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Vol. 8, Issue 10, 1845-1862, October 1997
HSP Research Institute, Kyoto Research Park, Shimogyo-ku, Kyoto 600, Japan
Submitted December 6, 1996; Accepted July 21, 1997  |
ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions ("ER stress"). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR in Saccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing of HAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin-proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little or no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stress-induced mRNA splicing, leading to activation of the UPR.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Newly synthesized secretory and transmembrane proteins traverse
the endoplasmic reticulum (ER), where they fold into
correct tertiary and quaternary structures. The productive folding
process of these proteins is assisted by molecular chaperones and
folding enzymes localized in the ER (reviewed by Gething and Sambrook, 1992
; Helenius et al., 1992). When unfolded proteins are
accumulated in the ER by a variety of physiological or environmental
stress conditions ("ER stress"), synthesis of these chaperones and
enzymes is known to be induced at the level of transcription (Lee,
1987; Kozutsumi et al., 1988). This means that eukaryotic
cells possess an intracellular signaling pathway from the ER to the
nucleus, called the unfolded protein response (UPR) pathway (reviewed
by McMillan et al., 1994; Shamu et al., 1994;
Pahl and Baeuerle, 1997).
In Saccharomyces cerevisiae, several components of the UPR
have been identified. Genetic approach identified the
ERN1/IRE1 gene that encodes a transmembrane protein kinase
Ern1p localized in the ER (Cox et al., 1993; Mori et
al., 1993). Ern1p spans the ER membrane (or possibly the nuclear
membrane, which is continuous to the ER membrane) once, with its
N-terminal half located inside microsomes and with its C-terminal half
carrying essential protein kinase activity on the cytoplasmic side
(Mori et al., 1993). The N-terminal domain of Ern1p is
likely to sense the accumulation of unfolded proteins and activates the
protein kinase domain through its own oligomerization and
autophosphorylation (Shamu and Walter, 1996; Welihinda and Kaufman,
1996), although the mechanism remains unknown. Thus, the information
about changes in the ER lumen is transmitted to the cytosolic
compartment by Ern1p and then to the nucleus. In the nucleus, each of
the target genes of the UPR is thought to contain a
cis-acting element, termed the unfolded protein-response
element (UPRE) as an upstream activator sequence (Mori et
al., 1992; Kohno et al., 1993; Partaledis and Berlin, 1993). UPRE was proposed to be a binding site of putative unfolded protein-response factor (UPRF; Mori et al., 1992). So far
six ER lumenal proteins have been shown to be regulated by the UPR, including two essential proteins: Kar2p, a member of the heat shock
protein 70 family (Normington et al., 1989; Rose et
al., 1989), and Pdi1p, protein disulfide isomerase (LaMantia
et al., 1991; Tachikawa et al., 1991).
UPRE was originally identified as a 22-bp sequence that is necessary
and sufficient for the induction of Kar2p by ER stress (Mori et
al., 1992). We conducted extensive mutational analysis and showed
that UPRE contains a partial palindrome with a spacer of 1 nucleotide
(nt; CAGCGTG) that is essential for its
function (Mori et al., 1996). This sequence resembles the
E-box consensus (CANNTG) to which trans-acting factors
containing a basic region as a DNA-binding domain are known to bind as
a homo- or heterodimer (Hurst, 1995; Littlewood and Evan, 1995).
However, the presence of a single C residue between CAG and GTG is
characteristic of UPRE and this one-base spacing is critical for the
response to ER stress. In addition, the sequence GAA located upstream
of the partial palindrome was found to be important for the activity of
UPRE. Thus, we proposed that yeast UPRF is composed of total three
trans-acting polypeptides (a dimer of a basic
region-containing protein[s] plus a protein recognizing the sequence
GAA; Mori et al., 1996).
We and others recently identified the
HAC1/ERN4 gene as an essential component of
the UPR (Cox and Walter, 1996; Mori et al., 1996; Nikawa
et al., 1996). The nonessential HAC1 gene encodes a basic-leucine zipper (bZIP) protein (Hac1p) of 230 aa. Haploid cells
lacking Hac1p (hac1
) are unable to induce transcription of any of the target genes tested and exhibit sensitivity to ER stress.
By using electrophoretic mobility shift assays, Hac1p was shown to bind
specifically to UPRE (Cox and Walter, 1996; Mori et al.,
1996). In addition, we demonstrated that Hac1p recognizes the
palindromic sequence separated by 1 nt in UPRE both in vivo and in
vitro. These results led us to conclude that Hac1p represents a major
component of the putative transcription factor UPRF responsible for the
UPR (Mori et al., 1996).
Cox and Walter (1996) further analyzed the mechanism of activation of
Hac1p and reported that the UPR is regulated by an unconventional type
of mRNA splicing that is induced by Ern1p-mediated signaling from the
ER: internal 252 nt were removed by the splicing from constitutively
expressed HAC1 precursor mRNA (pre-mRNA) to produce mature
mRNA in response to ER stress in the wild-type but not in the
ern1 strain. Moreover, the expression of the intron-less HAC1 cDNA constitutively activated the UPR even in the
ern1 strain. This splicing is unique in that the
sequences around the splice sites do not match the consensus for
conventional splicing (Kreivi and Lamond, 1996). Another interesting
feature is that the open reading frame switches: the splicing causes
replacement of the C-terminal portion of Hac1p due to the location of
the 5
splice site inside the coding region. HAC1 pre-mRNA
encodes a protein of 230 aa, whereas mature mRNA encodes a protein of
238 aa, although these two proteins are supposed to share an identical
N-terminal 220 aa.
