Heat Shock Induces Preferential Translation of ERGIC-53 and Affects Its Recycling Pathway
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m401860200
ISSN1083-351X
AutoresCarmen Spatuzza, Maurizio Renna, Raffaella Faraonio, Giorgia Cardinali, Gianluca Martire, Stefano Bonatti, Paolo Remondelli,
Tópico(s)RNA regulation and disease
ResumoERGIC-53 is a lectin-like transport receptor protein, which recirculates between the ER and the Golgi complex and is required for the intracellular transport of a restricted number of glycoproteins. We show in this article that ERGIC-53 accumulates during the heat shock response. However, at variance with the unfolded protein response, which results in enhanced transcription of ERGIC-53 mRNA, heat shock leads only to enhanced translation of ERGIC-53 mRNA. In addition, the half-life of the protein does not change during heat shock. Therefore, distinct signal pathways of the cell stress response modulate the ERGIC-53 protein level. Heat shock also affects the recycling pathway of ERGIC-53. The protein rapidly redistributes in a more peripheral area of the cell, in a vesicular compartment that has a lighter sedimentation density on sucrose gradient in comparison to the compartment that contains the majority of ERGIC-53 at 37 °C. This effect is specific, as no apparent reorganization of the endoplasmic reticulum, intermediate compartment and Golgi complex is morphologically detectable in the cells exposed to heat shock. Moreover, the anterograde transport of two unrelated reporter proteins is not affected. Interestingly, MCFD2, which interacts with ERGIC-53 to form a complex required for the ER-to-Golgi transport of specific proteins, is regulated similarly to ERGIC-53 in response to cell stress. These results support the view that ERGIC-53 alone, or in association with MCFD2, plays important functions during cellular response to stress conditions. ERGIC-53 is a lectin-like transport receptor protein, which recirculates between the ER and the Golgi complex and is required for the intracellular transport of a restricted number of glycoproteins. We show in this article that ERGIC-53 accumulates during the heat shock response. However, at variance with the unfolded protein response, which results in enhanced transcription of ERGIC-53 mRNA, heat shock leads only to enhanced translation of ERGIC-53 mRNA. In addition, the half-life of the protein does not change during heat shock. Therefore, distinct signal pathways of the cell stress response modulate the ERGIC-53 protein level. Heat shock also affects the recycling pathway of ERGIC-53. The protein rapidly redistributes in a more peripheral area of the cell, in a vesicular compartment that has a lighter sedimentation density on sucrose gradient in comparison to the compartment that contains the majority of ERGIC-53 at 37 °C. This effect is specific, as no apparent reorganization of the endoplasmic reticulum, intermediate compartment and Golgi complex is morphologically detectable in the cells exposed to heat shock. Moreover, the anterograde transport of two unrelated reporter proteins is not affected. Interestingly, MCFD2, which interacts with ERGIC-53 to form a complex required for the ER-to-Golgi transport of specific proteins, is regulated similarly to ERGIC-53 in response to cell stress. These results support the view that ERGIC-53 alone, or in association with MCFD2, plays important functions during cellular response to stress conditions. Cells and tissues sense and respond to environmental stress by activating the expression of stress genes encoding molecular chaperones and enzymes that repair and remove cell damage (1Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1850) Google Scholar, 2Morimoto R.I. Kline M.P. Bimston D.N. Cotto J.J. Essay Biochem. 1997; 32: 17-29PubMed Google Scholar, 3Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2749) Google Scholar). The heat shock response is the most conserved and efficient defense from stress described in living organisms (1Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1850) Google Scholar). In normal conditions, mammalian cells express constitutive heat shock (HS) 1The abbreviations used are: HS, heat shock; HSp, HS protein; TG, thapsigargin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UPR, unfolded protein response; QC, quality control; ERGIC, ER-to-Golgi intermediate compartment; ERSE, ER-stress response element; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; IRES, internal ribosomal entry sites; RT, reverse transcriptase. 