spo7 cells (JCY565)
were transformed with the original suppressor clones 62 (containing NUP157-T) or 146 (containing NIC96-T), an empty
vector, and plasmids containing SPO7 or PAH1. Transformants were tested by serial dilution on rich media (YPD) for
the ability to grow at 30˚C and 37˚C. (B) spo7 cells (JCY565) were transformed with an empty vector, a centromerebased plasmid containing the SPO7 gene, suppressor clone 62, and suppressor clone 146. All strains were then
transformed with a centromere-based plasmid containing the nucleoplasmic protein Pus1p fused to GFP. Cells were
then grown to mid-log phase and fixed with paraformaldehyde for fluorescence microscopy. The percentage of cells
exhibiting abnormal nuclear shape is graphed. (C) spo7 nup188 cells containing a URA3-marked SPO7 plasmid
(KW796) were transformed with an empty vector, a LEU2-marked SPO7 plasmid, and the original suppressor clones 62
and 146. Cells were then plated by serial dilution on plates containing 5-FOA or control (SC-LEU) media.
K. L. Witkin et al.
7 SI
FIGURE S6.—pom152 does not restore viability to all SPB deletions. Deletion alleles of various SPB mutants were
maintained with a wild-type copy of each SPB gene on a URA3-marked plasmid. Strains containing the indicated
deletions were plated by serial dilution on rich media (YPD) or 5-FOA containing plates. The strains used were:
SLJ001, SLJ2389, SLJ4262, SLJ4263, SLJ4264, SLJ4265, SLJ4270, SLJ4271, SLJ4272, SLJ4273, SLJ4274, SLJ4275,
SLJ4276, SLJ4277, SLJ4278, SLJ4279, SLJ4280, SLJ4281, SLJ4282, SLJ4283.
8 SI
K. L. Witkin et al.
FIGURE S7.—Spindle length distribution in cells lacking Mps3p. Wild-type cells (SLJ4336), pom152 cells (SLJ4338),
and the mps3 pom152 double mutants (SLJ4340) were examined by immunofluorescence using antibodies against the
SPB (anti-Tub4p antibodies) and microtubules (anti-a-tubulin antibodies). In cells containing 2 SPBs, the length of the
spindle was measured by determining the distance between the two anti-Tub4p spots. Shown is a dot-plot of spindle
lengths for each genotype. Average spindle length is indicated below the plot. For wild-type, pom152, and pom152
mps3, n=81, 92, and 50 respectively.
K. L. Witkin et al.
9 SI
FILE S1
Supporting Methods
Cytological techniques
For alpha-factor arrest, cells grown at 30˚C to mid-log phase were treated with 20 μM alpha-factor (Zymo Research,
Orange, CA) until fully arrested (approximately three hours), washed three times in fresh media, and then released into
fresh media at 37˚C. Immunofluorescence in Supplemental Figure S3 was performed as described previously
(CAMPBELL et al. 2006), except that monoclonal antibodies against alpha-tubulin (Sigma Aldrich, St. Louis, MO) were
used at 1:750 dilution. Short spindles were defined as those that traversed a spherical nucleus, while long spindles
traversed an hourglass nucleus with two separated DNA masses. To determine nuclear morphology, cells containing a
pPUS1-GFP-URA plasmid (CAMPBELL et al. 2006) were grown to mid-log phase, fixed in 4% paraformaldehyde for
one hour at 23˚C, and then observed using a Nikon Eclipse E800 fluorescent microscope.
