Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120
Heidelberg, Germany
Present address: Division of Immunogenetics, German Cancer Research Center, Im
Neuenheimer Feld 280, D-69120, Heidelberg, Germany
Present address: Max-Planck-Institut für Immunbiologie, Stübeweg 51,
D-79108 Freiburg, Germany
* Author for correspondence (e-mail: haasi{at}immunbio.mpg.de )
Accepted 19 March 2002
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Summary |
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Key words: Cellular stress, Endoplasmic reticulum, Polysome profile, Tunicamycin, Thapsigargin, UPR
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Introduction |
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In addition to these functions, BiP seems to fulfill a crucial role in the
unfolded protein response (UPR). This response was first characterized as an
upregulation of BiP at the transcriptional level, triggered by the
accumulation of misfolded proteins in the ER
(Kozutsumi et al., 1988;
Kohno et al., 1993
). Array
hybridization experiments revealed 208 UPR target genes in the yeast
Saccharomyces cerevisiae affecting multiple ER and secretory pathway
functions (Travers et al.,
2000
). The mediator of the UPR pathway in S. cerevisiae
is Ire1p (Cox et al., 1993
;
Mori et al., 1993
), an
ER-resident transmembrane kinase/nuclease. Activation of Ire1p, as induced for
instance by tunicamycin treatment, which inhibits N-linked glycosylation,
leads to the production of a transcription factor, Hac1p, that in turn drives
the transcriptional response (reviewed in
Chapman et al., 1998
).
In mammals, the ER stress response is more diverse. Two homologues of IRE1
were identified (Tirasophon et al.,
1998; Wang et al.,
1998
), which are most probably involved in transcriptional
activation of UPR target genes. An additional transmembrane ER-resident
kinase, PERK, not present in yeast, has a lumenal domain similar to that of
IRE1 (Harding et al., 1999
).
This kinase is responsible for the phosphorylation of the translational
initiation factor eIF2
(Harding et
al., 1999
), which interferes with the formation of an active 43S
translation-initiation complex
(Hinnebusch, 1994
). The
resulting inhibition of global protein synthesis
(Brostrom and Brostrom, 1998
;
Prostko et al., 1992
) is
required for cells to survive ER stress
(Harding et al., 2000b
).
Furthermore, ER stress also causes induction of growth arrest and programmed
cell death in many cell types (Larsson et
al., 1993
; Nakashima et al.,
1993
), most probably because of downstream effects of the
activated transcription factor CHOP/GADD153
(Wang et al., 1996
;
Zinszner et al., 1998
).
Accumulating evidence supports the idea that BiP plays the key role in the
UPR, not only as an ER-chaperone but also as an ER-stress sensor
(Dorner et al., 1992;
Leborgne-Castel et al., 1999
;
Little and Lee, 1995
;
Morris et al., 1997
). Most
strikingly, the lumenal domains of both IRE1 and PERK were shown to form a
stable complex with BiP, and release of BiP binding correlated with both
perturbation of protein folding in the ER and activation of the transmembrane
kinases (Bertolotti et al.,
2000
).
If BiP indeed has stress sensor function, BiP levels are critical for UPR
induction. For instance, too high levels of BiP would delay or prevent UPR,
whereas too low levels could cause a premature or prolonged UPR. Thus, a
mechanism is needed that controls the amount of BiP. Furthermore, this
mechanism should have a certain buffering capacity to prevent UPR induction by
transient and/or small perturbances. Some reports point to
post-transcriptional control of BiP expression
(Lam et al., 1992;
Leborgne-Castel et al., 1999
;
Ulatowski et al., 1993
). In
concert with classical transcriptional regulation, a translational mechanism
controlling BiP expression could increase the efficiency of the ER stress
response.
We have investigated the regulation of BiP expression in unstressed and
stressed cells. Using the tetracycline-sensitive (tet-off) expression system
(Gossen and Bujard, 1992), we
introduced a construct encoding heterologous mouse BiP into human HeLa cells.
This experimental system allowed us to investigate translation of BiP in cells
in which BiP transcript levels were increased in the absence of stress. This
system also allowed us to expose cells to ER stress and investigate
translation of the same amount of mouse BiP transcript as in unstressed cells,
because the heterologous BiP mRNA is not under UPR control. In addition, we
performed polysome analyses to monitor the ribosome loading of BiP mRNA under
normal and ER-stress conditions. Taken together, our data reveal the existence
of a regulation mechanism that acts in a stress-dependent manner and
specifically controls BiP expression at a translational level.
