Article |
2 Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Suita City, Osaka 565-0871, Japan
3 Discovery Research Lab, TANABE SEIYAKU Co., Ltd., Osaka City, Osaka 532-0031, Japan
4 Skirball Institute, New York University School of Medicine, New York, NY 10016
5 Departments of Surgery, Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032
6 CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
Address correspondence to Dr. Osamu Hori, Department of Neuroanatomy (Anatomy III), Kanazawa University, School of Medicine, 13-1 Takara-Machi, Kanazawa City, Ishikawa, 920-8640, Japan. Tel.: 81-76-265-2162. Fax: 81-76-234-4222. E-mail: osamuh{at}nanat.m.kanazawa-u.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: hypoxia; ER stress; protein synthesis; ATP-dependent protease; molecular chaperone
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ER is the organelle in which membrane and secretory proteins achieve correct folding and oligomerization. Once cells are exposed to stresses such as glucose starvation, inhibition of protein glycosylation, disturbance of Ca2+ homeostasis (ER stress), or oxygen deprivation, unfolded proteins accumulate in the ER and eukaryotic cells respond by several mechanisms: (a) transcriptional induction; (b) translational attenuation; and (c) degradation (Mori, 2000). Attenuation of protein synthesis in response to ER stress occurs to lessen the load of protein entering the ER, and this pathway requires an activation of the ER-resident protein kinase, PERK (Harding et al., 1999). Although general suppression of protein synthesis improved cell viability under ER stress (Harding et al., 2000), the detailed mechanisms involved, especially at the level of translational products destined for particular organelles, have not been elucidated.
Here we describe a novel signaling pathway from the ER to mitochondria through suppression of protein synthesis. Under ER stress, expression and assembly of cytochrome c oxidase (COX)*are disturbed, whereas mitochondrial ATP-dependent proteases/chaperones are induced, at least in part, to improve assembly of these complexes, and, potentially, to sustain mitochondrial function.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of Lon in response to cell stress
Northern blot analysis using total RNA (10 µg) from cultured astrocytes subjected to oxygen deprivation showed an increase in the level of Lon transcript after 1622 h (4.5-fold increase at the latter time point), and a subsequent decrease in Lon mRNA within 4 h of reoxygenation (Fig. 1 A). The temporal pattern of Lon mRNA expression in hypoxia paralleled that of GRP78 mRNA, a molecular chaperone in the ER, although GRP78 was induced to a greater extent than Lon (Fig. 1 A). Prompted by the results of these studies, the expression of Lon under conditions of hypoxia or other stimuli was investigated in general cell lines. When HeLa cells were exposed to hypoxia, Lon mRNA level was increased by 3.03.5-fold (unpublished data). Treatment of HeLa cells with tunicamycin (Tm), brefeldin A (BFA) or thapsigargin (Tg), all of which cause accumulation of unfolded polypeptides in the ER (ER stress), resulted in 3.84.3-fold increases in Lon transcript levels (Fig. 1 B, lanes 14). In contrast, heat shock (HS) or sodium arsenite (AS) had no effect (Fig. 1 B, lanes 57). Modulation of the expression of Lon mRNA in response to ER stress was compared with that of other molecular chaperones associated with the ER or mitochondria (GRP78, GRP75/mtHSP70, and HSP60), and with another mitochondrial ATP-dependent protease, Yme1. Transcripts of GRP75/mtHSP75, but not HSP60, were enhanced moderately (twofold increase) in response to ER stress, consistent with a previous report (Mizzen et al., 1989). In addition, Yme1 mRNA was also enhanced by ER stress (3.64.5-fold increase; Fig. 1 B).
|
To analyze Lon expression at the protein level, Western blotting was performed using anti-Lon antibody (#665) generated against an 18-aa Lon-derived polypeptide, which recognizes both human and rat Lon antigens. Immunoblotting of cultured cell extracts with this antibody displayed an immunoreactive band with Mr. of 100 kD as well as a band of
64 kD (Fig. 1 E). The appearance of the 100- kD band, but not the 64-kD band, was blocked by an excess amount of Lon peptide immunogen (Fig. 1 F), suggesting that only the 100-kD band represented Lon antigen. ER stress-induced elevation of Lon mRNA was accompanied by a 3.13.5-fold increase in the level of Lon antigen in HeLa cells (Fig. 1 E) and in astrocytes (unpublished data).
