From the HSP Research Institute, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan
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ABSTRACT |
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When unfolded proteins are accumulated in the endoplasmic reticulum (ER), an intracellular signaling pathway termed the unfolded protein response (UPR) is activated to induce transcription of ER-localized molecular chaperones and folding enzymes in the nucleus. In Saccharomyces cerevisiae, at least six lumenal proteins including essential Kar2p and Pdi1p are known to be regulated by the UPR. We and others recently demonstrated that the basic-leucine zipper protein Hac1p/Ern4p functions as a trans-acting factor responsible for the UPR. Hac1p binds directly to the cis-acting unfolded protein response element (UPRE) responsible for Kar2p induction. Moreover, we showed that the KAR2 UPRE contains an E box-like palindrome separated by one nucleotide (CAGCGTG) that is essential for its function. We report here that the promoter regions of each of five target proteins (Kar2p, Pdi1p, Eug1p, Fkb2p, and Lhs1p) contain a single UPRE sequence that is necessary and sufficient for induction and that binds specifically to Hac1p in vitro. All of the five functional UPRE sequences identified contain a palindromic sequence that has, in four cases, a spacer of one C nucleotide. This unique characteristic of UPRE explains why only a specific set of proteins are induced in the UPR to cope with ER stress.
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INTRODUCTION |
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Eukaryotic cells possess multiple intracellular signaling pathways from the endoplasmic reticulum (ER)1 to the nucleus (1). One of these, the unfolded protein response (UPR), is conserved from yeast to man and is activated by accumulation of unfolded proteins in the ER under a variety of stress conditions ("ER stress"), resulting in transcriptional induction of molecular chaperones and folding enzymes localized in the ER (2-5). The UPR is thought to be activated primarily to cope with deleterious effects of accumulated unfolded proteins. In addition, the possible linkage of UPR with phospholipid biosynthesis, especially with inositol metabolism, and its involvement in ER homeostasis have also been suggested (6-9).
Budding yeast Saccharomyces cerevisiae is an excellent model system in which to study the molecular mechanism of the UPR and has led to the identification of three genes as essential components of the UPR: the IRE1/ERN1 gene encoding a transmembrane protein kinase localized in the ER (6, 7), the HAC1/ERN4 gene encoding a basic-leucine zipper protein (10-12), and the RLG1 gene encoding tRNA ligase localized in the nucleus (13).
In contrast, all the known target proteins of the UPR are localized in
the ER, where they assist folding and assembly of newly synthesized
secretory and transmembrane proteins as molecular chaperones or folding
enzymes (14, 15). In S. cerevisiae, six lumenal proteins
have been shown to be regulated by the UPR: Kar2p, an Escherichia
coli DnaK homolog (16, 17); Lhs1p/Ssi1p/Cer1p, a member of the
Hsp70 subfamily (18-21); Scj1p, an E. coli DnaJ homolog
(22); Pdi1p, protein-disulfide isomerase (6, 23, 24); Eug1p, a
multicopy suppressor of the pdi1 strain (25); and Fkb2p,
peptidyl-prolyl cis-trans isomerase (26). Genes
encoding each of these ER stress-inducible proteins are thought to
contain an upstream activator sequence, termed the unfolded protein
response element (UPRE), in their promoter region (26-28). UPRE was
originally identified as a cis-acting element necessary for
transcriptional induction of Kar2p by ER stress, and the 22-base pair
sequence is sufficient to confer inducibility on a heterologous
promoter such as the yeast CYC1 promoter (27, 28). UPRE was
proposed to be a binding site of a putative transcription factor
termed unfolded protein response factor (UPRF) (27). A similar sequence found in the FKB2 promoter also conferred inducibility on
the CYC1-lacZ gene (26). However, whether induction of other
ER stress-inducible proteins is mediated by a sequence similar to KAR2 and FKB2 UPREs remains to be investigated.
Moreover, it is important to address the question of why only a limited
set of proteins are transcriptionally induced in response to ER
stress.
