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INTRODUCTION |
The endoplasmic reticulum
(ER)1 is a membranous network
structure where secretory and trans-membrane proteins are processed by
disulfide (S-S) bond formation, glycosylation, folding, and oligomerization. Perturbation of ER function results in the
accumulation of unfolded proteins in the ER, because only properly
folded proteins transit the secretory pathway. The accumulation of
unfolded proteins in the ER induces a stress pathway known as the
unfolded protein response (UPR) that functions to reduce the ER stress
situation (1). The activated UPR results in an increased
protein-folding capacity of the ER by up-regulating the transcription
of genes encoding ER-resident molecular chaperones, enzymes that
facilitate folding and assembly of proteins, and components of the
protein degradative machinery (2-4). In mammalian cells, induction of UPR also reduces protein synthesis by down-regulating translation to
reduce folding load for newly synthesized proteins. Growth arrest
and/or the apoptotic cascade is also induced in response to the ER
stress (5-7). Experimentally, the UPR can be induced by inhibiting
protein glycosylation with tunicamycin (TM) treatment or by preventing
disulfide bond formation with the addition of reducing agents, such as
-mercaptoethanol, to the growth medium. Glycosylation is the
covalent addition of an oligosaccharide core structure to side-chain
NH2 groups of selective asparagine residues in the protein.
Many conditions, including overexpression of ER membrane proteins or
mutant proteins, also induce the UPR (1, 8).
In yeast, the accumulation of unfolded proteins in the ER activates
Ire1p, an ER-transmembrane serine/threonine kinase similar in structure
to mammalian growth factor receptor kinases. The amino terminus of
Ire1p, located in the ER lumen, serves as a sensor to detect the
accumulation of unfolded proteins in the ER. The carboxyl-terminal
kinase and endoribonuclease domains function in the cytoplasm and/or
nucleoplasm to activate downstream signaling events in the pathway (4,
9). When activated, Ire1p oligomerizes and becomes
trans-autophosphorylated by neighboring Ire1p molecules (10).
Activated Ire1p triggers synthesis of the basic leucine zipper
transcription factor Hac1p (homology to
ATF/CREB) by cleaving a 252-nucleotide intron
from unspliced HAC1u mRNA (u = uninduced) (11, 12). This intron attenuates
HAC1u mRNA translation by base-pairing
interaction between the intron and the 5'-untranslated region (13-15).
Although HAC1u mRNA is recruited into
functional polyribosomes in the cytosol, the ribosomes stall during
elongation and do not produce Hac1pu (13, 14). Upon ER
stress, the RNase activity of Ire1p initiates the
spliceosome-independent HAC1u mRNA splicing
event (16, 17). This reaction is similar to tRNA splicing, where the
ends of the tRNA intron are cleaved independently (18). Indeed, tRNA
ligase, RLG1, involved in pre-tRNA splicing catalyzes ligation of
HAC1 mRNA resulting in the production of HAC1i mRNA (i = UPR-induced), which is translatable. The unspliced HAC1u mRNA is predicted to encode a protein
of 230 amino acids (Hac1pu), with the 10 COOH-terminal
amino acids encoded by the first part of the intron. Splicing of the
intron replaces this 10-amino acid tail with a novel 18-amino acid tail
encoded by the second exon, generating the 238-amino acid protein,
Hac1pu. This change increases the transcriptional
activation potential of Hac1pi (19). Both
Hac1pu and Hac1pi proteins have short
half-lives of approximately 2 min (13, 14). Therefore, the activity of
the UPR is not controlled by the difference in the stability of
Hac1pu and Hac1pi (13) but by regulated
splicing of HAC1i mRNA. Constitutive
expression of HAC1i mRNA results in
unregulated expression of Hac1pi and the constitutive
transcriptional induction of all known target genes.
The majority of the UPR-induced genes share a common upstream
cis-acting element in their promoters, the unfolded protein response
element (UPRE), that is required to stimulate their transcription upon
induction of the UPR (20-23). Hac1p is the transcription factor that
binds to the UPRE to activate transcription of UPR target genes.
Although the UPRE is necessary and sufficient for increased gene
expression in response to the UPR, it is not required for basal level
expression. All of the functional UPRE sequences identified contain a
palindromic sequence that has, in most cases, a spacer of one C
nucleotide. For example, the KAR2 UPRE contains the
palindromic sequence, CAGCGTG.
In addition to its role in the UPR, the Hac1p transcription factor also
regulates cell differentiation in response to nutrient availability and
functions as a positive regulator of the inositol biosynthetic pathway
(24, 25). Free inositol is a precursor in the synthesis of
phosphatidylinositol, one of the major glycerophospholipids of the ER
membrane in yeast. It has been proposed that Hac1p activates transcription of genes that are required for de novo
phospholipid synthesis including INO1 (encoding
inositol-1-phosphate synthase), CHO1 (encoding
phosphatidylserine synthase), and OPI3 (encoding phospholipid methyltransferase required for de novo
phosphatidylcholine synthesis) by antagonizing Opi1p repressor activity
(25). Indeed, hac1
cells are inositol auxotrophs, as a
result of inability to induce transcription of the INO1 gene
upon inositol starvation.
The Kar2p/BiP protein is located in the lumen of the ER, where one of
its functions is to facilitate the translocation of secretory
precursors of a number of proteins (invertase, carboxypeptidase Y, and
-factor) into the lumen of the ER (26). Kar2p is also required for
nuclear fusion during mating (27). However, it is not clear how this
function relates to its translocation role. The failure in karyogamy
results in division of the unfused nuclei mitotically in the zygote,
which buds off mostly mononucleated haploid cells called cytoductants
(28). Some of the cytoductants recovered from kar2 crosses,
although displaying the phenotype of one of the parental haploid
strains, become diploids by self-diploidization (29). In addition,
Kar2p inhibits UPR activation by binding to Ire1p and preventing its
dimerization/oligomerization (30).