The results of the above in vitro studies were extended to the in vivo setting using the rat middle cerebral artery (MCA) occlusion model. 8 h after MCA occlusion, Northern blotting of total RNA extracted from the ischemic brain showed increased Lon transcript level, compared with the RNA from the brain after a sham procedure (Fig. 2 A). In situ hybridization indicated increased Lon mRNA level in the ipsilateral cerebral cortex in response to ischemia, especially in neurons (Fig. 2 B). Western blotting with anti-Lon antibody 665 also confirmed the enhancement of Lon antigen in the ischemic rat brain (Fig. 2 C).
|
|
|
When wild-type or mutant forms (S845N and K519N) of Lon were transiently transfected into 293T cells, wild-type and S845N Lon antigens were detected as a single band in the mitochondrial matrix fraction (Fig. 5 A). however, K519N Lon antigen was detected in both mitochondrial matrix and cytosol fractions, and there was a poorly defined smear of FLAG immunoreactivity in the upper portion of the membrane, suggesting that mitochondrial import and oligomerization of K519N Lon may be impaired (Fig. 5 A). Therefore, wild-type and S845N Lon were used for further studies. Transfection efficiency into 293T cells was 70%, and the level of Lon antigen was 4-5-fold higher in transfectants compared with mock-transfected and nontransfected controls (Fig. 5 B). Resistance of COX I and II antigens, isolated from mitochondria of 293T cells, to degradation by trypsin was employed to monitor their assembly into COX. After transfection, cells were treated with Tm and protease degradation were performed. Trypsin resistance of COX II was enhanced in 293T cells transfected with the plasmid encoding wild-type or S845N compared to mock-transfected controls (Fig. 5 C). Quantative analysis of data from three experiments with Tm-treated cultures demonstrated that the percentages of COX II antigen resistant to tyrpsin (250 µg/ml) were 52, 76, and 19% in wild-type, S845N, and mock-transfected cells, respectively (Fig. 5 D).
|
Based on these observations, we assessed the trypsin resistance of COX II in wild-type and S845N Lon-transfected cells using stably transfected HeLa cells. The level of Lon antigen was 3.54.5 times higher in stable transfectants expressing each of the Lon constructs as compared with mock-transfected controls (Fig. 6 A). Trypsin digestion assays, performed after each clone was subjected to Tm treatment, revealed that resistance of COX II to trypsin proteolysis was enhanced in transfectants overexpressing wild-type and S845N Lon (Fig. 6 B). The percentages of COX II antigen resistant to trypsin (250µg/ml) were 41, 56, 48, and 17% in wild-type, S845N-1, S548N-2 (the latter represent two different clones expressing the S845N variant), and mock-transfected clones, respectively (Fig. 6 C). To exclude the possibility that enhanced trypsin resistance of COX II was due to the aggregation of unassembled (free) COX II, the binding of COX II to COX I, which occurs at an early stage in COX synthesis (Nijtmans et al., 1998), was assessed by immunoprecipitation followed by Western blotting (Fig. 6 D). The percentage of COX II bound to COX I (lanes 2, 4, 6, 8) compared with total COX II (lanes 3, 5, 7, and 9) was increased in clones overexpressing Lon (32, 40, 30, and 16% in wild-type, S845N-1, S845N-2, and mock-transfected clones, respectively; Fig. 6 E). Immunoprecipitation of mitochondria with nonimmune IgG did not show COX II antigen (Fig. 6 D, lane 1).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we first confirmed the increased expression of Lon in cells subjected to hypoxia or ER stress using several approaches: (a) increased Lon promoter activation, based on enhanced luciferase activity following transient transfection with human or murine Lon promoterreporter constructs; (b) increased mRNA level with no change in its stability; and (c) increased Lon antigen level. Another mitochondrial ATP-dependent protease, Yme1, and the molecular chaperone GRP75/mitochondrial HSP70, also displayed increased transcript levels in response to ER stress (Fig. 1 B). Taken together, these observations suggested the existence of a connection, direct and/or indirect, between ER stress and mitochondrial properties.