We recently conducted extensive mutational analysis of KAR2 UPRE to characterize its fine structure-function relationship (11). For convenience, we have numbered the nucleotides in UPRE, with nucleotide 1 the guanine at the 5'-end and nucleotide 22 the adenine at the 3'-end (see Fig. 1). Among the 22 nucleotides in KAR2 UPRE, nucleotides 10-12 (CAG) and 14-16 (GTG) were most critical for its function; point mutation of any of these nucleotides abolished the response to ER stress almost completely. The sequences CAG and GTG are reminiscent of the E box consensus (CANNTG), to which trans-acting factors containing a basic region as a DNA-binding domain would bind (29, 30). However, KAR2 UPRE contained a single C residue between the half-sites (CAGCGTG) and this one-base spacing was critical for the response to ER stress. This unique spacing appeared to distinguish KAR2 UPRE from other cis-acting elements recognized by basic region-containing trans-acting factors (11).
We and others recently identified the basic-leucine zipper protein Hac1p/Ern4p as a transcription factor responsible for the UPR in S. cerevisiae; haploid cells lacking Hac1p were unable to induce transcription of any of the target proteins tested and exhibited sensitivity to ER stress (10-12). Hac1p was shown to bind specifically to the KAR2 UPRE using electrophoretic mobility shift assays (10, 11). More importantly, we demonstrated that Hac1p recognizes the palindrome separated by a one-nucleotide spacer in KAR2 UPRE both in vivo and in vitro (11). Furthermore, we showed that Hac1p is involved in induction of all known target proteins (11, 31). This indicated that, as in the case of KAR2 UPRE, the promoters of other target proteins might contain a palindromic sequence with a spacer of one nucleotide recognized directly by Hac1p.
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EXPERIMENTAL PROCEDURES |
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Strains and Microbiological Techniques--
The yeast strains
used in this study were KMY1005 (MAT leu2-3, 112 ura3-52
his3-
200 trp1-
901 lys2-801), KMY1015 (KMY1005 ern1
::TRP1), and KMY2005 (MAT
leu2-3,
112 ura3-52 his3-
200 trp1-
901 lys2-801 sec-53-6) (11).
The SCJ1 deletion (scj1
) strain SNY1025
(MAT
leu2-3, 112 ura3-52 his3-
200 trp1-
901 lys2-801 suc2-
9 scj1
::TRP1) and its parental strain SEY6210
(32) were generous gifts of Drs. S. Nishikawa and T. Endo (Nagoya
University). The compositions of rich broth medium (YPD) and synthetic
complete medium used for selection of transformants such as SC(
Ura)
have been described (33). Tunicamycin was obtained from Sigma (T-7765) and used at a concentration of 5 µg/ml throughout the experiments. Yeast cells were transformed by the lithium acetate method (34).
Construction of Reporter Plasmids--
Recombinant DNA
techniques were carried out as described (35). The reporter plasmids
pSCZ2 and pMCZ2 were constructed by modifying the CYC1-lacZ
fusion gene of the multicopy vector pLG-178 (36), a derivative of
pLG670-Z (37), which is often utilized to assess activities of
cis-acting elements in S. cerevisiae (38). Because the lacZ reporter gene in pLG670-Z as well as
pLG
-178 is actually a translational fusion of lacI and
lacZ (38), we transferred the CYC1 portion in
pLG
-178 into another lacZ-containing vector in frame.
Thus, inserted into pUC118 was the 0.25-kb
XhoI-BamHI fragment of the CYC1-lacZ
fusion gene in pLG
-178, which contained the entire CYC1
promoter and 5'-terminal several nucleotides of the
CYC1-coding region located upstream of the BamHI
site (36, 37). From the resulting plasmid, one nucleotide C between the start ATG codon and the BamHI site was deleted by
site-directed mutagenesis (39) so that the ATG codon would be in frame
to the lacZ-coding sequence embedded in pSEYc102 (a
CEN4-ARS1-based single-copy vector) or pSEY101 (a
2-µm-based multicopy vector), both containing the URA3
selectable marker (27). The 0.25-kb EcoRI (derived from the
multicloning site of pUC118)-BamHI fragment of the
mutagenized CYC1 gene was inserted between the
EcoRI and BamHI sites of pSEYc102 and pSEY101 to
create the single-copy vector pSCZ2 and multicopy vector pMCZ2,
respectively. pMCZ2 possesses unique EcoRI and
XhoI sites upstream of the CYC1 promoter for inserting oligonucleotides, whereas pLG
-178 contains only one XhoI site. Various double-stranded, synthetic
oligonucleotides whose 5'- and 3'-termini are complementary to
protruding termini generated by EcoRI and XhoI,
respectively, were inserted between the EcoRI and
XhoI sites of pMCZ2.