Although the role for HAC1 in the UPR pathway has been
extensively characterized, no defects relating to ploidy control have been associated with hac1 deficiency. In wild-type cells, an
increase in ploidy occurs only as the inevitable conclusion to the
mating process and results in the formation of alloploid (increase in ploidy arising from the combination of genetically distinct chromosome sets) a/
diploids. Mitotically growing cells maintain the
same ploidy by precise regulation of chromosome duplication and
separation. The fidelity of these processes is dependent on precise
chromosome duplication and segregation (once-per-cell cycle
replication), spindle pole body duplication and separation, nucleus
migration, cytokinesis, etc. (31-33). However, mutations conferring
defects in these processes results in autopolyploidy (increase in
ploidy that originates by the multiplication of one basic set of
chromosomes) (34, 35). In this paper, we characterized the behavior of
hac1 mutants in the sporulation-proficient SK1 genetic
background. Hac1p is required for the maintenance of ploidy. Inhibition
of the UPR pathway results in autodiploidization and overexpression of
Kar2p/BiP suppresses this phenotype, suggesting that functional UPR is
required for ploidy maintenance.
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EXPERIMENTAL PROCEDURES |
Strains and Media
The genotypes and strain backgrounds of yeast strains used in
this study are listed in Table I. Liquid and solid media (36) and
synthetic complete medium lacking inositol were prepared as described
(37). For induction of the UPR pathway, tunicamycin (purchased from
Roche Molecular Biochemicals) was added to liquid medium or solid
medium (1 µg/ml). When necessary, early log phase cell cultures
(A600
0.1-0.2) were treated with TM for
4 h to induce the UPR. Standard genetic methods were used for
mating, diploid selection, and tetrad analysis, unless otherwise
indicated (36). Yeast stains were transformed with plasmids or linear DNA fragments using a modified lithium acetate procedure (38).
Plasmid Construction
HAC1 Constructs--
Clones containing the genomic
HAC1 locus were derived from YEp24-HAC1, which
contains an 11.5-kb genomic Sau3AI fragment spanning the
HAC1 locus and its flanking regions. pRS414-HAC1
and pRS424-HAC1 were created by subcloning a HAC1
containing a 2.46-kb StuI-NaeI fragment of
YEp24-HAC1 into the SmaI site of the
TRP1-based plasmids pRS414 and pRS424, respectively (39,
43). The integrity of the HAC1 gene was confirmed by
sequencing, and functionality was verified by complementation of the
hac1 phenotype. pRS416-hac1
1 was created by
cloning the 1.6-kb EcoRI fragment of YEp24-HAC1 into the EcoRI site of pRS416; the COOH-terminal 34 bp of
HAC1 including the stop codon derived from the second exon
is not included on this plasmid. pRS416-hac1
2 was created
by deleting the 0.45-kb SalI fragment from
pRS416-hac1
1, which removes most of the HAC1 coding region.
KAR2 Constructs--
pRS425-KAR2 was created by
subcloning the 3.1-kb PvuII-NotI
KAR2-containing fragment of pMR109 (a gift from Dr. Mark
Rose, Princeton University, Princeton, NJ) into the
SmaI-NotI sites of pRS425. Function on the gene
was verified by its ability to complement the growth defect of a
kar2
ts allele (MS192) at 37 °C (data not shown).
Creation of Mutant Alleles in Yeast
Construction of the hac1
100::URA3 and
hac1
200::LEU2 Null Alleles--
To create the
hac1
100::URA3 null alleles, a 1.4-kb
SpeI-NheI fragment containing the URA3
gene (from YEp24) was subcloned into the SpeI site of
pRS416-hac1
2 to create
pRS416-hac1
100::URA3. Only the first
8 NH2-terminal amino acids of HAC1 can be
expressed from this allele. To create the
hac1
200::LEU2 mutant allele, a DNA
fragment containing the LEU2 gene (a 2.2-kb genomic fragment of XhoI-SalI digest) was subcloned into the
XhoI-SalI sites of the hac1 allele
harbored on pRS416-hac1
2 to create
pRS416-hac1
200::LEU2. Both the
transcription and translation start sites as well as 78% of the coding
region of HAC1 gene are absent from this allele. Two
wild-type a/
diploid strains from different genetic backgrounds (LNY3, derived from the SK1 genetic background; LNY435, derived from the W303 genetic background) were transformed with linear
DNA fragments containing either
hac1
100::URA3 (2.6-kb KpnI-XbaI fragment of
pRS416-hac1
100::URA3) or
hac1
200::LEU2 (3.2-kb
ApaI-XbaI fragment of
pRS416-hac1
200::LEU2), and gene replacement was selected for uracil or leucine prototrophy,
respectively. Six independent transformants (three from each strain),
in which one copy of HAC1 was replaced with either
hac1
100::URA3 or
hac1
200::LEU2, were sporulated and
subjected to tetrad analysis.
Construction of the ire1
100::TRP1 Allele--
To
create the ire1
100::TRP1 null
allele, a 5-kb linear DNA fragment (EcoRI digest of pCS160,
a gift from Dr. Peter Walter, University of California, San Francisco,
CA), which contains 0.35 and 0.25 kb, respectively, of 5'- and
3'-IRE1 genomic sequences flanking both ends and the yeast
TRP1-selectable marker, was used to perform a one-step gene
replacement by transforming the diploid strain LNY3 (wild-type, SK1) to
tryptophan prototrophy. The integrity of the gene replacement was
confirmed by genomic PCR (data not shown). The
ire1
100::TRP1 heterozygotes were
sporulated and subjected to tetrad analysis on medium containing 30 µg/ml inositol, because ire1 mutants are inositol
auxotrophs (40).
-Galactosidase Assay
Quantitative
-galactosidase assays were carried out by
harvesting cultures grown to mid-log phase as described (41).
Sporulation and Tetrad Analysis
Strains were grown overnight on YPD plates and then
replica-plated to sporulation plates and incubated at 30 °C. To test
sporulation ability of hac1 autodiploid cells,
hac1 cells were transformed with plasmids that carrying the
opposite MAT allele. LNY686 and LNY688 were transformed with pAV123
(carrying MATa) and pAV115 (carrying
MAT
), respectively. Transformants were selected on
SC
Leu medium, sporulated, and dissected on YPD plate.