Importantly, only ER-stressed cells produced detectable amounts of
Hac1p, which was shown by immunoblotting to contain 18 aa encoded by the second exon and thus translated from mature mRNA.
Three lines of evidence led to the proposal (Cox and Walter, 1996) that
although 230aa-Hac1p was constitutively translated from pre-mRNA, it
was highly unstable and rapidly degraded by the ubiquitin-proteasome
pathway, whereas 238aa-Hac1p produced from mature mRNA was stable.
First, a majority of pre-mRNA and mature mRNA was shown to be
associated with polysomes, suggesting that they both could be
translated. Second, 
-galactosidase expressed from a reporter gene
monitoring the UPR was constitutively elevated in mutants defective in
some of the ubiquitin conjugating enzymes. Third, when the last 10 aa
present in 230aa-Hac1p was deleted, the resulting 220aa-Hac1p
constitutively activated the UPR as effectively as 238aa-Hac1p produced
from the intron-less HAC1 cDNA. They inferred from these
results that the combination of the N-terminal portion of 220 aa and
the 10 aa, but not the last 18 aa present in 238aa-Hac1p, made Hac1p
highly unstable.
We independently identified the regulated splicing of HAC1 pre-mRNA in response to ER stress, as described in the initial sections of this article. We then present data focusing on the question of why only Hac1p translated from mature mRNA is detected in cell extracts. Contrary to the previous proposal cited above, the differential protein stability is unlikely to be the main mechanism regulating the expression of Hac1p. The evidence presented below clearly indicates that the presence of intron prevents pre-mRNA from being translated: namely, Hac1p is synthesized only after the mRNA splicing takes place. Accordingly, we propose an alternative model for the regulated expression of Hac1p leading to activation of the UPR in S. cerevisiae.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Strains and Microbiological Techniques
The yeast strains used in this study are listed in Table
1. The compositions of rich broth medium
(YPD) and synthetic complete medium used for selection of transformants
such as SC(
Ura, Leu) have been described (Sherman et al.,
1986). Tunicamycin was obtained from Sigma (catalogue no. T-7765) and
used at a concentration of 5 µg/ml throughout the experiments. Yeast
cells were transformed by the lithium acetate method (Ito et
al., 1983).
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Construction of Plasmids
Recombinant DNA techniques were carried out as described
(Sambrook et al., 1989). Reporter plasmids, such as pSCZ-Y
and pMCZ-Y (the UPRE-CYC1-lacZ gene carried on a single-copy
vector and a multicopy vector, respectively), and expression plasmids
for the ERN1 gene, such as YCp-ERN1 and YEp-ERN1 (Cen and 2 µm, respectively, in Figure 3), were described previously (Mori
et al., 1996). YCp-HAC1 used herein (also referred as
YCp-HAC1WT) was previously described as YCp-ERN4, which contains the
LEU2-selectable marker (Mori et al., 1996).
YCp-HAC1HN or HA in Figure 2 was constructed by deleting the
0.67-kb
HindIII818-EcoNI1492
fragment or 0.33-kb
HindIII818-AscI1144
fragment from YCp-HAC1WT, respectively. YCp-HAC1intron (also referred as 238-type) was constructed by replacing the 0.87-kb MluI272-AscI1144
fragment in YCp-HAC1WT with the corresponding fragment of the reverse
transcriptase (RT)- coupled polymerase chain reaction (PCR) product
obtained from tunicamycin-treated cells (Figure 4B), thus lacking the
intron of the 252 nt. The intron-less HAC1 gene was also
inserted into a single-copy vector YCp50 containing the URA3
selectable marker (Rose and Broach, 1991). YCp-HAC1-220-type and
230-type in Figures 6, 7, and 10 were constructed by site-directed mutagenesis (Kunkel, 1985). A XbaI site was introduced at nt
640 in YCp-HAC1WT or YCp-HAC1intron [designated WT(XbaI)
or intron(XbaI), respectively, in Figure 9] by replacing
the MfeI fragment (residues 632-645) with a double-stranded
oligonucleotide (AATTGATTCTAGA, XbaI site
underlined). Three mutant versions of the HAC1 gene described in Figure 9 (Ala221Stop, Ala221Stop, and Asn216Stop) were
constructed by replacing the 0.18-kb
XbaI640-HindIII818
fragment with the corresponding fragment of the product obtained by
PCR-mediated mutagenesis after its sequence had been confirmed.
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Preparation of Cell Extracts
Yeast cell extracts for electrophoretic mobility shift assays
and immunoblotting were prepared from approximately 100 ml of midlogarithmic phase cultures (OD600 ~ 0.8) as
described (Mori et al., 1992) with some modifications.
Breakage buffer [200 mM Tris(hydroxymethyl)aminomethane (Tris)-HCl,
pH8.0, 10 mM MgCl2, 10% glycerol, and 0.5 mM
dithiothreitol] contained various protease inhibitors (2 µM
pepstatinA, 2 µM leupeptin, 2 mg/l chymostatin, 2 mM benzamidine, and
1 mM phenylmethylsulfonyl fluoride). Total proteins extracted as
described (Mori et al., 1992) were precipitated by a final
concentration of 2.6 M (NH4)2SO4 by
using 4 M instead of saturated
(NH4)2SO4 to obtain whole cell
extracts. When proteins in extracts were fractionated, proteins were
first precipitated by 2.0 M
(NH4)2SO4 (final concentration) to
obtain fraction A. Proteins that remained in the supernatant were then
precipitated by 2.6 M (NH4)2SO4 to
obtain fraction B. Precipitated proteins were dissolved in ~200 µl
of buffer and dialyzed extensively against dialysis buffer (20 mM
HEPES, pH 7.9, 50 mM KCl, 0.25 mM EDTA, 10% glycerol, and 0.5 mM
dithiothreitol) at 4°C. The protein concentration in extracts ranged
between 2 and 6 mg/ml, as determined by the Bio-Rad protein assay kit
(Bio-Rad, Richmond, CA). Bovine serum albumin was used as a standard.
Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays were carried out as
described (Mori et al., 1996). When competition experiments
were performed, unlabeled competitor DNA was included in the binding buffer before addition of cell extracts.
Immunoblotting
Fifty micrograms of proteins in cell extracts per lane were
subjected to SDS-PAGE using 12% (for Hac1p) or 8% gel (for Kar2p and
Pdi1p) and transferred to a Hybond-ECL nitrocellulose filter (Amersham,
Arlington Heights, IL). Rainbow colored protein molecular weight
markers (Amersham) were used as size markers. Rabbit polyclonal antisera against Kar2p (kindly provided by Dr. D. Williams, University of Toronto) or Pdi1p (kindly provided by Dr. H. Tachikawa, Tokyo University of Agriculture and Technology) were used at a 1:5000 dilution. Rabbit polyclonal antibodies were raised against the maltose-binding protein-Hac1p (230-type) fusion protein expressed and
purified from Escherichia coli cells (Mori et
al., 1996). After antibodies against maltose-binding protein were
removed from the antiserum by affinity chromatography through
maltose-binding protein immobilized on CH-Sepharose 4B (Pharmacia,
Piscataway, NJ), antibodies specific to Hac1p were purified with
CH-Sepharose 4B to which the fusion protein had been coupled.
Anti-Hac1p antibodies were dialyzed against phosphate-buffered saline
and the resulting solution of OD280 ~ 0.8 was used at a
1:2000 dilution. ECL Western blotting detection kit (Amersham) was used
to detect each antigen.
Northern Blot Hybridization Analysis
Northern blot hybridization analysis was carried out as
described previously (Mori et al., 1993, 1996). Total RNAs
were subjected to 1.5% agarose gel electrophoresis containing
formaldehyde. The DNA probe for the yeast LHS1/SSI1/CER1
(Baxter et al., 1996; Craven et al., 1996;
Hamilton and Flynn, 1996) or UBC4 (Seufert and Jentsch, 1990) was prepared by PCR.
-Galactosidase Assays
Assays for -galactosidase activity in yeast cell extracts
were carried out as described previously (Mori et al.,
1993).
Pulse-Chase Analysis of Hac1p
The method described by Franzusoff et al. (1991) was
basically used. Various transformants were grown at 30°C to
midlogarithmic phase in minimal medium containing 100 µM
(NH4)2SO4, 2% glucose, and other
nutrients as required. Cells were harvested at 8 OD600 units by centrifugation, washed once with distilled water, and resuspended in 2.6 ml of minimal medium containing 2% glucose and
other nutrients but lacking
(NH4)2SO4. After shaking at 30°C for 30 min, cells were pulse-labeled for 5 min at 30°C with 400 µCi
(14.8 MBq) of EXPRE35S35S protein labeling mix
(DuPont, Wilmington, DE) and chased by adding a 0.01 volume of
concentrated chase solution [100 mM
(NH4)2SO4, 0.3% cysteine, and
0.4% methionine]. An aliquot (650 µl) was removed and mixed with 7 ml of ice-cold 0.1 M Tris-HCl, pH 8.0, and 10 mM NaN3 to
terminate the labeling. Cells were recovered by centrifugation, washed
once with 1 ml of ice-cold 0.1 M Tris-HCl, pH 8.0, 10 mM NaN3, and resuspended in 200 µl of TBS/1% SDS containing
1 mM phenylmethylsulfonyl fluoride and 5 µg/ml leupeptin. Cells were disrupted by vigorous agitation with acid-washed glass beads
(0.425-0.6 mm in diameter, Sigma, St. Louis, MO) for four 30-s periods
at intervals of 90 s on ice. The resulting lysate was freed of
glass beads, boiled for 5 min, and after addition of 800 µl of
TBS/2% Triton X-100, clarified by centrifugation at 14,000 rpm for 10 min. The supernatant was treated with 10 µl each of purified
anti-Hac1p antibodies and anti-Pdi1p antibodies as an internal control.
After standing overnight at 4°C, 50 µl of 50% protein A-Sepharose
4 fast flow (Pharmacia) in TBS were added and the mixture was rotated for 2 h at room temperature. The resin was then washed as
described by Franzusoff et al. (1991), and the
immunoprecipitates were subjected to SDS-PAGE (12% gel). The amounts
of 35S-labeled Hac1p and Pdi1p were analyzed by using
BioImaging Analyzer BAS-2000 (Fuji Photo Film, Stamford, CT).
