The Unfolded Protein Response Is Required for Haploid Tolerance in Yeast*

Kyungho LeeDagger §, Lenore Neigeborn, and Randal J. KaufmanDagger ||

From the Dagger  Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109 and § Rutgers College, New Brunswick, New Jersey 08901

Received for publication, October 11, 2002, and in revised form, January 21, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HAC1 encodes a transcription factor that mediates the unfolded protein response (UPR) in Saccharomyces cerevisiae. We characterized hac1Delta mutants in the sporulation-proficient SK1 genetic background and found a novel function for HAC1 in haploid tolerance. hac1Delta spore clones contain a diploid DNA content as determined by fluorescence-activated cell sorting and genetic analyses. Autodiploidization of hac1 spore clones occurred after germination; hac1 spores were born haploid, but efficiently generated diploid progeny during the subsequent mitotic division. Once the hac1 mutant acquired a diploid DNA content, no further ploidy increase was observed. Interestingly, the increase in genome content following meiosis was not a general property associated with hac1 spore clones; instead, it was restricted to an inability to tolerate the haploid state. Genetic analyses involving the UPR target gene KAR2 and the UPR regulator IRE1 revealed that autodiploidization associated with hac1 mutants is a consequence of its role in the UPR pathway. Inhibition of the UPR pathway induces autodiploidization, and constitutive activation of UPR target genes suppresses this response.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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, hac1Delta 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 alpha -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/alpha 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 congruent  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-hac1Delta 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-hac1Delta 2 was created by deleting the 0.45-kb SalI fragment from pRS416-hac1Delta 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 hac1Delta 100::URA3 and hac1Delta 200::LEU2 Null Alleles-- To create the hac1Delta 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-hac1Delta 2 to create pRS416-hac1Delta 100::URA3. Only the first 8 NH2-terminal amino acids of HAC1 can be expressed from this allele. To create the hac1Delta 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-hac1Delta 2 to create pRS416-hac1Delta 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/alpha 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 hac1Delta 100::URA3 (2.6-kb KpnI-XbaI fragment of pRS416-hac1Delta 100::URA3) or hac1Delta 200::LEU2 (3.2-kb ApaI-XbaI fragment of pRS416-hac1Delta 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 hac1Delta 100::URA3 or hac1Delta 200::LEU2, were sporulated and subjected to tetrad analysis.

Construction of the ire1Delta 100::TRP1 Allele-- To create the ire1Delta 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 ire1Delta 100::TRP1 heterozygotes were sporulated and subjected to tetrad analysis on medium containing 30 µg/ml inositol, because ire1 mutants are inositol auxotrophs (40).

beta -Galactosidase Assay

Quantitative beta -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 MATalpha ), 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

hac1Delta 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 hac1Delta 100::URA3 allele (see "Experimental Procedures") (42). Sporulation and subsequent tetrad analysis produced four viable spores, with the hac1Delta 100::URA3 disruption allele segregating 2:2. However, spores carrying the hac1Delta 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 hac1Delta 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/alpha hac1Delta 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.   hac1Delta 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 hac1Delta 100::URA3 (Ura+). B, WT, LNY1, and hac1Delta 100::URA3, LNY688 viewed under differential contrast interference microscopy (magnification, ×1000) using a Nikon Eclipse E800 microscope.

Several lines of evidence indicate that this deletion was not a neomorphic allele. First, the hac1Delta 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 hac1Delta allele, hac1Delta 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 hac1Delta 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 hac1Delta 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 hac1Delta 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, hac1Delta 100::URA3 mutants in either background did not grow (Fig. 2A). Furthermore, tunicamycin-stimulated induction of a UPRE-dependent beta -galactosidase reporter gene was defective in hac1Delta 100::URA3 mutants derived from both SK1 and W303 genetic backgrounds (data not shown). Wild-type W303 strains were inositol prototrophs; however, isogenic hac1Delta 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 hac1Delta 100::URA3 (Fig. 2B, top right). Thus, both W303 and SK1 hac1Delta 100::URA3 strains are inositol auxotrophs.