Northern Probes
The following 32P-labeled probes were made using the
Random Prime labeling system (Roche Molecular Biochemicals): a 0.79-kb SpeI- HindIII fragment of HAC1, a
1.6-kb PCR fragment of KAR2 corresponding to bases 9-1645
of the coding region, an 1.1-kb PCR fragment of INO1
corresponding to bases 265-1336 of the coding region, and a 0.46-kb
PCR fragment of ACT1 corresponding to bases 974-1434 of the
coding region. Autoradiographs were visualized with a PhosphorImager
(Amersham Biosciences).
PCR and Primers
Standard PCR methods were used. Primers used for PCR are:
INO502 (5'-GGAATTATGCTCATTGGGTTAGGTG-3') and INO302
(5'-ACATCAACTCACTGTAATACTCGTC-3') for INO1 fragment
amplification, KAR501 (5'-CAACAGACTAAGCG-3') and KAR301
(5'-CGTTAGTGATGGTG-3') for KAR2 fragment amplification, ACT974 (5'-CTTCGAACAAGAAATGC-3') and ACT1924 (5'-AGAAACACTTGTGGTG-3') for ACT1 fragment amplification, and HAC5'IN
(5'-CAACTCGAGAAATGAATAC-3') and HAC3'IN
(5'-GTGTTCTTGTTCACTGTAG-3') for HAC1 fragment amplification.
All inserts were sequenced by the dideoxy method using the reagents and
methods suggested by the Sequenase kit (Amersham Biosciences).
Fluorescence-activated Cell Sorting (FACS) Analysis
Cells were fixed in 70% ethanol for 6 h at room
temperature with rotation. Fixed cells were sequentially treated with
0.25 mg/ml RNase (Sigma) in 50 mM Tris-HCl, pH 7.8, for
12 h at 37 °C and 1.9 mg/ml pepsin (Sigma) for 30 min at
37 °C and then stained with 50 mg/ml propidium iodide (Sigma) in 180 mM Tris-HCl, pH 7.5, 190 mM NaCl, 70 mM MgCl2 for 30 min. Just before FACS analysis, cell suspensions were diluted 1:25 with 50 mM Tris-HCl, pH
7.8, and sonicated for 1 min to break up clumps. Total fluorescence intensities were measured by quantitative flow cytometry using an Epics
Profile II Cytometer (Coulter Electronics). 20,000 counts were read per
sample, and the resulting DNA content was determined and plotted on log scale.
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RESULTS |
hac1
Mutations Confer Unique, Background-dependent
Phenotypes in SK1 Strains--
To generate a hac1 null
allele, one of the copies of HAC1 in the wild-type SK1
diploid LNY3 (Table I) was replaced with
a hac1
100::URA3 allele (see
"Experimental Procedures") (42). Sporulation and subsequent tetrad
analysis produced four viable spores, with the
hac1
100::URA3 disruption allele
segregating 2:2. However, spores carrying the
hac1
100::URA3 mutation cosegregated with slow growth, thus forming only small colonies on YPD medium after
4 days at 30 °C (Fig. 1A).
Microscopic examination revealed that the
hac1
100::URA3 mutant yeast were
larger, were somewhat elongated, and contained large vacuoles compared
with wild-type cells (Fig. 1B). To verify that slow colony
growth and increased cell size were a direct result of the
hac1 deficiency, a wild-type HAC1 gene on a
centromere-based plasmid pRS414-HAC1 was introduced into
LNY542 (a/
hac1
100::URA3/+) (40).
After sporulation and tetrad analysis, we found that the growth rate
and cell size of both wild-type and hac1 spore clones
harboring pRS414-HAC1 were comparable, indicating that these
phenotypes were a consequence of hac1 deficiency.
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Table I
Yeast strains used in this study
All strains were preexisting in this lab or created for this study
except the kar2 complementation tester MS192 which was
kindly provided by Dr. Mark Rose (Princeton University).
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Fig. 1.
hac1 100::URA3 mutants
display slow colony formation and increased cell size.
A, tetrads derived from LNY542 were dissected on YPD medium
and incubated at 30 °C for 4 days. Following photography the spore
clones were scored for genetic markers. The large colonies are
HAC1 (Ura ), and the small colonies are
hac1 100::URA3 (Ura+).
B, WT, LNY1, and
hac1 100::URA3, LNY688 viewed under
differential contrast interference microscopy (magnification, ×1000)
using a Nikon Eclipse E800 microscope.
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Several lines of evidence indicate that this deletion was not a
neomorphic allele. First, the
hac1
100::URA3 allele removed most of
the coding region including the entire DNA binding domain and leucine
zipper, rendering it unlikely that any residual activity remained.
Second, a second hac1
allele,
hac1
200::LEU2 (LNY545, Table I),
which extended the deleted region to remove both the initiator codon
and the promoter, produced phenotypes identical to those conferred by
hac1
100::URA3 (data not shown).
Furthermore, both alleles were fully recessive to wild type, which is
inconsistent with expression of a neomorphic mutation. Finally, we
created hac1
100::URA3 strains in the
W303 genetic background and found that these strains did not exhibit
the SK-specific phenotypes, but were defective for the UPR (see below).
To confirm that Hac1p activity was eliminated, we analyzed the SK1 and
W303 hac1
100::URA3 stains for
phenotypes known to be conferred by hac1 null mutations.
hac1 mutants are inositol auxotrophs and are defective in
the UPR. To monitor the UPR, we assayed for growth in the presence of
tunicamycin (44). Whereas wild-type strains from both genetic
backgrounds were able to form colonies on medium containing 1 µg/ml
tunicamycin, hac1
100::URA3 mutants
in either background did not grow (Fig.
2A). Furthermore, tunicamycin-stimulated induction of a UPRE-dependent
-galactosidase reporter gene was defective in
hac1
100::URA3 mutants derived from
both SK1 and W303 genetic backgrounds (data not shown). Wild-type W303
strains were inositol prototrophs; however, isogenic
hac1
100::URA3 mutants grew very
poorly in the absence of inositol, as previously described (Fig.