Other Techniques
RT-PCR was carried out using SUPERSCRIPT preamplification system (Life Technologies, BRL, Gaithersburg, MD) and Takara Ex Taq (Takara, Berkeley, CA). Hac1p was translated in vitro using TNT coupled wheat germ extract system (Promega, Madison, WI) and appropriate HAC1-derived DNAs as templates.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Induction of Hac1p by Tunicamycin
The wild-type (ERN+) and the
hac1 strains were grown in rich medium (YPD) and
incubated for 1 h in the presence or absence of tunicamycin, known
to elicit ER stress by inhibiting N-glycosylation of newly
synthesized proteins in the ER (Elbein, 1981; Kozutsumi et
al., 1988). Whole cell extracts were prepared, and electrophoretic mobility shift assays were carried out to determine whether
UPRE-specific DNA-binding activity was affected by unfolded proteins
accumulated in the ER. When the wild-type UPRE designated UPRE(Y) was
used as a 32P-labeled probe, specific binding activity was
detected only with extracts prepared from tunicamycin-treated
ERN+ strain (Figure
1Aa, lane 2). This
binding was specific because it was not detected when a mutant version
of UPRE designated UPRE(Tv10) was used as a probe (lane 6). UPRE(Tv10)
contains a single transversion at an essential nucleotide in the
palindromic sequence that almost completely abolishes the function of
UPRE (Mori et al., 1996). In addition, extracts from the
hac1 strain did not exhibit any specific binding to
UPRE(Y) (lane 4), indicating that the binding obtained in lane 2 represents UPRF of which Hac1p functions as a major component. In
contrast, when the cAMP-response element (CRE) was used as a probe,
similar amounts of specific binding activity [marked as CREBP in
Figure 1Aa] were detected in extracts prepared from the
ERN+ or hac1 strain that had been
treated or untreated with tunicamycin, confirming that Hac1p plays
little or no role in cellular CRE-binding activity, contrary to the
previous report (Nojima et al., 1994). These results
indicated that DNA-binding activity of UPRF was induced by ER stress.
Previously, UPRE-specific DNA-binding activity was detected
constitutively (Mori et al., 1992), but the assay condition
used was not optimal because 10 mM MgCl2 was found to be
required for specific binding (Mori et al., 1996). Because significant amounts of nonspecific binding (indicated by * and **) were
also detected, proteins in extracts were fractionated by differential
precipitation with (NH4)2SO4 as
described in MATERIALS AND METHODS. As shown in Figure 1Ab, UPRF was
recovered in fraction A (lane 10), whereas nonspecific binding** was
recovered in fraction B (lanes 13-16). By using fraction A from
tunicamycin-treated ERN+ strain, specific
binding of UPRF to 32P-labeled UPRE(Y) was
competed by 50- or 250-fold molar excess of various UPRE-like sequences
(Figure 1B). The ability of a mutant UPRE to compete the specific
binding correlated very well with its ability to mediate the UPR in
vivo (Mori et al., 1996).
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Next we performed immunoblotting analysis to detect Hac1p in cell extracts with purified anti-Hac1p antibodies. As shown in Figure 1Ac, Hac1p was detected only in extracts of tunicamycin-treated ERN+ cells (lane 10), indicating that not only DNA-binding activity but also Hac1p itself was induced by ER stress. Hac1p migrated as a single band of 41 kDa in SDS-PAGE (12% gel), which is appreciably larger than its calculated molecular weight 26,045 but identical with the position of Hac1p translated in vitro (see Figure 6). A high content of charged residues may account for this aberrant behavior.
Induction of Hac1p by Tunicamycin Requires the 3 Untranslated
Region (UTR) of HAC1 mRNA
When the ERN+ strain was grown in synthetic
medium selective for plasmid maintenance, a small amount of Hac1p was
produced but the amount was markedly enhanced by treatment with
tunicamycin for 1 h (Figure 2, lanes
1 and 2; immunoblot). The relatively high basal expression
of Hac1p, as compared with cells grown in broth, probably results from
increased amounts of unfolded proteins in the ER: it was not detected
in the ern1 strain lacking functional Ern1p, a key enzyme
in the signal transduction across the ER membrane (Figure 2, lane 3).
This is consistent with the approximately 2.5-fold higher
-galactosidase expressed from the UPRE-CYC1-lacZ reporter
gene in the ERN+ strain as compared with the
ern1 or hac1 strain grown in the absence of
tunicamycin (Figure 2, compare bars 1 and 3 or 5). The treatment of the
ERN+ strain with tunicamycin for 3 h caused
an approximately 10-fold induction of -galactosidase under these
conditions (Figure 2, bar 2). The induction of both Hac1p and
-galactosidase depended entirely on the function of Ern1p (Figure 2,
lane 4) and Hac1p (Figure 2, lane 6). Introduction of the wild-type
HAC1 gene into the hac1 strain fully restored
the induction of both proteins (Figure 2, lanes 7 and 8).
Interestingly, deletions downstream of the Hac1p-coding region (HN
and HA, Figure 2, lanes 9-12) abolished the induction of both
proteins, indicating that the induction of Hac1p requires 3UTR of the
mRNA. This also suggested that the induction of Hac1p occurs
posttranscriptionally rather than transcriptionally, consistent with
the absence of UPRE in the promoter region.
Induction of the 1.2-kb HAC1 mRNA by Tunicamycin
We then used Northern blot hybridization to determine whether ER
stress affected the amount or size of mRNA transcribed from the
chromosomal HAC1 gene. In the ERN+
strain, 1.4-kb HAC1 mRNA was expressed even in the absence
of tunicamycin (Figure 3A). Transcription
of the KAR2 gene, a target of the UPR, dramatically
increased within 1 h after addition of tunicamycin. At this time,
the amount of 1.4-kb HAC1 mRNA decreased and instead, the
smaller mRNA (1.2 kb) was observed. The smaller mRNA failed to appear
in the ern1 strain carrying a vector alone (V) but did
appear when the ERN1 gene was provided by a single-copy plasmid (Cen). The overexpression of the ERN1 gene from a
multicopy vector (2 µm) is known to activate the UPR (Mori et
al., 1993; Shamu and Walter, 1996): the amount of KAR2
mRNA increased by twofold in the absence of tunicamycin. Under these
conditions, the smaller HAC1 mRNA was detectable in the
absence of tunicamycin. Thus, the induction of 1.2-kb HAC1
mRNA by ER stress correlated well with the cellular UPR.