COX provided a useful model system in which to study the effects of ER stress on mitochondrial proteins because of its multisubunit structure, comprised of both mitochondrial- (I, II, and III) and nuclear-derived (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII in human) components, and as expression/assembly of COX complex has been studied extensively (Nijtmans et al., 1998). Although the enzymatic core of COX consists of subunits I and II (mitochondrial-derived), COX IV (nuclear-derived), is essential for assembly of the enzyme (Dowhan et al., 1985) and COX II and III, but not COX I, were rapidly degraded in COX IV-deleted yeast (Nakai et al., 1994). In this setting, Yme1, also referred to Osd1, was required for the degradation of COX II (Nakai et al., 1995). Although yeast Lon did not affect the degradation of COX subunits, as a molecular chaperone, it promoted the assembly of COX complex (Rep et al., 1996).
In the current study, we demonstrated that ER stress reduced steady-state levels of nuclear-derived COX IV and V subunits, and caused rapid degradation of COX II, the latter of which was likely due to accumulation of free COX II subunits (Fig. 3). In contrast, the apparent stability of COX I under the same conditions may reflect its interaction with heme A (Wielburski and Nelson, 1984). Treatment of cells with cycloheximide mimicked the altered expression of COX subunits with ER stress, and enhanced expressions of Lon and Yme1 at the mRNA level (Fig. 4, AC). Induction of Lon or GRP75/mtHSP70 was not observed in cells without PERK, a protein kinase that medicates suppression of protein synthesis in response to ER stress (Harding et al., 1999; Fig. 4 D). These observations suggested that suppression of cytosolic protein synthesis under ER stress is the common denominator linking these events.
Overexpression of wild-type Lon or a proteolytically inactive Lon mutant facilitates assembly of COX II into COX I-containing complexes under ER stress in experimental systems with transiently or stably transfected cells (Figs. 5 and 6). From the vantage point of maintenance of mitochondrial function, stably transfected HeLa cells overexpressing the proteolytically inactive Lon mutant were relatively resistant to changes in mitochondrial membrane potential induced by brefeldin A or hypoxia (Fig. 7).
Under apoptotic conditions, there have been several reports concerning mechanisms of communication between the ER and mitochondria via Ca2+ (Häcki et al., 2000; Nakamura et al., 2000). However, the results of our present study provided insight into a pathway involving ER stress in which suppression of cytosolic protein synthesis has a central role. Several recent findings have emphasized the potentially important role of mitochondrial ATP-dependent proteases or molecular chaperones in pathological-stressful conditions. Expression and activity of Lon were increased in rats bearing Zajdela hepatoma or in T3-treated hypothyroid animals (Luciakova et al., 1999). Mutations in Paraplegin, another mitochondrial ATP-dependent protease, cause hereditary spastic paraplegia (Casari et al., 1998). Preservation of the mitochondrial electron transport chain, by ischemic preconditioning or overexpression of HSP60 and HSP10, facilitated restoration of energy stores and cellular functions after reperfusion (Kobara et al., 1996; Lin et al., 2001).