2B) (25, 45). Surprisingly, wild-type SK1 strains were
significantly compromised for growth in the absence of inositol.
However, this limited growth capacity was completely abolished in the
presence of hac1
100::URA3 (Fig.
2B, top right). Thus, both W303 and
SK1 hac1
100::URA3 strains are
inositol auxotrophs.

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Fig. 2.
hac1 100::URA3
are defective in the UPR. A, hac1
mutants are tunicamycin-sensitive. SK1 WT, LNY2; SK1
hac1 100::URA3, LNY686; W303 WT, LNY315;
and W303 hac1 100::URA3, LNY625 streaked for
single colonies on a YPD plate supplemented with (right) or
without (left) 1 µg/ml tunicamycin and incubated at
30 °C for 3 days. B,
hac1 100::URA3 and SK1 strains are inositol
auxotrophs. Cell suspensions of the indicated strains were serially
diluted in 100-fold increments and spotted onto synthetic complete
medium with (+) and without ( ) supplemental inositol and incubated at
30 °C for 2 days. Top left, W303 WT, LNY315;
W303 hac1 100::URA3, LNY625. Top
right, SK1 WT, LNY2; SK1
hac1 100::URA3, LNY686. Bottom, SK1
WT (LNY2) strains were transformed with pRS425-KAR2
(pKAR2) or pRS425 (Vector). C,
Northern analysis of hac1 100::URA3
versus wild-type strains. Strains of the indicated genotype
(SK1 WT, LNY2; SK1 hac1, LNY686; W303 WT, LNY315; W303
hac1, LNY625) were grown in YPD to early-log phase, and
aliquots were incubated in the presence (+) or absence ( ) of 1 µg/ml TM. Total RNA was prepared for Northern blot analysis and
probed with KAR2, INO1, and ACT1
probes. The arrows indicate the position of cognate
transcript; the arrows labeled u and s
denote the unspliced and spliced forms of the HAC1 message,
respectively.
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Northern blot analysis was used to directly monitor the UPR (Fig.
2C). Under normal growth conditions, the unspliced form of
HAC1 mRNA was detectable in wild-type strains and the
spliced form accumulated after UPR induction by tunicamycin treatment (11, 14). As expected, HAC1-derived message was not
detectable under any conditions in the
hac1
100::URA3 mutants.
Tunicamycin-induced expression of KAR2, a UPR target gene,
was abolished in the hac1
100::URA3 mutants compared with wild-type strains. Similarly, induction of
INO1 was defective in hac1 strains. The
phenotypes and expression patterns observed in our hac1
mutants demonstrate that these were indeed null alleles. In fact, this
study also revealed that INO1 induction in wild-type SK1
strains is very weak compared with that in W303 strains, and may
explain the poor growth of the SK1 strains in the absence of inositol.
It is possible that the inositol auxotrophy inherent in SK1 strains may
contribute to the slow colony growth and increased cell size associated
with SK1 hac1
mutants. However, this is not likely
because supplementing the medium with excess inositol did not suppress
the phenotype in the SK1 background (data not shown). In addition,
because both wild-type and hac1 mutant SK1 cells grow well
in the medium containing inositol (Fig. 2B), inositol
metabolism is likely not defective under these conditions. In addition,
overexpression of Kar2p did not rescue the poor growth of wild-type SK1
strains in the absence of inositol (Fig. 2B,
bottom). Therefore, it is unlikely that the phenotype we
report here is synthetic phenotype of the UPR and inositol biosynthetic pathways.
SK1 hac1
Mutants Behave Genetically as Diploids--
To further
characterize the SK1 hac1
100::URA3
mutants, we backcrossed the spore clones derived from tetrad analysis
of LNY3 (+/+) and LNY542
(hac1
100::URA3/+).
Tetrads derived from SK1 strains characteristically displayed high
viability (99%) and 2:2 segregation of all markers, as was the case
for LNY3 (Table II, line 1). Likewise,
diploid strains heterozygous for
hac1
100::URA3 (LNY542) that were
generated by one-step gene replacement also produced tetrads with four
viable spores (Fig. 1A). A total of 55 tetrads were
analyzed; 215 of the 220 spores were viable, and the heterozygous
markers (hac1
100::URA3 and
MAT) segregated 2:2, as expected (Table II, line 2). Unusual
genetic behavior was observed, however, upon tetrad analysis on strains
generated by cross of SK1
hac1
100::URA3 spore clones to
wild-type SK1 haploids (Table II, line 3). First, spore viability was
poor; only 146 of the expected 460 spores dissected formed colonies
(32%), and viability was found to be independent of the
HAC1 or MAT genotype. Furthermore, marker
frequency within the viable spores was not random. We obtained an
excess of prototrophs when scoring traits in which the
hac1
100::URA3 parent was
prototrophic (including
hac1
100::URA3 and TRP1,
which segregated independently in backcrosses subsequently performed to
specifically assess marker segregation). Rather than half of the viable
spores being auxotrophs and half prototrophs for a given marker, two
thirds of the viable spores were prototrophs. When examining
segregation of the MAT locus, we recovered an excess of
spores expressing the MAT allele derived from the
hac1
100::URA3 parent as well as
non-maters. Finally, growth and morphology characteristics of the spore
clones were highly variable and unpredictable. We observed severe
flocculance, cell shape abnormalities, colony morphology abnormalities,
and random cell death.
All the above properties are reminiscent of spore clone behavior
following meiosis of a triploid parent (46), suggesting that the
hac1
100::URA3 spore clones
originally contained a 2C genome content. Indeed, similar viability and
phenotypic results were obtained upon tetrad analysis on a triploid
generated by mating a wild-type MAT
strain to a wild-type
diploid homozygous at the MATa locus (Table II, line
4). It is important to note that tetrad analysis on wild-type strains
generated by backcross of the wild-type spores from LNY542
(a/
hac1
100::URA3/+) showed
normal viability (96%; 92 spores of an expected 96) and normal marker
segregation (Table II, line 5). Thus, the increase in ploidy associated
with the SK1 hac1
100::URA3 strains
was spore-clone autonomous and, therefore, did not result from
defective meiotic divisions.