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Materials and Methods |
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Luciferase assay
Luciferase assays were performed as described previously
(Gossen and Bujard, 1992). 79%
of Bil11 and 96% of Bil58 cells tested positive for luciferase expression as
confirmed by subcloning.
Cell lysis, western blotting and antibodies
Cell lysis and western blotting were performed as described before
(Chillaron and Haas, 2000).
Depending on the assay performed, various reagents were used to detect BiP: for specific immunoprecipitation of mouse BiP, a polyclonal antiserum was used that does not react with human BiP (a kind gift of L. Hendershot, Memphis, TN). To detect mouse but not human BiP in western blots, commercially available anti-Grp78 antiserum (PA1-014; 1:10.000; Affinity Bioreagents, Golden, CO) was used. When total BiP was analyzed, monoclonal anti BiP antibody (a kind gift of L. Hendershot, Memphis, TN) was used in immunoprecipitation and western blot experiments, because this antibody reacts with both mouse and human BiP.
Additional antibodies used in western blots were: anti-calreticulin (Affinity Bioreagents, Golden, CO; 1:2000), anti-calnexin, (a kind gift of E. Ivessa, Vienna, Austria), anti-Erp29 (a kind gift of S. Mkrtchian, Stockholm, Sweden), anti ERp72 (a kind gift of H.-D. Söling, Göttingen, Germany), anti-tubulin (kind gift of J. Wehland, Braunschweig, Germany) and HRP-conjugated secondary antibodies (BioRad, München, Germany). Signals were obtained using BM Chemiluminescence Blotting Substrate (Roche Diagnostics, Mannheim, Germany) and quantified by standard scanning densitometry using the NIH Image program version 1.6.
Synthesis of recombinant BiP-GST fusion protein
The amounts of mouse BiP and total BiP were quantified by use of defined
amounts of a recombinant BiP-GST fusion protein detected by both antibody
reagents. Recombinant BiP-GST was made with the pGEX fusion protein system
(Amrad Cooperation Ltd., Melbourne, Australia) and contains
glutathione-S-transferase of 26 kDa fused to the 23.8 kDa C-terminal portion
of mouse BiP.
Pulse chase analysis and immunoprecipitation
Cells were washed twice in PBS and incubated in methionine-free RPMI 1640
medium/10% dialyzed FCS (1 hour, 37°C) prior to labeling (1.5 hour,
37°C, 100 µCi ml-1 [35S]-methionine). Chase was
initiated by replacing the label medium with DMEM/FCS containing an excess of
methionine (4.5 mM). At various time points, cells were washed once with
ice-cold PBS and lysed as described above. Incorporation of radioactivity into
TCA-precipitated proteins was measured with a scintillation-counter (Beckman
LS 6000TA, Beckman, München, Germany). Immunoprecipitations of BiP were
performed from equal amounts of cell lysates using either a monoclonal
anti-BiP antibody detecting equally well human and mouse BiP or a rabbit
anti-BiP antiserum specifically reacting with mouse BiP (kind gifts of L.
Hendershot). Labeled proteins were separated by SDS-PAGE under reducing
conditions as described previously
(Knittler and Haas, 1992) and
visualized by autoradiography. Signals were quantified using a phosphoimager
(BAS1000, Imaging Screen Plate BASIII, Fuji, Tokyo, Japan, using MACBAS
version 1.0).
Determination of synthesis rates and induction of UPR
Cells were washed twice with PBS and cultured in methionine-free medium
(1.5 hour, 37°C) prior to the addition of 100 µCi ml-1
[35S]-methionine. UPR was induced by tunicamycin (1 µg/ml) or
thapsigargin (200 nM), which was present during starvation and labeling. For
actinomycin D (5 µg/ml), cells were treated 5 minutes before and during
labeling. Cell lysis, performed at the time points indicated, measurement of
radioactivity incorporated into proteins, immunoprecipitation and signal
quantification are all described above.