Because tunicamycin treatment may cause pleiotropic defects in cellular
metabolism, we also examined the effect of the temperature-sensitive sec53 mutation on HAC1 mRNA. At nonpermissive
temperature, sec53 cells accumulate full-length precursors
of secretory proteins that are abnormally glycosylated and malfolded in
the ER due to the defect in phosphomannomutase activity (Feldman
et al., 1987), leading to activation of the UPR (Normington
et al., 1989; Rose et al., 1989). As expected,
the 1.4-kb HAC1 mRNA was constitutively expressed in the
sec53, ERN+ strain grown at the permissive
temperature of 23°C (Figure 3B). Upon a shift to a seminonpermissive
temperature of 30°C, the smaller HAC1 mRNA as well as
KAR2 mRNA was markedly induced within 1 h. Neither
KAR2 mRNA nor the smaller HAC1 mRNA were induced
at 30°C in the sec53, ern1 strain. These results
strongly indicated that 1.2-kb HAC1 mRNA is induced only if
unfolded proteins are accumulated in the ER, regardless of the nature
of stress conditions employed.
Time-course experiments with the ERN+, SEC+ strain revealed that the amount of 1.4-kb mRNA decreased within 10 min after addition of tunicamycin, concomitant with the appearance of 1.2-kb mRNA (Figure 3C). The sum of the two mRNA species remained almost constant, suggesting that 1.4-kb mRNA was directly converted to 1.2-kb mRNA. On the other hand, the amounts of target mRNAs (KAR2 and PDI1) when normalized with that of ACT1 mRNA, increased only after 10-20 min. Thus, the appearance of the 1.2-kb HAC1 mRNA preceded the induction of target mRNAs, consistent with the notion that the induction of 1.2-kb HAC1 mRNA is required for the UPR.
ER Stress-induced Splicing of HAC1 mRNA
The 1.4-kb and 1.2-kb HAC1 mRNAs were then
compared, particularly with respect to the 3UTR, because a small
deletion downstream of the Hac1p-coding region abolished the induction
of Hac1p (HA, see Figure 2), and the size of truncated
HAC1 mRNA thus produced, in comparable amounts, remained
unchanged upon addition of tunicamycin. Total RNAs were extracted from
the ERN+ strain grown for 1 h in the
presence or absence of tunicamycin, and mRNAs were converted to cDNAs
by using an oligo(dT) primer and RT. HAC1 cDNA was amplified
by PCR using a 5 primer just upstream of the start ATG codon and each
of the 3 primers indicated in Figure 4A.
Only when the 3 primer 5 or 6 was used, RNAs from tunicamycin-treated
cells gave rise to an additional PCR product shorter, by approximately
250 nt, than those obtained from untreated cells (Figure 4B).
Nucleotide sequence of the shorter PCR product obtained with primers 1 and 5 was determined after inserting it into pT7 blue T-vector
(Novagen, Madison, WI) and found to lack 252 nt, from nt 662 to 913 (Figure 4A). The absence of this region from the 1.2-kb HAC1
mRNA was confirmed by Northern blot hybridization using the various
probes shown in Figure 4A. Both 1.4-kb and 1.2-kb mRNAs were detected
in total RNAs from tunicamycin-treated cells with probe A, D, or E,
whereas only the 1.4-kb mRNA was detected with probe B or C (Figure
4C). Evidently, HAC1 mRNA becomes spliced in response to ER
stress.
The putative 5 and 3 splice sites did not match the consensus
sequence found in S. cerevisiae and higher eukaryotes (GT-AG or AT-AC; Kreivi and Lamond, 1996). Inclusion of G661 but
not G913 in the 1.2-kb mRNA was established by mutating
either of these Gs to A and sequencing the resulting RT-PCR product
obtained from tunicamycin-treated cells. Since the 5 splice site was
located in the coding region, this unusual splicing was expected to
replace the C-terminal portion of Hac1p. Whereas 1.4-kb pre-mRNA should encode a protein of 230 aa, the 1.2-kb mature mRNA should encode a
protein of 238 aa, because the C-terminal 10 aa should be removed by
the splicing, and the remaining 220 aa should be fused to a stretch of
18 aa encoded by the sequence starting with nt 914 (Figure 4A).
Constitutive Activation of the UPR by the Intron-less HAC1 Gene
We then prepared and expressed cDNA corresponding to the 1.2-kb
mRNA in the hac1 strain under the control of the
HAC1 promoter by using a single-copy expression plasmid
(YCp-HAC1intron) and compared its effects with those of the
wild-type gene (YCp-HAC1WT; Figure 5).
Total RNAs and total proteins were extracted from cells grown for
1 h in the presence or absence of tunicamycin. In
hac1 cells carrying YCp-HAC1WT, HAC1 pre-mRNA
was spliced and Hac1p was induced by tunicamycin as in the
ERN+ strain. As a result, transcription of all
of the six target genes (KAR2, LHS1/SSI1/CER1
[Baxter et al., 1996; Craven et al., 1996; Hamilton and Flynn, 1996], SCJ1 [Schlenstedt et
al., 1995], PDI1, EUG1 [Tachibana and
Stevens, 1992], FKB2 [Partaledis and Berlin, 1993]) was
well induced. In contrast, 1.2-kb HAC1 mRNA was
constitutively present in hac1 cells carrying
YCp-HAC1intron and was little affected by tunicamycin as expected.