Constructs to Determine Promoter Activity-- The 2.5-kb ApaI-PstI fragment containing the entire PDI1 gene in the plasmid pMTY17 (24) was cloned into pUC118, and a BamHI site was created immediately downstream of the start ATG codon (ATGGATCC- - -) by site-directed mutagenesis. The 0.44-kb ApaI-BamHI, 0.30-kb BstBI-BamHI, 0.21-kb SpeI-BamHI, or 0.09-kb MluI-BamHI fragment of the PDI1 promoter was inserted between the SmaI and BamHI sites of pSEYc102. Two point mutations were introduced to PDI1a UPRE by site-directed mutagenesis.
The plasmid pCT20 carrying both the EUG1 and FKB2 genes in tandem was kindly provided by Dr. T. H. Stevens (University of Oregon), and the 2.8-kb HindIII-SalI fragment was cloned into pUC119. The genomic LHS1 gene with franking regions was obtained by screening the yeast genomic library constructed on YEp13, a multicopy yeast vector (ATCC 37323, Ref. 40) and the 1.6-kb EcoRV fragment was cloned into pUC118. A BamHI site was created immediately downstream of the start ATG codon of EUG1, FKB2, or LHS1 (ATGGATCC- - -). The 0.19-kb HindIII-BamHI fragment of EUG1, 0.29-kb AccI-BamHI fragment of FKB2, or 0.27-kb BstXI-BamHI fragment of LHS1 was inserted between the SmaI and BamHI sites of pSEY101. Two point mutations were introduced to EUG1 UPRE, FKB2 UPRE, or LHS1 UPRE. The plasmid pPS177 carrying the entire SCJ1 gene was kindly provided by Dr. P. A. Silver (Harvard Medical School). The 3.0-kb KpnI-HindIII fragment was transferred to pRS316 (a CEN6-ARSH4-based single-copy vector containing the URA3 selectable marker, Ref. 41) to create pRS316-SCJ1. The 1.2-kb KpnI-SphI fragment of pPS177 was cloned into pUC118, and a BamHI site was created immediately downstream of the first or second ATG codon (ATGGATCC- - -). The KpnI-BamHI, StyI-BamHI, AflIII-BamHI, or HgaI-BamHI fragment was inserted between the SmaI and BamHI sites of pSEY101.Assays--
-Galactosidase assays and Northern blot
hybridization analysis were carried out as described previously (7, 11,
31). Electrophoretic mobility shift assays were performed as described (11). Cell extracts were prepared from the wild-type strain (KMY1005)
that had been grown in YPD medium to a mid-log phase and incubated for
1 h with tunicamycin, and proteins were fractionated by ammonium
sulfate according to our previous report (31). Hac1p of 238 amino acids
was translated in vitro using TNTTM-coupled wheat germ
extract system (Promega) and a template HAC1 DNA according to the manufacturer's instructions.
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RESULTS |
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KAR2 UPRE Confers Inducibility on the CYC1-lacZ Gene Regardless of
Its Orientation--
We constructed the reporter plasmids pSCZ2 (a
single-copy vector) and pMCZ2 (a multicopy vector) as described under
"Experimental Procedures." These plasmids contained a modified
version of the CYC1-lacZ gene of pLG-178 (36) and
provided a sensitive and convenient assay system for examining
cis-acting elements in S. cerevisiae. Using
pMCZ2, we showed that the KAR2 UPRE (previously referred to
as UPRE-Y, Ref. 27) contained a partial palindrome separated by one
nucleotide (Fig. 1); nucleotides 10-12
(CAG) and 14-16 (GTG) were most critical (boxed), and
nucleotides 8 (G), 13 (C), and 18 (C) were also important for UPRE
activity (11). In previous experiments to dissect the UPRE,
-galactosidase activity was measured in cells incubated in the
presence or absence of tunicamycin, which is known to elicit ER stress
by inhibiting N-glycosylation of newly synthesized proteins
in the ER (3, 42).
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Specific Binding of UPRE to UPRF in Cell Extracts and Hac1p
Translated in Vitro--
The cellular activity for specific binding to
UPRE, namely UPRF activity, was detected only in ER-stressed cells (10,
31); binding to the wild-type UPRE-Y was obtained with extracts
prepared from tunicamycin-treated cells (Fig.