Polysome analysis and isolation of total RNA
Cells at 50-60% confluency were treated for 90 minutes with tunicamycin (2
µg/ml) in DMSO or only the solvent. Cycloheximide (0.1 mg/ml) was added
during the last 3 minutes of treatment. The plates were washed twice with
ice-cold PBS containing cycloheximide. Cells from eight dishes were pooled,
lysed in 500 µl polysome extraction buffer (15 mM Tris-HCl [pH 7.4], 15 mM
MgCl2, 0.3 M NaCl, 1% Triton X-100, 0.1 mg/ml cycloheximide, 1
mg/ml heparin, 1 mM phenylmethysulfonyl fluoride, 10µg/ml leupeptin, 5
µg/ml chymostatin, 3 µg/ml elastatinal and 1µg/ml pepstatin) and
incubated on ice for 20 minutes. The lysates were cleared by centrifugation
(10 minutes, 12,000 g) and layered on top of a 10 ml 10-50%
sucrose gradient prepared in extraction buffer without protease inhibitors.
After a spin of 4 hours at 15,000 g in an SW41 rotor, the
absorbance at 254 nm was read across the gradient. The gradients were
fractionated into 16 samples of 650 µl, proteins were removed by
phenol/chloroform extraction, and RNA was precipitated with ethanol.
Poly A+ RNA isolation and northern blot analysis
Poly A+ RNA isolation was performed with Dynabeads mRNA DIRECT
Kit (Dynal A.S., Oslo, Norway). The RNA was denatured in formamide and
formaldehyde, separated on 1.2% denaturing agarose gels and blotted onto
Hybond-N membrane (Amersham Pharmacia Biotech, Uppsala, Sweden) as described
by the manufacturer. The two probes, covering position -5 to 1103 of mouse BiP
cDNA (in this region it has more than 92% identity with human BiP cDNA) and
the complete actin-coding sequence were radioactively labeled using a random
primer DNA labeling kit (Biorad, München, Germany). Hybridizations were
done according to standard procedures
(Sambrook et al., 1989), and
signal quantification was performed by phosphoimaging, as described above.
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Results |
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BiP expression is tightly controlled in unstressed cells
In the presence of tetracycline, transcription of the hetorologous sequence
was disabled. Upon removal of tetracycline, expression was turned on and
luciferase activity increased with similar kinetics to those described by
Gossen and Bujard (Gossen and Bujard,
1992) (K.G. and I.G.H., unpublished). The kinetics of mouse BiP
induction were investigated by a western blot analysis using anti-Grp78
antiserum, which recognizes only mouse BiP
(Fig. 1A). Mouse BiP was
detectable 24 hours after induction and only slowly increased thereafter.
Steady state levels were reached only after more than 96 hours of tetracycline
removal.
|
For detection of total BiP, we used a rat monoclonal anti-BiP antibody that recognizes mouse and human BiP equally well. The time-dependent increase in mouse BiP expression was not reflected by an increase in the amount of total BiP (Fig. 1B). Instead, the amount of total BiP stayed constant despite the additional expression of mouse BiP. To exclude the possibility that the amount of mouse BiP was too low to be detected as an increase in total BiP, we determined the amounts of BiP detected by the antibody reagents used. A BiP-GST fusion protein was generated that contains the epitopes recognized by both the monoclonal anti-BiP antibody and the anti-Grp78 antiserum. Using the BiP-GST fusion protein as a reference (Fig. 1C), we calculated the amount of mouse and of total BiP expressed in permanently activated HeLa cells. About 60-65% of total BiP consisted of mouse BiP, implying continuous displacement of human BiP by newly synthesized mouse BiP upon activation of mouse BiP expression (Fig. 1C).
These results reveal the existence of a cellular control mechanism
maintaining BiP at a constant level in unstressed cells, underlining the role
of BiP not only as a chaperone but also as stress sensor in the UPR. This
mechanism is specific for BiP because the levels of other ER chaperones, like
ERp29 (Mkrtchian et al.,
1998), ERp72 (Mazzarella et
al., 1990
), Calreticulin
(Smith and Koch, 1989
) and
Calnexin (Wada et al., 1994
)
were not affected by mouse BiP co-expression (K.G. and I.G.H.,
unpublished).
BiP expression is controlled at a translational level
To determine at which level BiP expression is controlled, BiP mRNA was
first examined. Poly A+-RNA was isolated from samples taken at
various time points after removal of tetracycline and analyzed by northern
blotting using a BiP cDNA probe that hybridizes with both human and mouse BiP
mRNA (Fig. 2A). In the presence
of tetracycline, only endogenous human BiP mRNA was detected. Bi-cistronic
transcripts containing the mouse BiP sequence reached steady state levels
within the first 24 hours of activation and remained constant thereafter.