1The abbreviations used are: HS, heat shock; HSp, HS protein; TG, thapsigargin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UPR, unfolded protein response; QC, quality control; ERGIC, ER-to-Golgi intermediate compartment; ERSE, ER-stress response element; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; IRES, internal ribosomal entry sites; RT, reverse transcriptase. genes encoding molecular chaperones, which control the folding and translocation of proteins across membranes of different cell compartments (4Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3077) Google Scholar). During hyperthermia, exposure to heavy metals, amino acid analogues, and several other agents, mammalian cells synthesize large amounts of inducible HS proteins, which remove or refold stress-denatured proteins (1Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1850) Google Scholar). Higher levels of stress-denatured proteins act as regulators of HS gene expression by binding the HSp70s, which interact with the HS transcription factors in the cytoplasm (5Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1509) Google Scholar, 6Santoro M.G. Biochem. Pharmacol. 2000; 59: 55-63Crossref PubMed Scopus (450) Google Scholar). Unbound HS factors are then able to move into the nucleus and activate transcription of inducible HS proteins by binding promoter sequences named heat shock elements, HSEs (5Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1509) Google Scholar, 6Santoro M.G. Biochem. Pharmacol. 2000; 59: 55-63Crossref PubMed Scopus (450) Google Scholar). In addition to transcriptional activation, some HS proteins are also preferentially translated in a cap- and 5′-end-independent manner, through internal mRNA elements termed internal ribosomal entry sites, IRESs (7Kim Y.K. Jang S.K. Biochem. Biophys. Res. Commun. 2002; 297: 224-231Crossref PubMed Scopus (50) Google Scholar) or by the mechanism of ribosome shunting (8Yueh A. Schneider R.J. Genes Dev. 1999; 14: 414-421Google Scholar). The endoplasmic reticulum (ER) shows an organelle-specific response to the accumulation of proteins misfolded by stress (9Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1912) Google Scholar). ER-resident proteins are specialized in the Quality Control (QC) of folding of newly synthesized proteins (10Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1647) Google Scholar). Genes of the QC machinery are under the transcriptional control of an interorganelle signal transduction pathway termed the unfolded protein response (UPR) (9Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1912) Google Scholar). UPR is conserved from yeast to man and is characteristically induced and regulated by the increase of unfolded or misfolded peptides into the ER lumen (9Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1912) Google Scholar, 11Patil C.K. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-356Crossref PubMed Scopus (669) Google Scholar). In yeast, the UPR results in the up-regulation of many genes involved in QC and also of Golgi-located glycosylating enzymes (12Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar). Conversely, in mammalian cells little is known about the stress response of protein located in post-ER compartments of the secretory pathway. Several glycoproteins exit from the ER through specific transport receptors (13Herrmann J.M. Malkus P. Schekman R. R. Trends Cell Biol. 1999; 9: 5-7Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). ERGIC-53 is a mammalian calcium-dependent lectin and is the most descriptive marker of the ER-to-Golgi intermediate compartment (ERGIC) (14Saraste J. Svensson K. J. Cell Sci. 1991; 100: 415-430Crossref PubMed Google Scholar, 15Klumperman J. Schweizer A. Clausen H. Tang B.L. Hong W. Oorschot V. Hauri H.P. J. Cell Sci. 1998; 111: 3411-3425PubMed Google Scholar), which is composed by