If the hac1
100::URA3 spore clones
were diploid, then restoration of an "even" number genome content
prior to meiosis should restore high spore viability and generate a
predictable pattern of marker segregations. To test this, we
backcrossed the hac1
100::URA3 strains to each other or to wild-type diploid strains that are homozygous at the MAT locus (Table II, lines 6 and 7). Both
combinations of crosses should generate tetraploid mating products that
produce viable, diploid spores following meiosis (46). In fact, this is
what we observed; spore viability from both types of crosses was 99%,
and marker segregation was consistent with the independent segregation
of two pairs of each chromosome during meiosis. This pattern was most
easily observed by examining behavior at the MAT locus; both
maters (a and
in equal numbers, totaling 27.7%) and
nonmaters (72.3%) were recovered. Furthermore, the nonmaters were
sporulation-competent (thus, they carry at least one
MATa allele and one MAT
allele). These
properties were reminiscent of spore clone behavior following meiosis
of a tetraploid parent (46), again suggesting that the original hac1
100::URA3 spore clones donated a
diploid genome content during mating.
SK1 hac1
Spore Clones Contain a Diploid DNA Content--
FACS
analysis verified that the
hac1
100::URA3 spore clones were
indeed diploid. The DNA content of unsynchronized, overnight cultures
of SK1 hac1
100::URA3 mutants was
compared with that of SK1 wild-type strains of defined ploidy (Fig.
3). Examination of the G1 and
G2 peaks of wild-type haploid and diploid strains defined
the normal 1C, 2C, and 4C DNA contents of SK1 strains (Fig.
3A, i and ii).
hac1
100::URA3 spore clones had a DNA
content indistinguishable from the wild-type diploid (Fig.
3A, iii), indicating that they were diploid.
Therefore, the genome appeared stably diploid, and this could explain
the increased cell size associated with
hac1
100::URA3 mutants. Diploid cells
are often larger than their haploid counterparts under normal growth
conditions (47, 48). Wild-type sister spores derived from the same
tetrad analysis that generated the
hac1
100::URA3 spore clones had the
normal, haploid DNA content (Fig. 3A, iv). Thus,
the increase in ploidy observed in the
hac1
100::URA3 strains was a
post-meiotic event. Stability of the ploidy increase was monitored by
analyzing the self-mating products between
hac1
100::URA3 spore clones and
products from mating with wild-type haploid strains (Fig.
3A, vi and vii); hac1
× WT haploid strains contained a triploid genome content, whereas
hac1
× hac1
strains contained a tetraploid
genome content. To demonstrate that the increased ploidy of
hac1
100::URA3 mutants was a
consequence of hac1 deficiency, a wild-type HAC1
gene on a centromere-based plasmid was introduced into the original
hac1
100::URA3 heterozygote (LNY542).
Upon sporulation and tetrad analysis both plasmid-containing
hac1
100::URA3 mutants and wild-type
sister spores had a haploid DNA content (Fig. 3B,
iii and iv). Thus, expression of HAC1
complemented the ploidy increase associated with
hac1
100::URA3 mutations in SK1
strains. Microscopic observation of
hac1
100::URA3 strains stained with
the nuclear dye 4,6-diamidino-2-phenylindole showed that each cell
contained only one nucleus and the nuclei of budding cells were
appropriately localized to the mother-daughter neck (data not shown).
Thus, the ploidy increase associated with SK1 hac1
mutants was not a consequence of defective nuclear migration or
division resulting in the generation of cells containing multiple nuclei.

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Fig. 3.
SK1 hac1 spore clones
contain a 2C DNA content. A, FACS analysis of SK1
strains of the indicated genotype was performed on non-synchronized,
stationary cultures grown in rich medium. The biphasic
peaks represent cells with G1 and G2
DNA content; the vertical axis represents cell
number, and the horizontal axis reflects DNA
content on a logarithmic scale. These data are representative examples;
identical results were obtained with all isogenic strains of a given
genotype. i, WT haploid, LNY1; ii, WT diploid,
LNY3; iii, hac1 100::URA3
spore clone, LNY686; iv, WT sister spore clone from same
tetrad as LNY686, LNY687; v, WT triploid, LNY645;
vi, mating product of
hac1 100::URA3 crossed to a WT
haploid, LNY553; vii, mating product of two
hac1 100::URA3 spore clones of
opposite mating type crossed together, LNY648. B,
HAC1 complements the ploidy increase defect associated with
SK1 hac1 100::URA3 mutants.
i, WT haploid, LNY1; ii, WT diploid, LNY3;
iii, plasmid-bearing WT spore clone derived from tetrad
analysis of LNY542 harboring pRS414-HAC1; iv,
plasmid-bearing hac1 100::URA3 spore
clone derived from same tetrad as iii.
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The Ploidy Increase in hac1 Mutants Occurs after
Germination--
Acquisition of an extra genome could result from
defective meiosis I or II division. However, nondisjunction during
either meiotic division could not account for the four viable spores detected, indicating that at least one genomic equivalent was appropriately apportioned to each spore. Furthermore, addition of the
extra genome could not precede the meiotic divisions because wild-type
sister spores were haploid. Hence, diploidization was hac1
100::URA3-specific and must
result from a post-meiotic event. It is possible that
hac1
100::URA3 nuclei undergo an
extra round of DNA replication that is not followed by mitotic
division, thus generating diploid spores that are packaged within the
ascus. In this case, the spores would be diploid prior to germination and all subsequent mitotic divisions would yield diploid cells. Alternatively, mitotic disjunction or defective nuclear migration during germination of hac1
100::URA3
spores could restrict all of the genetic material to either the mother
or daughter cell. In this case, one viable diploid cell per germination
division would be detected; the sister cell would contain no genetic
information and be dead. Finally, it is possible that the genome
content slowly increased over a period of many cell divisions during
colony formation. However, this model is unlikely because it cannot
explain the efficient gain of one extra genome.