Quantification of the signals revealed that the amount of mouse BiP
transcripts was about eight- to ten- fold that of human BiP transcripts.
Remarkably, transcriptional activation of the mouse sequence did not affect
the level of the endogenous BiP mRNA. These results show that control of BiP
expression levels is post-transcriptional.
|
To investigate the level of regulation in more detail, we next investigated the rate of BiP protein synthesis in control cells (BiLu33) expressing only endogenous human BiP (and firefly luciferase) and in activated transfectants (Bil11 and Bil58) additionally expressing mouse BiP. Note that the amount of BiP mRNA in control cells (BiLu33) is basically identical to BiP mRNA levels in unactivated Bil cells expressing endogenous human BiP only (K.G. and I.G.H., unpublished). Synthesis rates were visualized as a time-dependent increase in the amount of total labeled BiP immunoprecipitated with the monoclonal anti-BiP antibody, and conditions were chosen such that the cell lines investigated could be directly compared (Fig. 2B). Strikingly, the rates of BiP synthesis were identical in all cells investigated, whether or not mouse BiP was co-expressed. This indicates that BiP synthesis is independent of the actual amount of BiP transcripts in unstressed cells. Consistently, the half-life of total BiP was identical (28-33 hours) in activated transfectants and control cells (Fig. 2C). Furthermore, the half-life of mouse BiP did not differ from that of the human homolog as confirmed by a supplementary assay for the half-life of mouse BiP in the activated transfectants (K.G. and I.G.H., unpublished). In no case was secretion of BiP observed.
Altogether, these findings argue strongly for a mechanism controlling BiP expression at a translational level. Variations in BiP mRNA levels do not affect either the synthesis rate or the half-life of BiP, reaffirming that BiP expression is feedback regulated in unstressed cells.
UPR-mediated enhancement of BiP translation efficiency
Under conditions of ER stress, activation of PERK leads to inhibition of
eIF2-dependent translation initiation, resulting in downregulation of global
protein synthesis (Harding et al.,
1999). In addition, IRE1 activation leads to increased cellular
BiP mRNA levels, and more BiP protein is produced
(Cox et al., 1993
;
Kohno et al., 1993
;
Kozutsumi et al., 1988
;
Li et al., 1994
). However, the
results shown above clearly demonstrate that a mere enhancement of BiP
transcripts does not necessarily result in the production of more protein. If
more BiP is to be produced, the translational restraint present in unstressed
cells must be abrogated.
To characterize the novel regulation mechanism in more detail, we
investigated the effects of treating cells with the UPR-inducing drug
tunicamycin. As seen in Fig.
3A, the amount of endogenous BiP mRNA started to increase
approximately 2 hours after drug addition. After 8 hours, approximately 10
times the amount of transcripts present in unstressed HeLa cells was reached.
Shortly (60 or 90 minutes) after tunicamycin addition, we analyzed the rates
of synthesis of total BiP, mouse BiP and total cellular proteins in unstressed
and stressed cells. Tunicamycin has been reported to only moderately inhibit
protein synthesis in HeLA cells (Brostrom
and Brostrom, 1998). Consistently, we found that tunicamycin
treatment for 90 minutes lowered the subsequent incorporation of radioactive
methionine into TCA-precipitated material to approximately 80% of control
values (Fig. 3B). This
downregulation of global protein synthesis is a very rapid process in UPR and
is already detectable 5 minutes after tunicamycin addition (K.G. and I.G.H.,
unpublished). Inhibition of protein synthesis was also reflected by a decrease
in polysome size and an increase in free ribosomal subunits and monosomes (see
Fig. 5).
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In sharp contrast, we found BiP synthesis to be increased (by a factor of
two to four after 90 minutes of tunicamycin treatment compared with the
control values, Fig. 3C). These
findings were confirmed by an analysis performed under the same UPR conditions
as described above using a different HeLa cell transfectant producing a murine
immunoglobulin light chain, NS1. Synthesis of the
-light chain as well as that of total proteins decreased again to
approximately 80% of control values, whereas the rate of BiP protein synthesis
increased by a factor of two to four (K.G. and I.G.H., unpublished).