2A) but not from untreated cells (data not shown). This binding was specific because UPRF did not
bind to a point mutant of UPRE-Y designated Tv10, the activity of which
was less than 1% of UPRE-Y due to a transversion (C to A) at critical
nucleotide 10 (11). The faster migrating band marked by an
asterisk may represent a protein(s) recognizing sequences
outside of the palindrome. Similarly, binding to UPRE-Y but not to
UPRE-Tv10 was obtained with in vitro translated Hac1p (Fig.
2A), which was demonstrated previously to be a transcription factor responsible for the UPR (10, 11). When this UPRE-Y (KAR2 UPRE) was inserted into pMCZ2, 56-fold induction of
-galactosidase was observed after treatment of the wild-type cells
with tunicamycin at 30 °C for 3 h, whereas pMCZ2 alone caused
marginal (2.7-fold) induction (Fig. 3).
Thus,
-galactosidase assays with pMCZ2 as well as electrophoretic
mobility shift assays using cell extracts and Hac1p translated in
vitro allowed us to examine whether promoters of other ER
stress-inducible proteins also included a palindromic sequence directly
recognized by Hac1p.
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FKB2 UPRE Contains a Partial Palindrome Recognized by
Hac1p--
The FKB2 promoter was shown previously to
contain a cis-acting element that conferred inducibility by
ER stress on the CYC1-lacZ gene (26). We confirmed the
observation using the pMCZ2 system; -galactosidase was induced
10-fold by tunicamycin treatment, although the extent of induction was
much lower than that with KAR2 UPRE (Fig. 3). When aligned
with KAR2 UPRE, FKB2 UPRE was found to contain a
partial palindrome separated by one nucleotide (C), although the 3'
half was not well conserved. However, all three of the nucleotides
shown to be important for KAR2 UPRE activity at 8, 13, and
18 (dotted in Fig. 3) were conserved. This FKB2 UPRE competed for the specific binding between 32P-labeled
UPRE-Y (KAR2 UPRE) and UPRF in cell extracts or Hac1p translated in vitro, albeit less efficiently than
KAR2 UPRE (Fig. 2B), indicating that induction of
Fkb2p by ER stress is mediated, at least in part, by the interaction of
Hac1p with this FKB2 UPRE. No other potential UPRE sequences
were found in the 263 nucleotides between the translational start site
of FKB2 and the termination site of EUG1 known to
precede FKB2 (44). Indeed, this FKB2 UPRE is
necessary for the induction of Fkb2p (see below).
The PDI1 Promoter Contains a Functional UPRE--
Among the six
target proteins of the UPR so far known, only Pdi1p and Kar2p are
essential for vegetative growth of the cell. The presence of a
functional UPRE responsible for the induction of Pdi1p by ER stress was
proposed previously by sequence comparison, but its activity has not
been determined (5). We thus fused the 0.44-kb PDI1 promoter
region in frame to lacZ in the single-copy vector pSEYc102
as described under "Experimental Procedures." The PDI1
promoter responded to ER stress by inducing a 6-fold increase in
-galactosidase expression when cells were treated with tunicamycin
for 3 h (Fig. 4). The location of
the previously proposed UPRE (referred to here as PDI1b
UPRE, Ref. 5) is indicated in Fig. 4. Deletion of 0.14 kb between the
ApaI and BstBI sites showed little effect on the
promoter activity, whereas deletion of an additional 87 base pairs
containing PDI1b UPRE resulted in a decrease in basal
activity to nearly half of that of the 0.44-kb promoter without
affecting the extent of induction. In PDI1b UPRE, as shown
in Fig. 5, nucleotides 10-12 and 14-16
were not palindromic, although all three of the nucleotides important for UPRE activity at 8, 13, and 18 were identical to those in KAR2 and FKB2 UPREs. PDI1b UPRE hardly
competed for the binding of either UPRF in cell extracts or Hac1p
translated in vitro to 32P-labeled
KAR2 UPRE (Fig. 2B). When inserted into pMCZ2,
PDI1b UPRE increased basal activity by 4.3-fold but
affected the induction only slightly;
-galactosidase was induced
3.5-fold by tunicamycin treatment, whereas 2.4-fold induction was
observed with the vector alone (Fig. 5). These results raise doubts
about the role of PDI1b UPRE in the induction of Pdi1p. The
PDI1b UPRE with the surrounding sequence may provide a
binding site for some factor(s) important for basal expression of
Pdi1p.