To address these models, we analyzed the DNA content of the
hac1
100::URA3 spores themselves.
FACS analysis with dispersed spores was impracticable because of spore
wall autofluorescence and hydrophobic interactions (49). Therefore, the
ploidy of hac1
100::URA3 and
wild-type spores was determined, indirectly, following mating to
haploid tester strains. If the spores were haploid, then immediate
mating to wild-type haploids would yield diploids. Alternatively, if
the spores were diploid, then the mating products would be triploid.
Tetrads derived by sporulation of LNY542
(hac1
100::URA3/+) were dissected and
immediately allowed to mate to either MATa or
MAT
tester haploids that were micromanipulated into
position abutting the dissected spores. Mating products were identified
after incubation by prototrophic selection. FACS analysis revealed that
all mating products derived from crossing haploid testers to wild-type
or hac1
100::URA3 spores prior to
germination (mating initiated within 3 h of spore dissection) contained a diploid DNA content, indicating that both the wild-type and
hac1
100::URA3 spores were "born"
haploid (Fig. 4A,
i-iii). Thus, diploidization was not a consequence of an
unscheduled round of post-meiotic DNA replication in the nascent
hac1
100::URA3 spores.

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Fig. 4.
SK1
hac1 100::URA3 spores are
haploid-intolerant. A, SK1
hac1 100::URA3 spores are haploid and
generate diploid progeny during subsequent mitosis. Spores
(ii and iii) or mitotic spore clones
(v) derived from tetrad dissection of LNY542 were mated to
the haploid tester strain LNY178 (except where noted), and mating
products were isolated by prototrophic selection and subjected to FACS
analysis. Similar results were obtained with spores and clones of the
opposite mating type (using tester strain LNY174). i, WT
diploid, LNY3; ii, LNY1293, which is the mating product of
WT spore × LNY178; iii, LNY1295, which is the mating
product of hac1 100::URA3 × LNY178; iv, WT triploid, LNY645; v, LNY553, which
is the mating product of a
hac1 100::URA3 mitotic spore
clone × LNY1. B, SK1
hac1 100::URA3 spores are
haploid-intolerant. i, WT diploid, LNY3; ii,
hac1 100::URA3 tetraploid generated
by crossing two hac1 spore clones of opposite mating type,
LNY648; iii, representative spore clone derived from tetrad
analysis of LNY648, LNY1383 (identical results were obtained for all
spore clones derived from LNY648 and additional hac1
"homozygous" tetraploids); iv, representative spore
clone derived from tetrad analysis of LNY686 harboring a plasmid
carrying MATa. Identical results were obtained for all
spore clones derived from LNY686.
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Next, we monitored colony forming ability of cells generated soon after
germination of spores derived from LNY542
(hac1
100::URA3/+). Dissected spores
were permitted to germinate, and clusters containing two or three
adhered cells were micromanipulated apart over a period spanning seven
generations. Because of the strong adhesion properties of SK1 cells, it
was not possible to obtain single cells; cell separation via
micromanipulation was only possible after subsequent budding. Thus, we
were actually studying both mothers and daughters together. Once colony
formation was complete, markers were tested and genotypes were assigned
to the original spores. We found that both wild-type and
hac1
100::URA3 spores germinated
efficiently (6-7 h following ascus dissection) and mitotic division
rate was same for both wild-type and hac1
strains over two or three generations (data not shown). Thus,
mitotic nondisjunction during the germination division of
hac1
100::URA3 spores was not likely to be the
basis of the ploidy increase. However, after four or five generations,
viability (assessed by visible colony formation) of the
hac1
clones became erratic. Many of the cell clusters
failed to form colonies, indicating a high degree of random inviability
(data not shown). Considering that each cluster was composed of two or
three cells, failure to form a colony represents a dramatic defect. In
contrast, the wild-type clones continued to generate viable progeny. It
is possible that this random inviability was the cause of the apparent
slow growth phenotype associated with
hac1
100::URA3 mutants.
Interestingly, it is precisely at this stage in colony formation that
hac1
100::URA3 diploid cells were
identified (assayed indirectly by mating to haploid testers followed by
FACS analysis, as above). Indeed, hac1
100::URA3 cells derived from
subsequent generations (as early as 18 h following germination;
microcolony containing approximately
20 cells) generated triploid
mating products after crossing to tester haploids, demonstrating that
they were diploid at the time of mating (Fig. 4A,
iv and v). Wild-type clones at this stage, however, remained haploid. Thus, the results of the viability and FACS
analyses, taken together, indicated that SK1 hac1
spores were "born" haploid and became diploid during the subsequent
mitotic division(s).
SK1 hac1 Mutants Are
Haploid-intolerant--
hac1
100::URA3spore
clones derived from strains heterozygous for
hac1
100::URA3 (created by gene
replacement) contained a 2C DNA content. Significant populations of
cells containing higher ploidies were never observed in FACS analyses.
Thus, hac1
mutants experienced a ploidy increase from 1C
to 2C, but did not continue to aberrantly increase their genomic
content. Is this because the increase in genome content could only
occur following meiosis and spore germination, or were
hac1
100::URA3 mutants simply unable to
tolerate the haploid state? To test whether a hac1
deficiency would generate a genome duplication following spore
germination regardless of initial ploidy, the spore clones derived from
tetrad analysis of second generation
hac1
100::URA3 (diploid) × hac1
100::URA3 (diploid) and
hac1
100::URA3 (diploid) × hac1
200::LEU2 (diploid) matings were analyzed.
If genome duplication was routine for germinating hac1
spore clones, then the resultant colonies should be tetraploid: diploid
spores formed following meiosis, which then double their genome
content. However, the spore clones contained a DNA content indistinguishable from the wild-type diploid (Fig. 4B,
i and iii). Thus, no ploidy increase was
observed, indicating that genome duplication was not a general property
associated with hac1
100::URA3 spore clones;
instead, it was restricted to an increase from haploid to diploid,
suggesting that hac1
mutants were unable to tolerate the
haploid state. Indeed, when hac1
100::URA3
(MAT
) autodiploid strains were transformed with a plasmid
carrying MATa, it sporulated and formed viable
diploid spore clones (Fig. 4B, iv). Therefore, we
conclude that SK1 hac1
mutants were unable to tolerate
the haploid state.