The reverse effect of UPR on BiP translation and general protein synthesis
is seen when mouse BiP and firefly luciferase are compared. Although both
proteins are translated from the same transcript, mouse BiP translation
increased (note that the bi-cistronic construct is not under control of a UPR
sensitive promotor; Fig. 3D),
whereas luciferase activity dropped to 80% (K.G. and I.G.H., unpublished).
Interestingly, recovery of luciferase activity is observed 4 hours after
ongoing tunicamycin treatment (K.G. and I.G.H., unpublished). Recovery of
translation is also seen in measurements of total protein synthesis (K.G. and
I.G.H., unpublished) and was described previously for NIH 3T3 cells
(Brostrom and Brostrom,
1998).
Translational regulation is often mediated by elements located in the
5' and/or 3' UTR of the respective transcript
(Hershey, 1991;
Preiss and Hentze, 1999
).
Although mouse BiP lacks both of these sequences, this protein is subject to
feed-back control, indicating a control element located in the coding sequence
itself.
Our data show that the translational restraint acting in unstressed cells
is alleviated under UPR conditions. Translational upregulation is already seen
before transcript levels significantly rise. Furthermore, analysis of the
mouse protein demonstrates that the translational upregulation is indeed
uncoupled from the transcriptional response, because although the mouse BiP
transcript is not under UPR control, mouse BiP translation was increased
whereas synthesis of other proteins was downregulated. From these data, we
conclude that translational and transcriptional upregulation of BiP are
autonomous steps during UPR. In line with our data, enhanced BiP synthesis in
the absence of transcriptional upregulation was also observed with
glucocorticoid-treatment of S49 mouse lymphoma cells
(Lam et al., 1992).
Ron's group recently reported that the thapsigargin-induced increase in BiP
synthesis is not seen in the presence of the transcription inhibitor
actinomycin D (Harding et al.,
2000a). From this finding, it could be concluded that a
transcriptional increase in BiP mRNA is a prerequisite for upregulation of BiP
expression. Because our data indicate that translational upregulation of BiP
is not dependent on increased mRNA levels, we postulated that actinomycin D
might have a direct or indirect effect on BiP translation itself. To clarify
this issue, we analyzed the effect of actinomycin D on BiP synthesis in
thapsigargin-treated HeLa cells. Thapsigargin induces a more rapid and
stronger UPR than tunicamycin. BiP transcript levels in BiLu33 cells were
already elevated 30 minutes after thapsigargin addition (K.G. and I.G.H.,
unpublished), and the concomitant inhibition of protein synthesis was
approximately 50% in activated Bil11 cells
(Fig. 4A) as well as in BiLu 33
cells (K.G. and I.G.H., unpublished). We also analyzed the rate of BiP
translation in thapsigargin-treated cells in the absence
(Fig. 4A-C) or presence of
Actinomycin D (Fig. 4D-F),
which was added only 5 minutes prior to cell labeling. Under these conditions,
actinomycin D should not drastically affect BiP synthesis, because BiP
transcripts are already elevated. However, actinomycin D strikingly blocked
the thapsigargin-induced increase in BiP synthesis, both total (compare 4B and
E) and mouse (compare 4C and F) BiP. Thus, actinomycin D affects BiP
translation, either directly or indirectly, possibly by preventing
transcription of a short-lived protein required for translation of BiP.
|
To summarize, upregulation of BiP expression during UPR can be described as a two step process: (i) alleviation of the translational restraint present in unstressed cells resulting in increased translation efficiency of BiP mRNA and (ii) increased transcription of BiP from the well known classic UPR response.
Increased translation efficiency of BiP mRNA is probably caused by
increased ribosome transit
To gain more insight into the mechanism underlying control of BiP mRNA
translation, we next investigated polysome patterns in unstressed and stressed
cells by sucrose gradient centrifugation of solubilized HeLa
(Fig. 5A). Because of its
moderate stress-inducing effect, tunicamycin allowed us to compare cells that
stressed or unstressed contained the same amount of BiP
transcripts. UPR caused a drastic increase of monosomes and free ribosomal
subunits as was previously described
(Harding et al., 2000b). To
investigate how UPR affected ribosome association with specific transcripts,
total RNA isolated from the fractions was analyzed by northern blotting using
BiP and actin-specific probes (Fig.