Although the detailed mechanism underlying the cytoprotective effect(s) of Lon and the relevance of its protease activity under ER stress remain to be elucidated, our results emphasized the importance of the chaperone activity of this molecule for ensuring assembly of protein complexes and optimal functioning of mitochondria during the cellular response to environmental challenges.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ESD screening method for differentially expressed genes
The ESD screening for isolating genes upregulated in rat astrocytes exposed to hypoxia was performed as described (Suzuki et al., 1996). In brief, cDNA fragments derived from cultured astrocytes exposed to hypoxia for 22 h (H'-tracer) were equalized and subtracted with samples from normoxia (N-driver) or hypoxia (H-driver) up to three times (H'-N and H'-H, respectively). PCR was then carried out using [32P] dCTP (>3,000 Ci/mlM) and products were separated on a sequencing gel followed by autoradiography. Candidate DNA bands were cut out of the gel and cloned into pGEM-T vector (Promega). DNA sequencing was performed using a 377 DNA sequencer (Applied Biosystems) and differential expression of candidate genes in hypoxia versus normoxia was confirmed by Northern blotting using [32P]-radiolabeled cDNAs as probes. Sequence searches and comparisons were carried out using several databases, including the National Center for Biotechnology Information, FASTA, and BLAST molecular analysis systems, and the DNA Data Bank of Japan.
Cloning of rat Lon cDNA
One of the cDNA fragments obtained by ESD was 346 bp in length and 85% identical to human Lon. This fragment was used to screen a rat cDNA library (lambda ZapII cDNA library; Stratagene) and a cDNA of 2.97 kb was obtained spanning the entire open reading frame. Both strands were sequenced using a 377 DNA sequencer.
Northern blot analysis
Total RNA (10 µg), isolated from cultured astrocytes, HeLa cells, 293T cells or the ischemic rat brain was separated on agarose/formaldehyde (1%) gels and transferred onto Immobilon N membranes (Millipore). cDNA fragments for probes were generated as follows: rat Lon cDNA was obtained as described above; human GRP78, human GRP75/mitochondrial HSP70, human HSP60, and human Yme1 cDNAs were produced by PCR with specific primers. Each fragment was labeled with 32P by the random hexamer procedure (specific activity; 0.53 x 109 cpm/µg DNA) and was used to probe membranes with immobilized RNA. Primers used for PCR were: ATG GTA TTC TCC GAG TGA CA and TTG GCT TTA AAG TCT TCA AT for GRP78; GGA TGG CTG GAA TGG CCT TAG and CCA ACA AGT CGC TCC CCA TCT for GRP75/mtHSP70; ATG CTG TGG CCG TTA CAA TG and CTC CTG ATT TCC ACT GGA TT for HSP60; and AGA TAC AGT TCC TGA GCA TGA and GCT AGA GGA ACG TAA TGT ACT for Yme1. After washing in 2 x SSC and 0.5 x SDS for 1 h, membranes were subjected to autoradiography.
Construction of Lon promoterluciferase reporters and measurement of relative luciferase activity
A 608-bp fragment of the human Lon promoter (-584 to +24) was amplified by PCR using primers CAA CAT CAG CAT CGA CTT GG and ATA CTG GCG GCT CAC ACA ACT and cloned into Sac I-Hind III sites of pGL3 basic vector (Promega) after adding enzyme sites at both ends. Both strands were sequenced using a 377 DNA sequencer. Mouse Lon genomic DNA was cloned by screening a mouse BAC library using PCR primers AGC ACA GGC TAC GTG CGG CTC T and CTC GGA GGT CTC GTC GCT GCC A (Incyte Genomics). A 2.58-kb fragment (-2559 to +24; the numbers are tentative because the transcriptional start site was yet to be defined) was subcloned into Kpn I-Sma I sites of pGL3 basic vector after adding enzyme sites at both ends. Wild-type GRP78 promoter and a promoter mutant defective in the ERSE of GRP78 (-304 to +7) was inserted in pGL3 basic vector, provided by Dr. Mori (Kyoto University, Kyoto, Japan) (Yoshida et al., 1998). A reference plasmid (pRL-SV40) was purchased from Promega. Transfection was carried out by lipofection and, after 24 h, cells were exposed to Tm (2 µg/ml) or BFA (2.5 µg/ml) for 16 h to induce ER stress. Firefly and Renilla luciferase activities were measured using the Ascent system (Labsystems) and relative luciferase activity was obtained by dividing the former values by the latter. Fold induction was defined as the ratio of induced-to-basal levels of relative luciferase activity.