vesicular and tubular structures localized in the pre-Golgi region and involved in bidirectional protein transport at the ER-Golgi boundary. ERGIC-53 cycles between the ER and the Golgi complex and exports from the ER a small number of glycoproteins (16Hauri H.P. Appenzeller C. Kuhn F. Nufer O. FEBS Lett. 2000; 476: 32-37Crossref PubMed Scopus (131) Google Scholar, 17Hauri H.P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-59614Crossref PubMed Google Scholar). It interacts with cathepsin-Z-related proteins and is required for the intracellular trafficking of the lysosomal enzyme cathepsin C (18Vollenweider F. Kappeler F. Itin C. Hauri H.P. J. Cell Biol. 1998; 142: 377-389Crossref PubMed Scopus (133) Google Scholar, 19Appenzeller C. Andersson H. Kappeler F. Hauri H.P. Nat. Cell Biol. 1999; 1: 330-334Crossref PubMed Scopus (267) Google Scholar) and for the efficient secretion of coagulation factors V and VIII (20Moussalli M. Pipe S.W. Hauri H.P. Nichols W.C. Ginsburg D. Kaufman R.J. J. Biol. Chem. 1999; 274: 32539-32542Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Indeed, mutations in the ERGIC-53 gene (LMAN1) are responsible for the inherited bleeding disorder referred to as combined deficiency of FV and FVIII (FVFVIIID), in which the secretion of FV and FVIII is impaired (21Nichols W.C. Seligsohn U. Zivelin A. Terry V.H. Hertel C.E. Wheatley M.A. Moussalli M.J. Hauri H.P. Ciavarella N. Kaufman R.J. Ginsburg D. Cell. 1998; 93: 61-70Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 22Neerman-Arbez M. Johnson K.M. Morris M.A. McVey J.H. Peyvandi F. Nichols W.C. Ginsburg D. Rossier C. Antonarakis S.E. Tuddenham E.G. Blood. 1999; 93: 2253-2260Crossref PubMed Google Scholar). However, in about 30% of the FVFVIIID patients, the disorder is generated by mutations in other gene having functional properties similar to ERGIC-53 (21Nichols W.C. Seligsohn U. Zivelin A. Terry V.H. Hertel C.E. Wheatley M.A. Moussalli M.J. Hauri H.P. Ciavarella N. Kaufman R.J. Ginsburg D. Cell. 1998; 93: 61-70Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). It has been recently shown that mutations in the multiple coagulation factor deficiency 2 gene (MCFD2) cause a FVFVIIID phenotype that is indistinguishable from that generated by mutations in LMAN1 (23Zhang B. Cunningham M.A. Nichols W.C. Bernat J.A. Seligsohn U. Pipe S.W. McVey J.H. Schulte-Overberg U. de Bosch N.B. Ruiz-Saez A. White G.C. Tuddenham E.G. Kaufman R.J. Ginsburg D. Nat. Genet. 2003; 34: 220-225Crossref PubMed Scopus (215) Google Scholar). Interestingly, MCFD2 encodes a protein that interacts with ERGIC-53 and, thus, it was suggested that the two proteins form a specific cargo receptor complex that is necessary for the ER-to-Golgi transport of specific proteins (23Zhang B. Cunningham M.A. Nichols W.C. Bernat J.A. Seligsohn U. Pipe S.W. McVey J.H. Schulte-Overberg U. de Bosch N.B. Ruiz-Saez A. White G.C. Tuddenham E.G. Kaufman R.J. Ginsburg D. Nat. Genet. 2003; 34: 220-225Crossref PubMed Scopus (215) Google Scholar). The specific function of ERGIC-53 in the export from the ER of several glycoproteins prompted us to investigate its role during cell stress. The evidence presented in this paper shows that both expression level and recycling pathway of the protein are modulated by heat shock. While this work was in progress, it was shown that ERGIC-53 is transcriptionally activated during UPR (24Nyfeler B. Nufer O. Matsui T. Mori K. Hauri H.P. Biochem. Biophys. Res. Commun. 2003; 304: 599-604Crossref PubMed Scopus (29) Google Scholar). Our results confirm this observation and, interestingly, show that the accumulation of ERGIC-53 during heat shock is driven only by enhanced translation of its mRNA. Moreover, we found that MCFD2 is regulated similarly to ERGIC-53 in response to cell stress, thus supporting the importance of the role of these two proteins in stress conditions. Antibodies—The following antibodies were used: rabbit polyclonal antibody anti-ERGIC-53 (α-CT) raised against a synthetic peptide corresponding to the last 12 amino acids of the C-terminal end of the cytosolic domain of ERGIC-53 (immunoblots, immunoprecipitation, and immunofluorescence assays); mouse monoclonal anti-ERGIC-53 antibody (immunofluorescence analysis) (25Schweizer A. Fransen J.A. Bachi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (373) Google Scholar); rabbit polyclonal anti-GM130 (26Marra P. Maffucci T. Daniele T. Tullio G.D. Ikehara Y. Chan E.K. Luini A. Beznoussenko G. Mironov A. De Matteis M.A. Nat. Cell Biol. 2001; 3: 1101-1113Crossref PubMed Scopus (121) Google Scholar); rabbit polyclonal anti-giantin (Covance); goat polyclonal anti-Sec23, donkey anti-goat, and mouse monoclonal anti-α-tubulin (Santa Cruz Biotechnology); mouse monoclonal antibody recognizing MCFD2 (immunoblot and immunofluorescence) (23Zhang B. Cunningham M.A. Nichols W.C. Bernat J.A. Seligsohn U. Pipe S.W. McVey J.H. Schulte-Overberg U. de Bosch N.B. Ruiz-Saez A. White G.C. Tuddenham E.G. Kaufman R.J. Ginsburg D. Nat. Genet. 2003; 34: 220-225Crossref PubMed Scopus (215) Google Scholar); rabbit polyclonal anti-cal-reticulin and rabbit polyclonal antibody anti-HSp70 (Stressgen); peroxidase-conjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich). Cell Culture Heat Shock and Drug Treatment—All experiments were performed with the human hepatoma Huh7 cell line. Cells were routinely grown at 37 °C in Dulbecco's modified Eagle's medium, 10% fetal calf serum in a humidified atmosphere with 5% CO2. For heat shock experiments actively growing cells were fed with medium preincubated at 42 °C and transferred to a 42 °C preset incubator, while control cells were maintained at 37 °C. Cells were incubated with genistein and quercetin (Sigma-Aldrich) for the same times as HS treatment and control cells were cultured in the presence of the same amount of Me2SO (Sigma-Aldrich). Thapsigargin (TG) (Sigma-Aldrich) was used at concentrations ranging between 300 and 500 nm. Western Blot Analysis—Cells were lysed in B-buffer containing 10 mm Tris-HCl pH 7.4, 150 mm NaCl, 1 mm EDTA, pH 8.0, and 1% SDS. Proteins were separated by 10% SDS-PAGE and then transferred on Protran nitrocellulose membranes (Schleicher & Schuell). Filters were stained with ponceau red to check that equal amounts of proteins were transferred and blocked overnight in phosphate-buffered saline containing 10% nonfat dry milk and 0.5% Tween-20. Membranes were incubated with dilutions of rabbit polyclonal anti-ERGIC-53, anti-HSp70, anti-MCFD2, and anti-α-tubulin antibody for 2 h. Anti-rabbit or anti-mouse IgG horseradish peroxidase conjugated was used as a second antibody. To visualize bands membranes were developed employing the ECL reaction (Amersham Biosciences), which was performed according to the instructions of the manufacturer. In Western blot experiments we used the concentration of 10 μg/lane, which was chosen by titrating the amount of protein extract that gave signals in the linear range by the ECL method. Anti-α-tubulin antibody was used to normalize for equal amounts of proteins and calculate the relative induction ratio. Densitometry of autoradiographs was performed by the NIH Image program and values obtained were the mean ± S.D. of three independent experiments. RNA Extraction, Northern Blot, and Real Time RT-PCR Analyses— Total RNA was extracted by using the TRIzol (Invitrogen) according to the instructions of the manufacturer. 20 μg/lane of total RNA was fractionated on a formaldehyde-1% agarose gel and transferred on Hybond N+ nylon strips (Amersham Biosciences). Filters were hybridized with a 32P-labeled human ERGIC-53 SacII/HindIII fragment (27Sarnataro S. Caporaso M.G. Bonatti S. Remondelli P. Biochim. Biophys. Acta. 1999; 1447: 334-340Crossref PubMed Scopus (6) Google Scholar). 32P-labeled human HSp70 cDNA probe (Stressgen) was used as marker of HS response. GAPDH transcripts were detected by 32P-labeled EcoRI/EcoRI human cDNA fragment that was used as equal loading control probe. Serial dilutions corresponding to 0.02–2 μg of total RNA were reverse-transcribed (Invitrogen Life Technologies, Inc.) and real time RT-PCR was performed using iCycler Apparatus (Bio-Rad). Forty PCR amplification cycles of 60 s (15 s, 95 °C; 45 s, 60 °C) were run and amplification rates were monitored by the Sybr Green method. For PCR amplification the following primers were used: GAPDH-forward: GAA GGT GAA GGT CGG AGT C; GAPDH-reverse: GAA GAT GGT GAT GGG ATT TC; ERGIC-53-forward: GGG CAG CAT GGG CAG ATT AC; ERGIC-53-reverse: CAT AGA CGC CTC CAG CAG AGC; GRP94-forward: TCC GCC TTC CTT GTA GCA GAT A; GRP94-reverse: TGT TTC CTC TTG GGT CAG CAA T; MCFD2-forward: TGC ATG ATT ATG ATG GCA ATA ATT T; MCFD2-reverse: CAT TAG TGG TGC CTG TTC ACT CC. Transfection, Radioactive Labeling, and Immunoprecipitation of Huh7 Cell Extract—For metabolic labeling Huh7 cells were preincubated 1 h in methionine/cysteine-free Dulbecco's modified Eagle's medium, 0.5% fetal calf serum. Thereafter, cells were incubated for 2 h in the same culture medium supplemented with 100 μCi/ml [35S]methionine/cysteine labeling mix (PE Life Sciences). 4 × 107 cpm of cell lysates were incubated with undiluted polyclonal rabbit anti-ERGIC-53 antibody in ice-cold buffer overnight. Cells were chased for variable times with labeling medium containing 5-fold excess cold cysteine/methionine. Expression vectors carrying the cDNAs encoding CD8α (28Migliaccio G. Zurzolo C. Nitsch L. Obici S. Lotti L.V. Torrisi M.R. Pascale M.C. Leone A. Bonatti S. Eur. J. Cell Biol. 1990; 52: 291-296PubMed Google Scholar) and AP-CD8 (29Mottola G. Jourdan N. Castaldo G. Malagolini N. Lahm A. Serafini-Cessi F. Migliaccio G. Bonatti S. J. Biol. Chem. 2000; 275: 24070-24079Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) were transfected in Huh7 cells cultured as described above. Immunoprecipitation, SDS-PAGE and fluorography, were performed as detailed previously (28Migliaccio G. Zurzolo C. Nitsch L. Obici S. Lotti L.V. Torrisi M.R. Pascale M.C. Leone A. Bonatti S. Eur. J. Cell Biol. 1990; 52: 291-296PubMed Google Scholar). Indirect Immunofluorescence—Huh7 cells were grown on glass coverslips. Following incubation at 42 °C, cells were fixed with 4% formaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS. Only in the case of the goat polyclonal anti-Sec23 antibody the cells were fixed in methanol at –20 °C for 1 min, followed by 1 min at –20 °C in acetone and 5 min in PBS-glycine 0.1 m at room temperature. Cells were labeled with the appropriate antibody and with fluorescein or Texas Red-conjugated secondary antibodies. Coverslips were mounted in Moviol and viewed by epifluorescence on a Zeiss axiomatic photomicroscope with a ×63 planar objective. Results of quantitative analysis of ERGIC-53 distribution in normal and heat-shocked cells were performed by analyzing 150 cells/time point and values obtained were the mean ± S.D. of three independent experiments. Electron Microscopy—For conventional thin sections electron microscopy, samples were fixed with 2% glutaraldehyde in PBS buffer at room temperature. Samples were postfixed in 1% osmium tetroxide in veronal acetate buffer (pH 7.4) for 1 h at 25 °C, stained with 0.1% tannic acid in the same buffer for 30 min at 25 °C and with uranyl acetate (5 μg/ml) for 1 h at 25 °C, dehydrated in acetone, and embedded in Epon 812. Thin sections were examined unstained or poststained with uranyl acetate and lead hydroxide. Discontinuous Sucrose Gradients—Cells (7–10 × 107) were homogenized by 10 strokes in a Wheaton glass homogenizer in a buffer containing HEPES/KOH pH 7.3 20 mm, sucrose 120 mm. Postnuclear supernatant (PNS) was obtained by centrifugation at 500 × g for 5 min in a microcentrifuge and loaded on the top of a discontinuous sucrose gradient (15, 20, 25, 30, 35, 40, 45, % w/v) made up in the same buffer. The gradient was spun in a SW 50.1 rotor for 1 h at 43,000 rpm in a Beckman ultracentrifuge and 13 fractions were collected from the bottom of the tube with a peristaltic pump. Fractions were trichloroacetic acid-precipitated, and proteins were separated by SDS-PAGE, transferred to ECL membranes and subjected to Western blot analyses (30Erra M.C. Iodice L. Lotti L.V. Bonatti S. Cell Biol. Int. 1999; 23: 571-577Crossref PubMed Scopus (9) Google Scholar). Cells Accumulate ERGIC-53 in Response to HS—To evaluate the expression level of ERGIC-53 in response to HS, the amount of ERGIC-53 in Huh7 cells cultured for different times at 42 °C was measured by Western blot analysis and compared with the level of HSp70, marker for HS response, and α-tubulin, marker for the constitutively expressed gene (Fig. 