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Fig. 2.   hac1Delta 100::URA3 are defective in the UPR. A, hac1 mutants are tunicamycin-sensitive. SK1 WT, LNY2; SK1 hac1Delta 100::URA3, LNY686; W303 WT, LNY315; and W303 hac1Delta 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, hac1Delta 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 hac1Delta 100::URA3, LNY625. Top right, SK1 WT, LNY2; SK1 hac1Delta 100::URA3, LNY686. Bottom, SK1 WT (LNY2) strains were transformed with pRS425-KAR2 (pKAR2) or pRS425 (Vector). C, Northern analysis of hac1Delta 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.

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 hac1Delta 100::URA3 mutants. Tunicamycin-induced expression of KAR2, a UPR target gene, was abolished in the hac1Delta 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 hac1Delta 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 hac1Delta 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 hac1Delta Mutants Behave Genetically as Diploids-- To further characterize the SK1 hac1Delta 100::URA3 mutants, we backcrossed the spore clones derived from tetrad analysis of LNY3 (+/+) and LNY542 (hac1Delta 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 hac1Delta 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 (hac1Delta 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 hac1Delta 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 hac1Delta 100::URA3 parent was prototrophic (including hac1Delta 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 hac1Delta 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.


                              
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Table II
hac1Delta spore clones display aberrant genetic behavior

All the above properties are reminiscent of spore clone behavior following meiosis of a triploid parent (46), suggesting that the hac1Delta 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 MATalpha 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/alpha hac1Delta 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 hac1Delta 100::URA3 strains was spore-clone autonomous and, therefore, did not result from defective meiotic divisions.

If the hac1Delta 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 hac1Delta 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 alpha  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 MATalpha allele). These properties were reminiscent of spore clone behavior following meiosis of a tetraploid parent (46), again suggesting that the original hac1Delta 100::URA3 spore clones donated a diploid genome content during mating.

SK1 hac1Delta Spore Clones Contain a Diploid DNA Content-- FACS analysis verified that the hac1Delta 100::URA3 spore clones were indeed diploid. The DNA content of unsynchronized, overnight cultures of SK1 hac1Delta 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). hac1Delta 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 hac1Delta 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 hac1Delta 100::URA3 spore clones had the normal, haploid DNA content (Fig. 3A, iv). Thus, the increase in ploidy observed in the hac1Delta 100::URA3 strains was a post-meiotic event. Stability of the ploidy increase was monitored by analyzing the self-mating products between hac1Delta 100::URA3 spore clones and products from mating with wild-type haploid strains (Fig. 3A, vi and vii); hac1Delta  × WT haploid strains contained a triploid genome content, whereas hac1Delta  × hac1Delta strains contained a tetraploid genome content. To demonstrate that the increased ploidy of hac1Delta 100::URA3 mutants was a consequence of hac1 deficiency, a wild-type HAC1 gene on a centromere-based plasmid was introduced into the original hac1Delta 100::URA3 heterozygote (LNY542). Upon sporulation and tetrad analysis both plasmid-containing hac1Delta 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 hac1Delta 100::URA3 mutations in SK1 strains. Microscopic observation of hac1Delta 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 hac1Delta 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, hac1Delta 100::URA3 spore clone, LNY686; iv, WT sister spore clone from same tetrad as LNY686, LNY687; v, WT triploid, LNY645; vi, mating product of hac1Delta 100::URA3 crossed to a WT haploid, LNY553; vii, mating product of two hac1Delta 100::URA3 spore clones of opposite mating type crossed together, LNY648. B, HAC1 complements the ploidy increase defect associated with SK1 hac1Delta 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 hac1Delta 100::URA3 spore clone derived from same tetrad as iii.