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Characterization of the EUG1 and FKB2 Promoters--
When the
sequence proposed to be responsible for the induction of Eug1p by ER
stress in the previous study (25) was inserted into pMCZ2, this
putative EUG1 UPRE increased basal activity by 4.8-fold but
conferred only slight inducibility (3.4-fold increase by tunicamycin
versus 2.4-fold increase with the vector alone) as in the
case of PDI1b UPRE (Fig. 5). However, this EUG1
UPRE competed for the specific binding between 32P-labeled
KAR2 UPRE and UPRF in cell extracts or Hac1p translated in vitro, albeit very weakly (Fig. 2B). Although
the palindromic sequence in EUG1 UPRE is not typical of
other functional UPREs, Hac1p seems to be capable of recognizing
EUG1 UPRE (see "Discussion"). Sequences responsible for
increased basal activity observed with EUG1 UPRE (Fig. 5)
may be able to be separated from the palindromic sequence exerting weak
UPRE activity. We found no other potential UPRE sequences in the 250 nucleotides located between the start ATG codon of Eug1p and the
termination codon of the preceding open reading frame (ORF) of 372 amino acids (D9719.22; Ref. 44). To determine the EUG1
promoter activity accurately, we used the multicopy vector pSEY101,
considering the previous finding that Eug1p is at least 10-fold less
abundant than Pdi1p (25). The EUG1 promoter conferred marked
induction (66-fold) of -galactosidase by tunicamycin (Fig.
6). However, it should be noted that it
showed very low basal activity (less than 1 units) and conferred only mild induction (4-5-fold) when inserted into the single-copy vector pSEYc102. Mutation of two nucleotides in the EUG1 UPRE
(CACGCGTG was changed to CACTCTTG) eliminated most of the response of
the EUG1 promoter to ER stress. Similarly, introduction of
two point mutations into the FKB2 UPRE mentioned above
(CAGCGCA was mutated to CATCTCA) made the FKB2 promoter
almost insensitive to ER stress (Fig. 6). Thus, the EUG1 and
FKB2 promoters each contain a single UPRE which are
primarily responsible for the induction of Eug1p and Fkb2p,
respectively.
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Identification of UPRE Responsible for the Induction of
Lhs1p--
The sequences proposed to be functional UPREs responsible
for the induction of Lhs1p by ER stress in the previous reports (18,
19) did not confer inducibility on the CYC1-lacZ gene (data
not shown). Instead, we found a sequence that contained the same
partial palindrome with a one-nucleotide spacer
(CAGCGTG) as KAR2 UPRE. This
LHS1 UPRE responded to ER stress by inducing a 29-fold
increase in -galactosidase expression when inserted into pMCZ2 (Fig.
5), although the orientation of the LHS1 UPRE used for
in vivo assay was opposite to that present in the endogenous LHS1 promoter. In addition, this LHS1 UPRE
competed for the specific binding between 32P-labeled
KAR2 UPRE and UPRF in cell extracts or Hac1p translated in vitro, albeit less efficiently than KAR2 UPRE
(Fig. 2B). As Lhs1p is thought to be expressed at a much
lower level than Kar2p (18), we also used the multicopy vector pSEY101
to analyze the LHS1 promoter. Twelve-fold induction of
-galactosidase by tunicamycin treatment was observed when the
LHS1 promoter was fused in frame to lacZ (Fig.
6), and this induction was abolished almost completely by two point
mutations introduced into the LHS1 UPRE redefined in this
study (CAGCGTG was changed to CATCTTG). We concluded that Lhs1p is
induced by ER stress through the interaction between Hac1p and this
LHS1 UPRE, which functions as a cis-acting
element that is necessary and sufficient for induction.
Apparent Absence of a Functional UPRE in the SCJ1
Promoter--
The sequence proposed previously to be a functional UPRE
responsible for the induction of Scj1p by ER stress (Ref. 22; indicated by the most downstream box f in Fig. 7)
did not confer significant inducibility on the CYC1-lacZ
gene when inserted into pMCZ2 (data not shown). Unlike the other
promoters of ER stress-inducible proteins, an ORF of 590 amino acids
(YM8261.07) was predicted immediately upstream of two putative TATA
sequences for SCJ1 (see Fig. 7 and also compare the
broken arrow in Fig. 9 with those in Fig. 6; see also Ref.