Generation of antibodies and immunoblotting
To obtain antibodies reactive with Lon, a peptide with the sequence CEKDDKDAIEEKFRERLKE was synthesized (an extra C was introduced at its NH2 terminus) and conjugated to keyhole limpet hemocyanin. Rabbits were immunized with this peptide by conventional methods. Once high titer antibodies were obtained, the IgG fraction was purified by chromatography on protein G columns (GIBCO BRL). For detection of Lon and other proteins, cultured astrocytes, HeLa cells, or 293T cells were lysed in buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 1% Deoxycholate, 1 mM PMSF, 1 µg/ml Aprotinin, 1 µg/ml leupeptin, and 1 µg/ml Pepstatin. Immunoblotting was performed using antibodies to Lon, FLAG (Sigma-Aldrich), KDEL (StressGen; Biotechnologies Corp.), and COX subunits I, II, IV, or Vb (Molecular Probes). Sites of primary antibody binding were determined by alkaline phosphatase-conjugated secondary antibodies or the ECL method.
Expression of Lon in the ischemic brain
Unilateral permanent MCA occlusion was performed in male Sprague-Dawley rats (250 g) as described (Yamaguchi et al., 1999). After 8 h of ischemia, rats were sacrificed and brains were frozen at 80°C. Northern blotting was performed to detect Lon mRNA as described above. Serial coronal sections were also cut and the distribution of Lon mRNA was examined by in situ hybridization using digoxigenin-labeled cRNA probes as described (Hori et al., 1995). In brief, sense and antisense riboprobes for Lon were in vitro transcribed from the rat Lon cDNA inserted into the pGEM T vector. After linearizing the vector with NcoI (for the sense probe) or NdeI (for the antisense probe), reaction mixtures were incubated with digoxigenin-UTP and SP6 or T7 RNA polymerase (Roche Diagnostics Corporation). Brain sections were then hybridized with either the sense or antisense probe. For detection of hybridized cRNA probes, alkaline phosphataseconjugated antibody to digoxigenin was used and the color was developed with NBT and X-phosphate solution. Lon antigen was also detected by Western blotting with antibody to Lon using brain homogenates after MCA occlusion (8 h) or sham operation (8 h) as described above.
Pulse-chase analysis of COX subunits. HeLa cells (5 x 106cells /condition) treated with Tm for 16 h, or maintained in medium alone (as control) were labeled with [35S]-methionine (200 µCi/ml; Amersham Pharmacia Biotech) for 30 min in methionine-free medium and chased for the indicated times (up to 8 h) in regular medium. Cell extracts in lysis buffer (see above) were immunoprecipitated with anti-COX subunit I, II, or IV antibodies, and subjected to SDS-PAGE followed by autoradiography.
Isolation of mitochondria and assembly of COX II
HeLa cells (107 cells) exposed to Tm or incubated in medium alone were homogenized by nitrogen bomb cavitation, and mitochondria were isolated by sequential centrifugation (Evans, 1992). After solubilizing mitochondria in 4% Na-cholate, 50 mM NaHPO4, 0.9% NaCl, and 1 mM EDTA, assembly of COX was assessed by monitoring trypsin resistance of COX I and II antigens as described (Rep et al., 1996). Binding of COX I and COX II subunits was monitored by immunoprecipitation of solubilized mitochondria (in 3% Na-cholate, 50 mM NaHPO4, 0.9% NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml Aprotinin, 1 µg/ml Leupeptin, and 1 µg/ml Pepstatin) with anti-COX I antibody followed by Western blotting with anti-COX II antibody.