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 hac1Delta 100::URA3-specific and must result from a post-meiotic event. It is possible that hac1Delta 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 hac1Delta 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 hac1Delta 100::URA3 spores themselves. FACS analysis with dispersed spores was impracticable because of spore wall autofluorescence and hydrophobic interactions (49). Therefore, the ploidy of hac1Delta 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 (hac1Delta 100::URA3/+) were dissected and immediately allowed to mate to either MATa or MATalpha 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 hac1Delta 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 hac1Delta 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 hac1Delta 100::URA3 spores.


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Fig. 4.   SK1 hac1Delta 100::URA3 spores are haploid-intolerant. A, SK1 hac1Delta 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 hac1Delta 100::URA3 × LNY178; iv, WT triploid, LNY645; v, LNY553, which is the mating product of a hac1Delta 100::URA3 mitotic spore clone × LNY1. B, SK1 hac1Delta 100::URA3 spores are haploid-intolerant. i, WT diploid, LNY3; ii, hac1Delta 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.

Next, we monitored colony forming ability of cells generated soon after germination of spores derived from LNY542 (hac1Delta 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 hac1Delta 100::URA3 spores germinated efficiently (6-7 h following ascus dissection) and mitotic division rate was same for both wild-type and hac1Delta strains over two or three generations (data not shown). Thus, mitotic nondisjunction during the germination division of hac1Delta 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 hac1Delta 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 hac1Delta 100::URA3 mutants. Interestingly, it is precisely at this stage in colony formation that hac1Delta 100::URA3 diploid cells were identified (assayed indirectly by mating to haploid testers followed by FACS analysis, as above). Indeed, hac1Delta 100::URA3 cells derived from subsequent generations (as early as 18 h following germination; microcolony containing approximately approx 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 hac1Delta spores were "born" haploid and became diploid during the subsequent mitotic division(s).

SK1 hac1 Mutants Are Haploid-intolerant-- hac1Delta 100::URA3spore clones derived from strains heterozygous for hac1Delta 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, hac1Delta 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 hac1Delta 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 hac1Delta 100::URA3 (diploid) × hac1Delta 100::URA3 (diploid) and hac1Delta 100::URA3 (diploid) × hac1Delta 200::LEU2 (diploid) matings were analyzed. If genome duplication was routine for germinating hac1Delta 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 hac1Delta 100::URA3 spore clones; instead, it was restricted to an increase from haploid to diploid, suggesting that hac1Delta mutants were unable to tolerate the haploid state. Indeed, when hac1Delta 100::URA3 (MATalpha ) 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 hac1Delta 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/alpha ire1Delta 100::TRP1/+, Table I) produced four viable spores with the ire1Delta 100::TRP1 disruption allele segregating 2:2. However, like hac1 mutants, spores carrying the ire1Delta 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 ire1Delta 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, ire1Delta 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, hac1Delta 100::URA3 harboring pRS425 (vector); iv, plasmid-bearing SK1 hac1Delta 100::URA3 derived by tetrad analysis of hac1Delta 100::URA3/+ (LNY542) that had been transformed with pRS425-KAR2 prior to sporulation, LNY1304.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, hac1Delta mutants displayed increased cell size, and behaved genetically as autodiploids. Second, FACS analysis verified that hac1Delta 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 hac1Delta 100::URA3 spore clones. Rather, it was restricted to an increase from haploid to diploid. Consistent with this observation, hac1Delta 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 hac1Delta 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 alpha -factor caused by a translocational block of the alpha -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.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: National Genome Research Inst., National Inst. of Health, Nokbun-dong, Eunpyung-ku, Seoul 122-701, Korea.

|| To whom correspondence should be addressed: Howard Hughes Medical Inst., MSRB II, Rm. 4570, 1150 W. Medical Center Dr., University of Michigan Medical Center, Ann Arbor, MI 48109. Tel.: 734-763-9037; Fax: 734-763-9323; E-mail: kaufmanr@umich.edu.

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M210475200

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; UPR, unfolded protein response; UPRE, unfolded protein response element; WT, wild-type; FACS, fluorescence-activated cell sorting; TM, tunicamycin.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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