The UPR Mediates Haploid Tolerance through KAR2--
If haploid
intolerance was consequence of a defective UPR, then SK1
ire1 mutants should display the same ploidy defect that hac1 mutants. Sporulation and subsequent tetrad analysis on
SK1 diploid strain LNY1408 (a/
ire1
100::TRP1/+, Table I) produced
four viable spores with the
ire1
100::TRP1 disruption allele
segregating 2:2. However, like hac1 mutants, spores carrying the ire1
100::TRP1 mutation
cosegregated with slow growth forming only small colonies on rich
medium. Microscopic observation revealed that the ire1
mutant cells were somewhat larger than the wild type (data not shown).
Finally, FACS analysis demonstrated
ire1
100::TRP1 spore clones contained
a DNA content indistinguishable from the wild-type diploid (Fig.
5A). Thus, IRE1 was
required for haploid tolerance in SK1 strains. Therefore, the growth,
morphology, and ploidy defects associated with SK1 hac1 (and
ire1) mutants are likely to be a novel manifestation of
a defective UPR.

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Fig. 5.
Haploid tolerance requires IRE1
and is mediated through Kar2p. Representative data from FACS
analysis are shown. A, i, SK1 WT haploid, LNY2;
ii, SK1 WT diploid, LNY3; iii,
ire1 100::TRP1 spore clone, LNY1411;
iv, WT sister spore clone from same tetrad as LNY1411,
LNY1410. B, i, SK1 WT haploid, LNY2 harboring
pRS425 (vector); ii, SK1 WT diploid, LNY3 harboring pRS425
(vector); iii,
hac1 100::URA3 harboring pRS425
(vector); iv, plasmid-bearing SK1
hac1 100::URA3 derived by tetrad
analysis of hac1 100::URA3/+ (LNY542)
that had been transformed with pRS425-KAR2 prior to sporulation,
LNY1304.
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UPR-dependent induction of the KAR2 gene,
encoding the ER resident protein chaperone BiP, requires splicing of
HAC1 mRNA (22, 50). Because Kar2p is a major
transcriptional target of Hac1p, we tested whether supplemental
KAR2 expression can suppress the haploid-intolerant
phenotype. hac1 strains carrying a high copy vector
expressing Kar2p displayed a haploid DNA content indicating that
hac1 is suppressed by Kar2p overexpression (Fig.
5B). In addition, both the morphology and growth defects
associated with hac1 were also suppressed (data not shown).
This result suggests that HAC1 mediates ploidy through
control of KAR2 expression in the SK1 genetic background.
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DISCUSSION |
During the course of characterizing the behavior of
hac1 mutants, we uncovered a novel phenotype associated with
hac1 deficiency in the sporulation-proficient SK1 genetic
background. In this study we elucidated five unique features of this
phenotype. First, hac1
mutants displayed increased cell
size, and behaved genetically as autodiploids. Second, FACS analysis
verified that hac1
100::URA3 spore
clones contain a diploid DNA content. Third, the autodiploidization event occurred after germination; hac1 spores were born
haploid and generated diploid progeny during the subsequent mitotic
division(s). Fourth, interestingly, genome duplication was not a
general property associated with
hac1
100::URA3 spore clones. Rather,
it was restricted to an increase from haploid to diploid. Consistent
with this observation, hac1
mutants were unable to
tolerate the haploid state. Finally, the growth, morphology, and ploidy
defects associated with hac1 and ire1 mutants
were suppressed by Kar2p overexpression and likely are a novel
manifestation of a defective UPR. The results support the conclusion
that the UPR is required to maintain the haploid state in the SK1
background. Taken together, our results argue that Hac1p controls
ploidy by affecting gene products whose expression is
UPR-dependent. The most likely explanation is that the UPR is required for the synthesis and/or localization and/or degradation of
one or more proteins required to maintain haploid tolerance and genomic
identity. The UPR can contribute to the maintenance of ploidy
indirectly by regulating protein translocation, because Kar2p is
required for the translocation of SRP-independent and SRP-dependent precursors into the yeast ER and Hac1p or
Ire1p overexpression suppresses mutants defective in COPII
vesicle formation (26, 51-53). Considering that Kar2p is required for
maintaining luminal ER-associated protein degradation substrates in a
retrotranslocation-competent state, the defect in SK1
hac1
mutant could be resulted from defective ER-associated protein degradation (54). Thus, disruption of the UPR
would reduce (or abolish) activities of these factors required to
maintain the haploid state.
There are several ways to achieve an increase in ploidy phenotype in
yeast. One simple explanation is the presence of multiple nuclei. This
state can result from a failure to undergo budding, nuclear migration
or cytokinesis and would yield bi- or multinucleated cells. This is
unlikely to be what happens in UPR mutants because multinucleated cells
are highly unstable and generate offspring with both increased and
decreased genomic contents. Furthermore, microscopic examination
revealed the presence of only one nucleus in the hac1
mutants. We cannot rule out that the original autodiploidization event
occurs as a result of the fusion of two haploid nuclei trapped within a
single cell. However, such a mechanism requires an explanation for how
and why exactly two nuclei happened to be in the same cell and why they
would fuse. Indeed, nuclear fusion in yeast efficiently occurs only in
pheromone-induced cells during the mating process, and there is no
evidence for unscheduled activity of this pathway (55). In addition,
considering that KAR2 complements a mutation that blocks
nuclear fusion (27) and KAR2 expression is up-regulated by
Hac1p, nuclear fusion is unlikely to be favored in hac1 null
mutants. Thus, a mechanism for autodiploidization involving an
intermediate that includes multiple nuclei is unlikely.