10 SI
K. L. Witkin et al.
TABLE S1
Strains used in supplemental information
Strain name
Genotype
JCY565
MATa ura3-1 trp1-1 leu2-3,112 spo7::NAT can1-100
KW332
MATa ura3-1 his3-11,15 trp1-1 mps3::NAT leu2::MPS32-64-LEU2 can1-100
KW334
MATa ura3-1 his3-11,15 trp1-1 mps3::NAT leu2::MPS32-64-LEU2 spo7::KAN
can1-100
KW336
MATa ura3-1 his3-11,15 trp1-1 mps3::NAT leu2::MPS375-150-LEU2 can1-100
KW338
MATa ura3-1 his3-11,15 trp1-1 mps3::NAT leu2::MPS375-150-LEU2
spo7::KAN can1-100
KW340
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 can1-100
KW343
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 spo7::NAT can1-100
KW459
MATa ura3-1 trp1-1 spo7::NAT can1-100
KW509
MAT ura3-1 trp1-1 spo7::NAT can1-100
KW514
MATa ura3-1 trp1-1 ade2-1 leu2::mps2-381-LEU2 mps2::KAN can1-100 pURA3-MPS2
KW515
MATa ura3-1 trp1-1 ade2-1 leu2::mps2-381-LEU2 mps2::KAN spo7::NAT can1-100 pURA3-MPS2
KW538
MAT ura3-1 trp1-1 spo7::NAT spc29-3 can1-100
KW542
MAT ura3-1 trp1-1 spc29-3 can1-100
KW543
MAT ura3-1 trp1-1 can1-100
KW559
MATa ura3-1 trp1-1 leu2-3,112 spo7::NAT spc42-11 can1-100
KW560
MATa ura3-1 trp1-1 leu2-3,112 spc42-11 can1-100
KW561
MATa ura3-1 trp1-1 leu2-3,112 spo7::NAT can1-100
KW562
MATa ura3-1 trp1-1 leu2-3,112 can1-100
KW570
MATa ura3-1 trp1-1 ade2-1 his3-11,15 leu2-3,112 kar117 spo7::NAT can1-100 pURA3-KAR1
KW571
MATa ura3-1 trp1-1 ade2-1 his3-11,15 leu2-3,112 kar117 can1-100
KW572
MATa ura3-1 trp1-1 ade2-1 leu2-3,112 spo7::NAT can1-100 pURA3-KAR1
KW573
MATa ura3-1 trp1-1 ade2-1 leu2-3,112 can1-100
KW594
MATa ura3-1 trp1-1 leu2-3,112 can1-100
KW595
MATa ura3-1 trp1-1 leu2-3,112 spo7::NAT can1-100
KW596
MATa ura3-1 leu2-3,112 ndc1::KAN ndc1-39-TRP1 can1-100
KW598
MATa ura3-1 leu2-3,112 ndc1::KAN ndc1-39-TRP1 spo7::NAT can1-100
KW693
MATa ura3-1 ade2-1 leu2-3,112 trp1-1 spo7::NAT can1-100
KW696
MATa ura3-1 ade2-1 leu2-3,112 trp1-1 can1-100
KW796
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 nup188::KAN spo7::NAT can1-100 pURA3-SPO7
OCF1533-28B
MATa ura3-1 trp1-1 can1-100
SLJ001
MATa bar1::hisG ura3-1 leu2-3,112 trp1-1 his3-11,15 ade2-1 can1-100
SLJ910
MATa mps3-1 ura3-1 ade2-1 leu2-3,112 his3-11,15 trp1-1 mps3-1 can1-100
pURA3-MPS3
SLJ2389
MAT pom152::HYGB ade2-1 ade3100 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100
K. L. Witkin et al.
SLJ4262
11 SI
MATa pom152::HYGMX mps3::NATMX ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1 lys2
pURA3-MPS3
SLJ4263
MAT pom152::HYGMX mps3::NATMX ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1
pURA3-MPS3
SLJ4264
MATa pom152::HYGMX spc42::LEU2 ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1
pURA3-SPC42
SLJ4265
MAT pom152::HYGMX spc42::LEU2 ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1
pURA3-SPC42
SLJ4270
MAT pom152::HYGMX mps2::KANMX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-MPS2
SLJ4271
MATa pom152::HYGMX mps2::KANMX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-MPS2
SLJ4272
MAT pom152::HYGMX kar1::KANMX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-KAR1
SLJ4273
MAT pom152::HYGMX kar1::KANMX ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1
pURA3-KAR1
SLJ4274
MATa pom152::HYGMX nbp1::HIS5MX ade2 ade3 his3 leu2 trp1 ura3 pURA3-ADE3-NBP1
SLJ4275
MAT pom152::HYGMX nbp1::HIS5MX ade2 ade3 his3 leu2 trp1 ura3 lys pURA3-ADE3-NBP1
SLJ4276
MATa pom152::HYGMX nud1::HIS5MX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-NUD1
SLJ4277
MAT pom152::HYGMX nud1::HIS5MX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-NUD1
SLJ4278
MAT mps3::NATMX ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1 lys2 pURA3-MPS3
SLJ4279
MATa spc42::LEU2 ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-SPC42
SLJ4280
MATa mps2::KANMX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-MPS2
SLJ4281
MATa kar1::KANMX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 lys2 pURA3-KAR1
SLJ4282
MATa nbp1::HIS5MX ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-ADE3-NBP1
SLJ4283
MAT nud1::HIS5MX ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 pURA3-NUD1
SLJ4336
MAT ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1
SLJ4338
MAT pom152::HYGB ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1
SLJ4340
MAT pom152::HYGB mps3::NATMX ade2-1 ade3100 his3-11,15 leu2-3,112 trp1-1 ura3-1