1). These experiments showed that the amount of ERGIC-53 increased with exposure time to HS, in a similar fashion to HSp70 (Fig. 1, A and C). Densitometry of autoradiographs showed that the increase in ERGIC-53 was detectable at 4 h of HS with a relative fold of induction of 2.68 ± 0.25 and that such values remained higher (2.86 ± 0.18) after 8 h of continuous HS. Interestingly, HSp70 presented similar rates of induction (2.6 ± 0.45 and 2.94 ± 0.40) at the same time points. Conversely, between 8 and 16 h at 42 °C the level of HSp70 stayed high, while the level of ERGIC-53 returned to steadiness (data not shown). Accumulation of ERGIC-53 was also measured during the cell response to transient HS (Fig. 1, B and D). In these experiments, the cells were first incubated for 30 (data not shown) or 60 min at 42 °C, then moved to 37 °C and analyzed at different time points. Interestingly, increased amounts of either ERGIC-53 (2.06 ± 0.51) or HSp70 (2.6 ± 0.56) appeared 4 h after exposure to transient HS. The ERGIC-53 level returned normal after 8 h, while HSp70 remained higher at 8 h (1.8 ± 0.2) and returned normal after 12 h exposure to transient HS (not shown). Finally, the cells were fully viable after HS treatment and could be normally cultured and passaged, and similar accumulation of ERGIC-53 in response to HS was detected in a monkey (CV1) cell line (data not shown). Differential Regulation of ERGIC-53 mRNA Expression in Response to HS or UPR—To test whether the increased amount of ERGIC-53 in response to HS was due to increased levels of ERGIC-53 mRNA, we performed Northern blot analysis. The results showed that the level of ERGIC-53 mRNA remained unchanged both during prolonged and short exposure of cells to HS (Fig. 2, A and B). As expected, the same RNA sample revealed a higher level of HSp70 mRNA and an unaltered level of GAPDH mRNA, a marker of housekeeping genes (Fig. 2, A and B). Conversely, cells expressed a higher level of ERGIC-53 mRNA in response to the ER calcium ATPase inhibitor thapsigargin (TG), a well-known inducer of UPR that was recently shown to enhance the transcription of ERGIC-53 (24Nyfeler B. Nufer O. Matsui T. Mori K. Hauri H.P. Biochem. Biophys. Res. Commun. 2003; 304: 599-604Crossref PubMed Scopus (29) Google Scholar). As shown in Fig. 2C, higher levels of ERGIC-53 mRNA were detected, with maximal intensity between 4 and 8 h of TG treatment. To conclusively prove that ERGIC-53 gene transcription is differentially regulated in response to HS or UPR, we switched to quantitative RT-PCR assays, a more accurate method than Northern blot. These assays confirmed that ERGIC-53 mRNA was stably expressed under both transient and prolonged HS (Fig. 2D). Conversely, higher levels of ERGIC-53 mRNA, and of the UPR marker GRP94, were detectable after 4 and 8 h of TG treatment (Fig. 2D). These results strongly suggested that UPR, but not HS, transcriptionally activates the expression of ERGIC-53. This conclusion was further confirmed by the finding that the accumulation of ERGIC-53 protein at 42 °C was not inhibited by quercetin, a drug which blocks the activation of HS transcription factors and therefore the heat-induced synthesis of heat shock proteins, or by genistein, which inhibits the up-regulation of glucose-regulated proteins (GRPs) and HSps (Fig. 3, A and B) (31Zhou Y. Lee A.S. J. Nat. Cancer Inst. 1998; 90: 381-388Crossref PubMed Scopus (130) Google Scholar, 32Nagai N. Nakai A. Nagata K. Biochem. Biophys. Res. Commun. 1995; 208: 1099-1105Crossref PubMed Scopus (129) Google Scholar). Conversely, both drugs were able to inhibit the accumulation of HSp70 protein in response to HS, and had no effect on the level of expression of α-tubulin (Fig. 3, A and B). Heat Shock Enhances Translation of ERGIC-53—The results reported above suggest that the accumulation of ERGIC-53 protein during HS could depend on an increased synthesis of ERGIC-53 protein, or a decreased degradation, or both. To address these questions, cells were pulse-labeled with [35S]methionine and cysteine in the presence or absence of HS and the amount of newly synthesized ERGIC-53 measured on SDS-PAGE after immunoprecipitation. As shown in Fig. 4B, increased ERGIC-53 synthesis