Submitochondrial fractionation
Crude fractionation of 293T cells (107cells) transfected with Lon cDNA was performed by sequential centrifugation as described above, and submitochondrial fractions were prepared as described previously (Schnaitman and Greenawalt, 1968). In brief, isolated mitochondria were dissolved in isolation buffer containing 2 mM Hepes, 220 mM D-mannitol, 70 mM sucrose, 0.5 mg/ml BSA, and 0.5% digitonin, and centrifuged at 8,000 g for 10 min. Both the supernatant and pellet were treated with Lubrol or 1% digitonin for 15 min on ice and centrifuged at 144,000 g for 1 h.
Plasmid construction and overexpression of Lon
Rat Lon cDNA encoding the complete open reading frame was tagged with FLAG epitope at the COOH terminus and cloned into pcDNA3.1(+) (Invitrogen) or pME18Sf+, a gift from Dr. Maruyama (Tokyo Medical and Dental University, Tokyo, Japan) containing the hygromycin resistance gene, provided from Dr. Ohishi (Osaka University, Osaka, Japan). Lon mutants were constructed using LA PCR in vitro mutagenesis primer set (Takara Bichemicals). Primers used for creating Lon K519N and S845N were AAT GCT GGT GTT GCC GCC CAC ACC TGG T and ATG GCC CTA ATG CAG GTT GCA CCA TT, respectively. All constructs were sequenced prior to transfection studies. Wild-type or mutant Lon was transiently transfected into 293T cells (4 µg of DNA/107cells) and stably transfected into HeLa cells (25 µg of DNA/107cells) by lipofection (Lipofectamine; GIBCO BRL) or electroporation (GenePulser II; Bio-Rad Laboratories), respectively. HeLa cells overexpressing Lon were selected with hygromycin (300 µg/ml; Sigma-Aldrich) for 2 wk.
Measurement of mitochondrial membrane potential. Mitochondrial membrane potential was assessed by microscopically (excitation wavelength, 510560 nm) after treating cells with MitosensorTM (CLONTECH Laboratories, Inc.). The number of cells showing positive signals in a mitochondria-derived dot-like pattern was counted and indicated as the percentage of total cells (100 cells). Statistical analysis was performed using Student's t test.
Laser densitometric analysis
Laser densitometric analysis was performed to standardize the results of Western and Northern blotting using Quality One software (pdi) as previously (Yamaguchi et al, 1999).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
Experiments in the Ron lab were supported by the National Institutes of Health (ES08681 and DK47119). D. Ron is an Ellison Medical Foundation Senior Scholar on Aging.
Submitted: 20 August 2001
Revised: 22 March 2002
Accepted: 25 May 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Casari, G., M. De Fusco, S. Ciarmatori, M. Zeviani, M. Mora, P. Fernandez, G. De Michele, A. Filla, S. Cocozza, R. Marconi, A. Dürr, B. Fontaine, and A. Ballabio. 1998. Spastic paraplegia and OXPHOS impairment caused by mutation in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell. 93:973983.[Medline]
Dowhan, W., C.R. Bibus, and G. Schatz. 1985. The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast. EMBO J. 4:179184.[Abstract]
Evans, W.H., 1992. Preparative Centrifugation. A Practical Approach. Rickwood, D., editor. Oxford University Press, Oxford, UK. 399 pp.
Häcki, J., L. Egger, L. Monney, S. Conus, T. Rossé, I. Fellay, and C. Borner. 2000. Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2. Oncogene. 19:22862295.[CrossRef][Medline]
Harding, H.P, I. Novoa, Y. Zhang, H. Zeng, R. Wek, M. Schapira, and D. Ron. 2000. Regulated translation inhibition controls stress-induced gene expression in mammalian cells. Mol. Cell. 6:10991108.[Medline]
Hori, O., M. Matsumoto, Y. Maeda, H. Ueda, T. Kinoshita, D. Stern, S. Ogawa, and T. Kamada. 1994. Metabolic and biosynthetic alteration in cultured astrocytes exposed to hypoxia/reoxygenation. J. Neurochem. 62:14891495.[Medline]
Hori, O., J. Bred, T. Slattery, R. Cao, J. Zhang, J.X. Chen, M. Nagashima, E.R. Lundh, S. Vijay, D. Nitechi, J. Morser, D. Stern, and A.M. Schmidt. 1995. The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. J. Biol. Chem. 270:2575225761.