5B). Signals were detected only in the polysome fractions,
indicating that all transcripts for the genes analyzed were associated with
ribosomes. In HeLa cells, actin mRNA (main peak in fraction 13/14) shifted to
lighter fractions after tunicamycin treatment (main peak in fraction 11/12)
indicating that actin transcripts were associated with fewer ribosomes during
UPR (Fig. 5B). A similar but
very weak shift was observed with human BiP mRNA, the majority of which
remained in the same fraction as without UPR (fraction 13/14;
Fig. 5B), showing a minor
influence of inhibition of translational initiation on human BiP mRNA.
These results clearly show that the overall polysome organization of the BiP-encoding transcript is not drastically affected by the UPR. Thus, the increased translation efficiency of BiP mRNA observed under UPR conditions is most probably caused by a higher ribosome transit rate on BiP transcripts.
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Discussion |
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Use of the tet-off activation system
(Gossen and Bujard, 1992)
allowed us to establish stable human cell lines capable of conditionally
overexpressing mouse BiP in the absence of cellular stress. Antibodies
directed to the C-terminus of rodent BiP specifically detected the protein
derived from the construct introduced, and a monoclonal antibody recognizing
both mouse and human proteins was used to quantify total cellular BiP. Firefly
luciferase, encoded downstream of mouse BiP, was used as a reporter to
indirectly quantify the bi-cistronic transcript. We show that the heterologous
mRNA reached maximal levels (approximately 10 times the amount of endogenous
BiP transcripts) 24 hours after transcriptional activation without affecting
endogenous BiP mRNA levels. Mouse BiP protein increased very slowly, and
steady state levels were reached only after more than 4 days of
transcriptional activation. Strikingly, total cellular BiP levels did not
increase. HeLa cells activated to produce mouse BiP transcripts maintain
protein expression at a constant level by slowly displacing human BiP by the
mouse protein. The slow increase in mouse BiP presumably reflects the low
turnover rate of the long-lived protein. Since endogenous human BiP is
degraded very slowly, mouse BiP can replace it only very slowly too. Under
steady state conditions, mouse BiP protein made up approximately 60% of total
cellular BiP, implying that human BiP transcripts translate into less protein
when mouse BiP is co-expressed. In unstressed cells, translation of mouse BiP
is evidently restricted as well, since the same amount of transcript is
translated more efficiently under stress conditions. It is true that the final
proportions of mouse to human BiP (65% and 35%, respectively, i.e.
2:1)
did not reflect the proportions of the respective transcripts present in
permanently activated cells (90% and 10%, respectively). However, this could
be related to the lack in the mouse BiP construct of the 5' untranslated
portion responsible for efficient 5' cap-dependent or -independent
translation initiation (Kozak,
1986
; Sarnow,
1989
). Taken together, our data show that BiP expression is
feed-back regulated by restricted translation of BiP mRNA in unstressed cells.
Reduced translation efficiency was not observed for other ER proteins,
indicating that the mechanism described here is BiP specific.
In other systems, overexpression of BiP has been reported
(Dorner et al., 1992). In
unstressed cells, this may be possible when transcript levels are high enough
to override the translational control mechanism presented here. Careful
studies in transgenic tobacco plants carrying additional wild-type BiP copies
showed that a 100- to 150-fold increase in BiP mRNA (in the system presented
here, BiP mRNA is increased approximately 10 fold) only led to a modest
five-fold increase in BiP protein
(Leborgne-Castel et al.,
1999
), pointing to a mechanism regulating BiP expression in this
system as well. When cellular BiP levels cannot be maintained by translational
control, degradation may additionally be induced, as indicated by lower
molecular mass bands probably representing BiP degradation products.
Interestingly, loss of BiP also seems to be counterbalanced. Yeast mutants in
p24 gene family members, implicated in vesicular transport, secrete
KDEL-bearing ER proteins such as BiP
(Marzioch et al., 1999
).
However, BiP export does not lead to a reduction in intracellular BiP levels.
It is tempting to speculate that the translation efficiency of BiP is enhanced
in these mutants.