45). We found no other potential UPRE sequences between the termination
codon of this ORF and the start ATG codon of Scj1p. By sequence
homology search, two sequences (indicated by boxes a and
d in Fig. 7) in which a partial palindrome was separated by
one C nucleotide (CAGCGTA) and a sequence
(box b) in which a perfect palindrome was separated by one C
nucleotide (CAGCCTG) were found within the
upstream ORF. When inserted into pMCZ2, however, these putative UPREs
did not confer inducibility on the CYC1-lacZ gene, probably
because two important nucleotides 8 and 18 were not conserved (see
"Discussion"). Only the results with one putative UPRE (box
d) are shown in Fig. 5. This putative SCJ1 UPRE
increased basal activity by 2.9-fold without conferring significant
inducibility. In addition, this sequence did not compete for the
specific binding of 32P-labeled KAR2 UPRE to
UPRF or Hac1p (Fig. 2B). We tested a total of six UPRE-like
sequences present in the upstream region (indicated by boxes
a-f in Fig. 7), but none were functional in -galactosidase induction (data not shown).
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DISCUSSION |
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Recent work conducted in two laboratories established that the UPR is mediated by a specific and direct interaction between the transcription factor Hac1p and cis-acting element UPRE (10, 11). Hac1p was shown to be responsible for the induction of all known target proteins (11, 31). However, only the promoters of Kar2p and Fkb2p were actually known to contain a functional UPRE (26, 27). The KAR2 and FKB2 UPRE sequences were so divergent (see Fig. 3) that it was difficult to assess essential features of the UPRE. Indeed, although putative UPREs were proposed previously to be present in the promoters of Pdi1p (5), Scj1p (22), Lhs1p (18, 19), and Eug1p (25), most of these were hardly active as shown in this study.
Our recent extensive mutational analysis of KAR2 UPRE shed some light on this issue (11). The results revealed that KAR2 UPRE contains an E box (CANNTG)-like palindromic sequence that provides a binding site for Hac1p. Interestingly, Hac1p exhibits a strong preference for a spacer of one nucleotide between the half-sites, apparently a characteristic specific to Hac1p. The lack of spacing reduced induction to 13% of the normal level, whereas increased spacing abolished induction almost completely. Hac1p also shows a preference for a specific spacer nucleotide in the order C > G > A > T. Furthermore, CAC is preferred to CAG as a half-site (11).2 Thus, the UPRE activity of seven nucleotides containing two half-sites with a one-nucleotide spacer (referred hereafter as "central seven nucleotides") is highest with CACCGTG and decreases in the order CACCGTG > CAGCGTG > CAGCCTG.
In this study, we demonstrated that each of the KAR2, PDI1, EUG1, FKB2, and LHS1 promoters contains a single UPRE that is necessary and sufficient for induction by ER stress. Importantly, the levels of each UPRE activity determined by in vivo (Fig. 5) and in vitro (Fig. 2B) analyses correlated roughly with those of mRNA induction (Fig. 5, top); KAR2 UPRE showed the highest level of activity, FKB2 UPRE the lowest, and PDI1, EUG1, and LHS1 UPREs showed intermediate activities. The correlation would be even more significant if we consider the orientation of LHS1 UPRE. When inserted in the opposite orientation, KAR2 UPRE showed slightly decreased activity (Fig. 1). By analogy, the degree of LHS1 mRNA induction may be lower than that expected from the LHS1 UPRE activity obtained with in vivo assay, and therefore PDI1, EUG1, and LHS1 mRNAs may show similar levels of induction (Fig. 5). In addition, the degree of KAR2 mRNA induction might be reduced by the high basal expression level of KAR2 mRNA due to the heat shock element and the GC-rich region present in the KAR2 promoter (27) but lacking in the other five promoters.
Transcriptional regulation of Scj1p was exceptional; the promoter
region apparently lacks any functional UPREs (Fig. 7) and is
insufficient for induction (Fig. 9). Interestingly, -galactosidase was expressed only when the lacZ-coding sequence was fused
immediately downstream of Met28 but not of
Met1, consistent with the previous report that Scj1p is
translated from the second methionine (22). If Scj1p was translated
from the first methionine, the first 25 amino acids would be predicted to function as a mitochondrial targeting signal (46), whereas if the
second methionine was used to initiate translation, the next ~20
amino acids would target Scj1p into the lumen of the ER (22).
Therefore, regardless of the mechanism, the preferential starting of
Scj1p translation at Met28 would ensure its function as an
ER-resident molecular chaperone. In fact, the absence of Scj1p caused
slight activation of the UPR; the level of KAR2 mRNA was
approximately 2-fold higher in tunicamycin-untreated scj1
cells than that in untreated SCJ+ cells
presumably because of increased amounts of unfolded proteins accumulated in the ER (Fig. 8).