was observed between the second and the fourth hour of continuous HS treatment, as well as between the second and fourth hour at 37 °C after transient HS (60 min at 42 °C). Conversely, no effect was revealed in the synthesis of tubulin that was used as internal control protein (data not shown). In addition, very similar amounts of ERGIC-53 protein were recovered at the end of 8 h chase performed either at 37 °C or at 42 °C aftera2h pulse at 37 °C (Fig. 4C). Therefore, the increased level of ERGIC-53 protein in response to HS was the result of enhanced translation of its mRNA. Analysis of the Putative Promoter and 5′-Untranslated Region of ERGIC-53—Our results suggested that the 5′ of the ERGIC-53 gene contained cis-acting elements regulating either transcription by UPR or enhanced translation during hyperthermia. Therefore, we analyzed the putative promoter and the 5′-untranslated region of ERGIC-53 found in the human genome data base (NCBI ref/NT_033908.1/Hs18_34063). Computer analysis to identify transcription factors binding sites (MatInspector, Genomatix Software Gmbh) revealed that the ERGIC-53 putative promoter (500 nt) contained two CCAAT motifs and five GC boxes (Fig. 5A). The main but not the only CCAAT-binding protein is CBF/NFY, while GC boxes are consensus binding sites for factors of the SP1 family. These sequences are encountered at very high frequency in many eukaryotic promoters, in which they mediate constitutive expression of genes (33Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (442) Google Scholar, 34Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1244) Google Scholar). Instead, the transcriptional control of UPR-responsive genes requires ER-stress response elements (ERSEs) (34Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1244) Google Scholar). ERSEs are structured in a tripartite configuration composed by the CCAAT box followed by a 9-bp spacer sequence containing a GGC triplet and a CCACG motif at the 3′-end (35Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (995) Google Scholar, 36Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (213) Google Scholar). Interestingly, we found that none of the CCAAT sequences found in the putative promoter region of ERGIC-53 gene fulfilled all the criteria expected for a functional ERSE sequence (36Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (213) Google Scholar). Moreover, as we expected, we found no binding sites for eukaryotic heat shock factors (i.e. HSE) and, interestingly, we did not find either sequences corresponding to known binding sites for the eukaryotic initiation factor TFIID (TATA box) or classic eukaryotic signals for transcription initiation (37Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2789) Google Scholar). We also analyzed the ERGIC-53 5′-untranslated region (21 nt) that was previously identified (38Schindler R. Itin C. Zerial M. Lottspeich F. Hauri H.P. Eur. J. Cell Biol. 1993; 61: 1-9PubMed Google Scholar). Interestingly, sequence analysis revealed a striking complementarity to 18 S rRNA (Fig. 5B). In addition, RNA structure prediction analysis (39Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (9968) Google Scholar) showed that the 5′-untranslated of ERGIC-53 might be able to form a stable RNA/RNA hybrid with 18 S rRNA (Fig. 5B). Since complementarity with the 18 S rRNA is an essential requirement for the preferential translation of viral and cellular genes during down-regulation of general translation, we suggest that ERGIC-53 could be preferentially translated by a ribosomal shunting mechanism in response to HS. Intracellular Redistribution of ERGIC-53 in Response to HS—To test whether HS could affect the intracellular trafficking of ERGIC-53 we assayed the intracellular localization of the lectin after different times of incubation at 42 °C by indirect immunofluorescence. At 37 °C, ERGIC-53 is localized in punctuate structures of variable size, dispersed throughout the cytosol, as well as in the central Golgi region which is concentrated perinuclearly (Fig. 6A, panel a). Conversely, incubation at 42 °C clearly generated a redistribution of ERGIC-53 in the cell perip
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