UPR is known to upregulate a number of target genes, including BiP. However, our findings definitively indicate that an increased amount of BiP transcripts is not sufficient to raise the protein levels, because, in unstressed cells, elevated transcript levels led neither to increased translation of BiP nor to increased protein levels. Consequently, the constraint limiting BiP expression in unstressed cells must be revoked when more BiP is needed. Indeed, under conditions of ER stress, we found BiP synthesis to be more efficient. Thus, an increase in BiP expression during UPR is possible by revoking the mechanism controlling BiP expression in unstressed cells rather than by the mere enhancement of BiP transcripts. Moreover, it seems that translational and transcriptional upregulation of BiP are independent events. Increased BiP translation rates upon UPR induction with both tunicamycin and thapsigargin could be detected before BiP mRNA levels rose. Even more strikingly, mouse BiP translation was enhanced under UPR conditions even though mouse BiP transcription is not under UPR control. Thus, in addition to the classical ER stress response, namely transcriptional upregulation of UPR genes, and downregulation of general protein synthesis, UPR also leads to a very rapid increase in the translation efficiency of BiP mRNA. The fast translational response allows the cell to adapt to small perturbations without inducing the transcriptional response. Only if a certain threshold value is reached is the final transcriptional upregulation of UPR genes turned on.
The increase of BiP translation efficiency under UPR conditions deserves
further discussion. As already mentioned, UPR also leads to PERK-mediated
phosphorylation of the translation initiation factor eIF2
(Harding et al., 1999
). Since
inactivation of a translation initiation factor seems to be a very general
mechanism to downregulate protein translation, it is difficult to explain how
BiP can evade this inhibition and even show increased translation efficiency.
It has been shown that an IRES element situated upstream of the initiation AUG
in BiP mRNA is involved in some alternative pathway of translation initiation
(Sarnow, 1989
). Further
examples for the involvement of 5' and 3' untranslated regions in
translational regulation have been described
(Hershey, 1991
;
Preiss and Hentze, 1999
). Our
experimental system allowed us to investigate the role of the 5' or
3' untranslated regions of BiP mRNA, as both regions are missing in the
mouse sequence employed. If elements in these regions were essential for
translational control, BiP would be expected to be downregulated upon UPR
induction as is any other cellular protein, for example, actin or luciferase.
However, this was clearly not the case. These findings suggest that
translational upregulation of BiP is not dependent on a cis-acting IRES
element or on other regions present in the non-coding portion.
BiP translation efficiency does not seem to be affected by inactivation of
the trans-acting initiation factor eIF2, although this elicits
disassembly of polysomes and a reduced rate of global protein synthesis
(Brostrom and Brostrom, 1998
;
Prostko et al., 1992
). Despite
the slightly decreased polysome size of BiP transcripts under UPR conditions,
BiP translation is clearly enhanced. These findings are consistent with a
model in which BiP expression is controlled at the level of translation
elongation. If the BiP translation elongation rate was low in unstressed
cells, enhancement of the elongation rate in stressed cells could allow faster
migration of ribosomes and the same or even a decreased amount of ribosomes
would be able to synthesize more BiP protein from the same amount of
transcript (protein synthesis rate=([number of active ribosomes]x[number
of amino acid residues])/[ribosome transit time];
(Hershey, 1991
)). In this
context, it is interesting to note that regulation of chicken reticulocyte
hsp70 mRNA translation is at the level of elongation
(Theodorakis et al.,
1988
).
There seem to be various translational mechanisms acting during UPR.
Another protein described as escaping stress-induced inhibition of translation
initiation is the transcription factor ATF4 (Harding et al., 2000). In the
absence of stress, ATF4 synthesis is inefficient because translation is
initiated at an out-of-frame ATG situated upstream of the real start codon.
Under UPR conditions, however, more ATF4 is produced because the ribosomes
initiate translation at the correct start codon, shifting ATF4 transcripts
into higher polysome fractions (Harding et
al., 2000a).
The translation elongation rate of BiP mRNA could be controlled either by
some structural motifs present in the coding region of BiP mRNA or by signals
in the protein itself. Control at the level of translocation, for instance, is
a possibility to slow down translation elongation. Interestingly, an event
that limits protein translocation into the ER seems to limit apolipoprotein B
synthesis in liver cells when the lipid supply for very low density
lipoprotein formation is insufficient
(Bonnardel and Davis, 1995).
Whatever the precise mechanism is, with regard to the many functions of BiP,
it is not surprising that even subtle changes in BiP concentrations have
deleterious effects on the cells. We propose that this mechanism is to ensure
on/off switch functions of BiP by allowing the cells to fine-tune BiP levels
in order to prevent untimely UPR.
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