This resulted in constitutive synthesis of Hac1p and constitutively
high levels of transcripts of all target genes (Figure 5A). The levels
of two target proteins (Kar2p and Pdi1p) were also constitutively
elevated in these cells (see Figure 6B,
lane 9 in the Inset). Cells carrying YCp-HAC1intron grew at
significantly slower rates than those carrying YCp-HAC1WT (doubling
time 3 h versus 2 h), indicating that excess synthesis of
these target proteins is toxic to the cell.
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Consistent with these results, -galactosidase expressed from
the UPRE-CYC1-lacZ reporter gene was markedly induced in the hac1 strain carrying YCp-HAC1WT (Figure 5B, lanes 11 and
12) as in the ERN+ strain (Figure 5B, lanes 1 and 2), and the induction depended on the function of Ern1p (Figure 5B,
lanes 5 and 6). In sharp contrast, -galactosidase was constitutively
expressed in the hac1 strain carrying YCp-HAC1intron
(Figure 5B, lanes 13 and 14). The high level expression in the latter
case did not require the function of Ern1p (Figure 5B, lanes 7 and 8).
Thus, the expression of 1.2-kb HAC1 mRNA lacking the intron
constitutively activates the UPR without involvement of the Ern1p
kinase. From these results, we concluded that the UPR is controlled by
the regulated splicing of HAC1 pre-mRNA.
Constitutive Expression of Hac1p of 238, 220, and 230 aa from Mutant Versions of the HAC1 Gene
The results so far presented are consistent with those reported
previously (Cox and Walter, 1996). Hac1p produced from the intron-less
HAC1 gene (referred as 238-type in Figure 6A) in the hac1 strain migrated at the position identical with that
of 238aa-Hac1p translated in vitro but faster than that of 230aa-Hac1p
translated in vitro (Figure 6C, lanes 9 and 10). The apparent anomaly
in migration may result from the fact that the last 10 aa of
230aa-Hac1p contain two basic and no acidic residues but the last 18 aa
of 238aa-Hac1p contain no basic and three acidic residues. This
provided additional confirmation of the previous observation cited
above that all of Hac1p so far detected in cell extracts was composed of 238 aa and thus translated from mature mRNA. Therefore, we next
focused on the question of why 230aa-Hac1p possibly synthesized from
pre-mRNA was not detected.
As mentioned earlier, Cox and Walter (1996) proposed that pre-mRNA is
also translated but its product of 230 aa is degraded as soon as it is
made due to the C-terminal 10 aa that confer extreme instability to the
otherwise stable N-terminal portion of 220 aa. However, the data on
differential stability among different types of Hac1p were lacking. We
thus tried to express the three types of Hac1p and test their stability
individually. To express 220aa-Hac1p, the 18-aa segment and the whole
intron were deleted from the HAC1 gene, and the resulting
plasmid was introduced into the hac1 strain (Figure 6A,
construct 220-type). This strain produced a large amount of protein
regardless of tunicamycin treatment (Figure 6C, lanes 11 and 12), which
migrated at the position identical with that of 220aa-Hac1p translated
in vitro. The construct 220-type also caused production of
-galactosidase in both ern1 (Figure 6B, bars 3 and 4)
and hac1 (Figure 6B, bars 11 and 12) strains, whose
activities were approximately 40% of those for 238aa-Hac1p (Figure 6B,
bars 1, 2, 9, and 10) with or without tunicamycin.
To express 230aa-Hac1p, we removed the region of nt 691-966 containing
the first stop codon, the majority of the intron, and the 18-aa segment
from the HAC1 gene (see Figure 4A) and transformed the
hac1 strain with the resulting plasmid (Figure 6A,
construct 230-type). Doublet protein bands were detected with or
without tunicamycin (Figure 6C, lanes 13 and 14) that migrated at
positions around that of 230aa-Hac1p translated in vitro. The results
were quite unexpected and suggested that the stability of 230aa-Hac1p may not differ appreciably from that of 238aa-Hac1p.
It was also noteworthy that the -galactosidase activity detected in
tunicamycin-untreated ern1 or hac1 cells
expressing 238aa-Hac1p (Figure 6B, bar 1 or 9) was approximately
12-fold higher than that in cells expressing 230aa-Hac1p (Figure 6B,
bar 5 or 13) when compared after subtracting the activity with vector alone (Figure 6B, bar 7 or 15), whereas the amount of 230aa-Hac1p detected by immunoblotting was nearly half as much as
238aa-Hac1p (Figure 6C, compare lanes 9 with 13). We therefore examined
proteins from tunicamycin-untreated hac1 cells by
immunoblotting with anti-Kar2p and anti-Pdi1p
antibodies: the levels of two target proteins were again found to be
well correlated with those of -galactosidase activity (Figure 6B,
inset). The anti-Hac1p antibodies used were raised against 230aa-Hac1p
fused to maltose-binding protein as described in MATERIALS AND METHODS
and appeared to recognize mainly the bZIP region (our unpublished
results). If we assume the same immunoreactivities of the two proteins,
the specific transcriptional activator activity of 238aa-Hac1p appeared to be severalfold higher than that of 230aa-Hac1p. Thus, the
splicing-mediated replacement of the C terminus may generate a
transcription factor with significantly higher activity. We are
currently characterizing transcriptional activation domains found in
Hac1p of 238, 220, and 230 aa.