All of the five functional UPREs identified contained a palindromic sequence (Fig. 5) and competed for the specific binding between 32P-labeled KAR2 UPRE and Hac1p with various efficiencies (Fig. 2B). Based on the results of analysis of a number of active and inactive UPRE-like sequences, we can now deduce some important features required for the interaction between Hac1p and UPRE.
First, Hac1p recognizes not only the central seven nucleotides but also certain surrounding sequences. The central seven nucleotides, CACCGTG or CAGCGTG, of PDI1a or LHS1 UPRE, respectively, may be expected to confer higher or equal activity, respectively, as compared with KAR2 UPRE containing CAGCGTG as discussed earlier. However, in vivo activities of PDI1a and LHS1 UPREs were much lower than that of KAR2 UPRE (Fig. 5). Since mutations of nucleotides 1-5 in PDI1a UPRE from CCAAT to GGAAC (KAR2 type) increased in vivo activity only slightly (data not shown), as was the case of FKB2 and FKB2' UPREs (Fig. 3), a "natural" transversion of the important nucleotide 18 seemed to be responsible for the weak activity of PDI1a UPRE observed; in the case of KAR2 UPRE, the same transversion reduced the activity to less than 20% (11). On the other hand, nucleotides 8 and 18 in LHS1 UPRE are transitions rather than transversions of those in KAR2 UPRE, perhaps explaining why LHS1 UPRE is more active than PDI1a UPRE.
Second, at least one half-site sequence must be CAG or CAC since PDI1b UPRE is virtually inactive although all three important nucleotides 8, 13 and 18 are conserved. Third, when one of the half-site sequences diverges from CAC or CAG, the three important nucleotides 8, 13, and 18 must be conserved, because FKB2 UPRE is active, whereas SCJ1 UPRE shown in Fig. 5 as well as other UPRE-like sequences shown in Fig. 7 are virtually inactive.
Finally, the case of EUG1 UPRE is exceptional, because its half-sites are separated by two nucleotides (CACGCGTG), which is usually inactive as mentioned earlier. However, this EUG1 UPRE competed for the binding between KAR2 UPRE and Hac1p, although very weakly (Fig. 2B). The presence of two overlapping half-sites with no spacing (CACGCG and CGCGTG) flanked by two important nucleotides (at positions 8 and 18) might explain the recognition of EUG1 UPRE by Hac1p.
In addition to the one-nucleotide spacing between the E box-like half-sites, sequences outside of the central seven nucleotides are important for UPRE recognition by Hac1p. This unique characteristics may distinguish UPRE from among various cis-acting elements recognized by basic region-containing transcription factors, and are likely to explain why transcription of only molecular chaperones and folding enzymes in the ER is induced when unfolded proteins are accumulated in the ER. Without this induction system, yeast cells cannot survive under ER stress conditions (6, 7, 11). Furthermore, increased synthesis of the target proteins of the UPR in the absence of excess unfolded proteins in the ER is also toxic to the cell (10, 31). Therefore, the UPR must be tightly regulated to meet the requirements of this organelle. Recently, we and others showed that Hac1p itself is induced by ER stress and the induction is mediated by unconventional splicing of HAC1 precursor mRNA (10, 13, 31, 47, 48). The unique features of UPRE revealed in this study suggest a basis for the specificity of the UPR; Hac1p induced under ER stress conditions may activate transcription of a limited set of proteins only necessary to cope with deleterious effects of unfolded proteins accumulated in the ER. The UPR, an intracellular signaling from the ER to the nucleus, appears to possess multiple distinguished characteristics among biological signal transduction systems.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. N. Nishikawa, T. Endo, T. H. Stevens, and P. A. Silver for providing yeast strains or plasmids. We thank Masako Nakayama, Mayumi Ueda, Hideaki Kanazawa, and Rika Takahashi for technical assistance.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed. Tel.: 81-75-315-8656;
Fax: 81-75-315-8659; E-mail: kazumori{at}hsp.co.jp.
1 The abbreviations used are: ER, endoplasmic reticulum; ORF, open reading frame; UPR, unfolded protein response; UPRE, unfolded protein response element; UPRF, unfolded protein response factor; kb, kilobase pair(s).
2 K. Mori, N. Ogawa, T. Kawahara, H. Yanagi, and T. Yura, unpublished results.
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