An alternative explanation is unscheduled cell fusion between progeny
cells. However, the frequency of cell fusion between cells of the same
mating type in yeast is extremely low. Even under circumstances in
which cell fusion is favored because of spheroplast formation, the
frequency of nuclear fusion following cell fusion is extremely low
(55). In this situation, the frequency of diploid formation resulting
in homozygosity at MAT was induced significantly if the
cells were pretreated with mating pheromone, suggesting that nuclear
fusion requires activation by conjugation-specific signals (55).
Therefore, the cell fusion model is viable only if Hac1p depletion in
haploid cells results in inappropriate stimulation of the mating
pathway in combination with an abnormal tendency for cell fusion.
Inconsistent with this model is the unusually low mating efficiency
between SK1 hac1 cells of opposite mating types (data not
shown), indicating that the mating response pathway is actually
reduced, not increased, in hac1 mutants. Indeed, null alleles of CER1/LHS1, a Hac1p target gene in the
UPR pathway, display decreased secretion of the mating pheromone
-factor caused by a translocational block of the
-factor
precursor during transit into the ER (56). Therefore, cell fusion is
unlikely to be the mechanism accounting for the ploidy increase in
hac1 cells.
A third mechanism permitting a ploidy increase is endomitosis:
chromosomal DNA replication in the absence of karyokinesis and
cytokinesis (57, 58). In yeast, overreplication can be induced by a
number of perturbations, including decreased CDC16, CDC27, or
G2 cyclin/p34Cdc28 activity or high
levels of specific components of the DNA replication machinery (35, 59,
60). In Saccharomyces cerevisiae, Cdc6p, a component of the
pre-replication complex, is required to limit DNA replication to once
per cell cycle (31, 61, 62). Consistent with this, overexpression of
Cdc18, the Schizosaccharomyces pombe homolog of
S. cerevisiae CDC6, drives cells into a cycle of
continuous DNA synthesis without mitosis (63). Expression of
Cdc18 is promoted by the S. pombe Cdc10p
transcription factor (64). This is of interest because ectopic
expression of S. cerevisiae HAC1 suppresses cdc10
S. pombe mutations (65),
providing a possible connection between the UPR and DNA replication
control. However, the connection is the opposite of what would be
expected based on the increased ploidy defect associated with the loss
of UPR function. Nevertheless, it is attractive to speculate that Hac1p
might interact with regulators of cell cycle gene expression in
S. cerevisiae.
A common defect leading to accumulation of cells with an increased
genomic content is failure to properly segregate replicated DNA at
mitosis. This could result from defects in nuclear migration/separation (33, 66, 67), chromosome duplication (59, 60), or spindle pole body
duplication or mitotic spindle formation (68, 69). In some cases
impaired nuclear division could lead to a failure in cytokinesis.
However, there is an incongruity inherent in attributing the
hac1-dependent ploidy increase to any mechanism
involving perturbation of the cell cycle. Usually, defects in the
latter manifest themselves as ever-increasing polyploidization and
eventual cell death because the faulty event recurs every cell cycle
(34). In contrast, hac1 mutants experience a ploidy increase
from 1C to 2C, but do not appear to continue to aberrantly increase
their genome content. Thus, endomitosis or failure in chromosome
segregation could only be an explanation for the hac1 mutant
phenotype if we consider that the defect resulting in the ploidy
increase is "suppressed" (or no longer inherent) in hac1
cells that have become diploid. Increased gene dosage of any gene(s)
that function(s) downstream of HAC1 in the UPR pathway,
ER-associated protein degradation, inositol biosynthetic pathway, or
other pathway would quell the motivating force for the ploidy increase
in hac1 haploid cells. Indeed, a stable gene
dosage-dependent ploidy increase was observed in mutants
harboring conditional alleles of MOB1, an essential yeast
gene required for completion of mitosis, cytokinesis, and maintenance
of ploidy (70, 71). mob1 conditional alleles confer a
genomic increase in ploidy at the permissive temperature. It is thought
that this increase is mediated through activity of the Mps1p protein
kinase, which controls spindle pole body duplication and mitotic
checkpoint regulation (70).
The ploidy increase associated with hac1 was genetic
background-dependent. The autodiploidization observed in SK1
strains was not observed in W303 hac1 mutants. There are
many examples of strain background-dependent phenotypes in
yeast, and these are usually a result of pathway redundancy and gene
expression polymorphisms. We found that wild-type SK1 strains grow
poorly in medium lacking inositol, and INO1 basal level
expression and induction with tunicamycin treatment is very weak in
rich medium. Thus, the SK1 hac1 mutant phenotype we observed
could be attributed to a combination of intrinsically poor
INO1 expression in combination with the UPR-mediated defect.
Because inositol is involved in many cellular pathways including lipid
metabolism, it is possible that some underlying genetic defect in the
secretory pathway or lipid metabolism might be synthetic with
hac1 in the SK1 genetic background. However, defects in at
least 31 different genes are known to generate inositol auxotrophy.
These genes play a role in a number of cellular processes including
transcription (SWI1, SWI2, SWI3, RPB1/RPO21/SUA8, RPB2, RPB4,
SRB2, ADA1, SUB1, SPT7, SPT15, SPT20/ADA5, BSD2, HAC1, INO2, INO4,
GCN5, SNFs), cell growth (SNF1, SUA5), cell cycle (BUR1/SGV1),
chromosome segregation (BUR2), chromosome silencing and exit from
mitosis (NET1), ATP import (SAC1), and others (IRE1, TRL1, UBC4, UBC5,
DOA4, SCS2, SCS3, CSE1), etc. (12, 72-83).
The phenotype of hac1 mutants observed in our genetic and
FACS analyses suggests that SK1 hac1 mutants are true
autodiploids. Our experiments cannot rule out the possibility that SK1
hac1 mutants are not perfectly diploid, i.e. they
may accumulate or lose chromosomes becoming monosomic, disomic, or
trisomic etc., and those cells that have attained the "true"
diploid state are selected for in our experiments by virtue of their
selective advantage. Furthermore, FACS analysis is not sensitive enough
to distinguish simple aneuploids (2C
1 or 2C+1) from true diploids.