Hac1p Is Highly Unstable
We then examined stability of three types of Hac1p after treating
cells with cycloheximide, an inhibitor of protein synthesis. Cycloheximide at 10 to 300 µg/ml completely inhibited the induction by tunicamycin of -galactosidase from the UPRE-CYC1-lacZ
reporter gene without affecting cell viability. Total proteins were
isolated from hac1 cells carrying each of the mutant
versions of the HAC1 gene described in Figure 6A at various
times after addition of 20 µg/ml cycloheximide and
immunoblotted with anti-Hac1p or anti-Kar2p antibodies
(Figure 7A). Whereas the amount of Kar2p
remained virtually unchanged, both the 238aa- and 230aa-Hac1p totally
disappeared within 5 min after addition of cycloheximide; the
220aa-Hac1p seemed to be slightly more stable than the others. A
separate experiment with hac1 cells expressing both
238aa- and 230aa-Hac1p showed that the amounts of both Hac1p decreased
with similar time course (half-life of 1 min or less), indicating that
both proteins are highly unstable in vivo in the absence of protein
synthesis.
To further examine possible differential stability, we conducted
pulse-chase experiments with [35S]methionine and
[35S]cysteine and hac1 cells that were made
to produce both 238aa- and 230aa-Hac1p (Figure 7B). We adopted this
approach particularly to avoid possible complications arising from
differential growth rates: hac1 cells expressing
238aa-Hac1p grew significantly more slowly than those expressing
230aa-Hac1p (doubling time 3 h versus 2 h), perhaps
reflecting the differential ability to transactivate the UPR. The
amount of pulse-labeled 238aa-Hac1p obtained by immunoprecipitation was
1.6-fold higher than that of 230aa-Hac1p, which might be due, at least
in part, to their differential mRNA levels (see Figure 6C). Both types
of Hac1p were chased by unlabeled amino acids with similar time course,
whereas Pdi1p remained virtually unchanged. The quantification of
radioactivities followed by correction with Pdi1p values revealed
essentially no difference in stability between 238aa- and 230aa-Hac1p,
the half-life being approximately 2 min. Thus, the differential protein
stability is unlikely to explain the absence of 230aa-Hac1p in cells
carrying the wild-type HAC1 gene.
Constitutive Activation of the UPR in ubc Mutants Results from Increased but Low Levels of Splicing
Constitutive activation of the UPR in mutants defective in some of
the ubiquitin-conjugating enzymes provided strong support to the
proposal by Cox and Walter (1996). The -galactosidase activities
expressed from the reporter plasmid were approximately 4.5-, 2-, 10-, 5-, and 4-fold higher in the ubc4, ubc5,
ubc45, ubc7, and
ubc8 strains, respectively, than in the wild-type (UBC+) and the increased basal activity in the
ubc45 strain was independent of the mRNA
splicing, suggesting stabilization of 230aa-Hac1p translated from
pre-mRNA in these mutants. Unfortunately, however, the presence of
stabilized 230aa-Hac1p in the mutant extracts was not shown directly.
We thus carried out similar experiments using the same set of strains
and the isogenic UBC+ strain (see Table 1).
These strains (MAT
) were transformed with the
UPRE-CYC1-lacZ reporter gene carried on either
the single-copy or multicopy vector, and -galactosidase activities
in the resulting transformants were determined in the absence of
tunicamycin.
Unexpectedly, -galactosidase activity in the
ubc4 strain was lower, rather than higher, than that in
the UBC+ strain (Figure
8A). We also examined the
ubc4 strain of opposite mating type
(MATa) but the results were almost identical. The
absence of UBC4 mRNA in the ubc4 strain was
confirmed by Northern blot hybridization (Figure 8B), and the levels of
two target proteins of the UPR (Kar2p and Pdi1p) in the
ubc4 strain were not significantly higher than those in
the UBC+ strain (see Figure 8B, bottom).
Currently, we have no explanation for this discrepancy. Similarly,
-galactosidase activity in the ubc45
double mutant was lower than that in the UBC+
strain (Figure 8A) unlike the previous report, although its extremely long doubling time (10-fold of control; Seufert and Jentsch, 1990) posed a question in interpreting the results. On the other hand, -galactosidase activities in the ubc5 and
ubc7 strains were 3.1- and 3.4-fold higher than that in
the UBC+ strain, respectively, when the
multicopy reporter plasmid was used (Figure 8A). However, the increase
was marginal when the single-copy reporter plasmid was used (1.4- and
1.6-fold, respectively), and the levels of Kar2p and Pdi1p were
enhanced only slightly (Figure 8B, bottom).
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We then asked which type of Hac1p was responsible for the weak but
constitutive activation of the UPR in the ubc5 and
ubc7 strains. As in the ERN+
strain, HAC1 pre-mRNA was spliced within 1 h after
addition of tunicamycin in the three ubc mutant as well as
UBC+ strains (Figure 8B). Total proteins were
isolated from cells grown in YPD medium to avoid small but significant
amounts of 238aa-Hac1p that would be produced when cells were grown in
synthetic medium without tunicamycin (see Figures 2 and 5). The normal
levels of 238aa-Hac1p were found after but not before addition of
tunicamycin in all the strains tested (Figure 8B). Upon longer exposure
of the film, low but significantly enhanced levels of 238aa- but not
230aa-Hac1p were detected in the ubc5 and
ubc7 strains in the absence of tunicamycin (Figure 8B,
bottom). This indicated that the splicing of HAC1 pre-mRNA
occurred constitutively at very low levels in these two strains, though
no appreciable amounts of mature mRNA were detected by Northern blot
hybridization. We then disrupted the ERN1 and
HAC1 loci in the ubc5 and ubc7
strains and examined their effects: not only the induction by
tunicamycin but also the increased basal expression of
-galactosidase were abolished (Figure 8C). These results
demonstrated that the constitutively activated UPR in some of the
ubc mutants results from enhanced splicing of
HAC1 pre-mRNA rather than stabilization of the pre-mRNA product.
Hac1p Is Translated Only from Mature mRNA
The results shown in Figures 7 and 8 clearly demonstrated
that differential protein degradation cannot be the main mechanism regulating the expression of Hac1p, contrary to the previous proposal (Cox and Walter, 1996). We then tested the alternative possibility that
the absence of 230aa-Hac1p in cell extracts is due to the lack of
translation of HAC1 pre-mRNA. We showed above that Hac1p of
220aa was expressed constitutively in the hac1 strain
carrying the mutant HAC1 gene lacking the entire intron and
the 18-aa segment (Figure 6, 220-type). It was then asked whether we
could similarly express 220aa-Hac1p from a mutant version of the
HAC1 gene containing the intron. For convenience in plasmid
construction, a XbaI site was introduced at nt 640 between
the two MfeI sites in YCp-HAC1WT and YCp-HAC1intron
[Figure 9, WT(XbaI) and
intron(XbaI), respectively] that changed
Leu214-Asp215 to Ser-Arg (see MATERIALS AND
METHODS). The two amino acid changes did not affect the level of either
HAC1 mRNA or Hac1p but slightly reduced the transcriptional
activator activity of 238aa-Hac1p; only slightly lower levels of
-galactosidase were produced from the
UPRE-CYC1-lacZ reporter gene in the hac1
strain (compare Figure 9, lanes 1-4, with Figure 5B, lanes 11-14).
Three mutants were constructed by using YCp-HAC1WT(XbaI).
The Ala221Stop mutant contained a stop codon at aa 221 instead of the
codon for Ala221 without affecting other nucleotide
sequences in the intron. When this mutant was introduced into the
hac1 strain, similar level of HAC1 pre-mRNA
was expressed as the wild-type without tunicamycin (Figure 9, compare
lane 1 with 5 and also lane 13 with 17). Strikingly, however, this
mutant mRNA was not translated to produce 220aa-Hac1p (Figure 9, lane
17). Addition of tunicamycin also failed to induce Hac1p (Figure 9,
lane 18), probably because cleavage of the mRNA at the 5 splice site
was inhibited due to the alteration of the 3 nt, causing degradation of
the mRNA cleaved only at the 3 splice site. These results should be
contrasted with those shown in Figure 6C, lanes 11 and 12, where a
large amount of 220aa-Hac1p was constitutively expressed from the
mutant HAC1 gene (intron 220-type). To examine synthesis rates of Hac1p, and Pdi1p as a control, cells were
pulse-labeled with [35S]methionine and
[35S]cysteine for 5 min and immunoprecipitates were
analyzed by SDS-PAGE. As shown in Figure
10, the labeled 220aa-Hac1p was
detected from hac1 cells carrying the intron 220-type
mutant but not from those carrying the Ala221Stop mutant; synthesis of
Hac1p from the latter mRNA was estimated to be less than 4% of that
from the intron-less mRNA.
To further examine the effects of splicing on Hac1p synthesis, we
constructed another mutant Ala221Stop by inserting 2 nt, TA, between
the codons for Pro220 and Ala221, so that a
stop codon was created immediately after Pro220 but the
nucleotide sequences at the 5 splice site remained identical from the
position 2. However, the results obtained with Ala221Stop (Figure 9,
lanes 7 and 8) were similar to those with Ala221Stop, indicating that
the sequence upstream of the position 2 is also important for
recognition of HAC1 pre-mRNA by putative endonuclease(s). We
thus made another construct in which a stop codon was created at aa 216 next to the XbaI site. In this case (Asn216Stop), the mutation site was placed far enough from the 5 splice site to allow
tunicamycin-inducible pre-mRNA splicing albeit at reduced efficiencies
(Figure 9, lanes 15 and 16). From this mutant mRNA, Hac1p of probably
215 aa migrating slightly faster than the 220aa-Hac1p translated in
vitro was produced in a tunicamycin-inducible manner. The pattern of
protein expression was indistinguishable from the case of 238aa-Hac1p
produced from the wild-type mRNA (Figure 9, lanes 13 and 14). These
results clearly demonstrated that ER stress-induced splicing of
HAC1 pre-mRNA can effectively lead to the synthesis of
Hac1p.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Hac1p is a trans-acting factor responsible for the UPR,
transcriptional induction of genes encoding ER-localized molecular chaperones and folding enzymes in response to ER stress, in S. cerevisiae (Cox and Walter, 1996; Mori et al., 1996;
Nikawa et al., 1996). Hac1p itself is also induced by ER
stress and the induction is mediated by ER stress-induced
unconventional mRNA splicing as reported previously (Cox and Walter,
1996) and further substantiated by the present study. In addition, we
demonstrated inclusion of G661 but not G913 in
mature mRNA (see Figure 4) and involvement of the splicing in induction
of all known target genes of the UPR (Figure 5). The results from two
laboratories agreed that only Hac1p translated from mature mRNA was
detected in extracts prepared from cells carrying the wild-type
HAC1 gene under a variety of conditions tested. However, the
model proposed by Cox and Walter (1996) to explain sig
). The
Asn216Stop mutant contains a stop codon TAG (double underlined) instead
of the codon for Asn216. The hac1