Kuwabara, K., M. Matsumoto, J. Ikeda, O. Hori, S. Ogawa, Y. Maeda, K. Kitagawa, N. Imuta, T. Kinoshita, D. Stern, H. Yanagi, and T. Kamada. 1996. Purification and characterization of a novel stress protein, the 150-kD oxygen-regulated protein (ORP150), from cultured rat astrocytes and its expression in ischemic mouse brain. J. Biol. Chem. 27:50255032.[CrossRef]
Lin, K.M., B. Lin, I.Y. Lian, R. Mestril, I.E., Scheffler, W.H. Dillmann. 2001. Combined and individual mitochondrial HSP60 and HSP10 expression in cardiac myocytes protects mitochondrial function and prevents apoptotic cell deaths induced by simulated ischemia-reoxygenation. Circulation. 103:17871792.
Liu H., R.C. Bowes III, B. van de Water, C. Sillence, J.F. Nagelkerke, and J.L. Stevens. 1997. Endoplasmic reticulum chaperons (GRP78 and calreticulin) prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells. J. Biol. Chem. 272:2175121759.
Luciakova, K., B. Sokolikova, M. Chloupkova, and B.D. Nelson. 1999. Enhanced mitochondrial biogenesis is associated with increased expression of the mitochondrial ATP-dependent Lon protease. FEBS Lett. 444:186188.[CrossRef][Medline]
Matsuo N., S. Ogawa, T. Takagi, D. Stern, A. Wanaka, and M. Tohyama. 1997. Cloning of a putative vesicle transport-related protein, RA410, from cultured rat astrocytes and its expression in ischemic rat brain. J. Biol. Chem. 272:1643816444.
Mizzen, L.A., C. Chang, J.I. Garrels, and W.J. Welch. 1989. Identification, characterization, and purification of two mammalian stress proteins present in mitochondria, grp75, a member of the hsp 70 family and hsp58, a homologue of the bacterial groEL protein. J. Biol. Chem. 264:2066420675.
Nakai, T., Y. Mera, T. Yasuhara, and A. Ohashi. 1994. Divalent metal ion-dependent mitochondrial degradation of unassembled subunit 2 and 3 of cytochrome c oxidase. J. Biochem. 116:756758.
Nakai, T., T. Yasuhara, Y. Fujiki, and A. Ohashi. 1995. Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria. Mol. Cell. Biol. 15:44414452.[Abstract]
Nakamura, K., E. Bossy-Wetzel, K. Burns, M.P. Fadel, M. Lozyk, I.S. Goping, M. Opas, C. Bleackley, D.R. Green, and M. Michalak. 2000. Changes in endoplasmic reticulum luminal environment after cell sensitivity to apoptosis. J. Cell Biol. 150:731740.
Nijtmans, L.G.J., J. Taanman, A.O. Muijsers, D. Speijer, and Van Den Bogert. 1998. Assembly of cytochrome-c oxidase in cultured human cells. Eur. J. Biochem. 254:389394.[Abstract]
Ozawa, K., K. Kuwabara, M. Tamatani, K. Takatsuji, Y. Tsukamoto, S. Kaneda, H. Yanagi, D. Stern, Y. Eguchi, Y. Tsujimoto, S. Ogawa, and M. Tohyama. 1999. 150 kD Oxygen-regulated protein (ORP150) suppressed hypoxia-induced apoptotic cell death. J. Biol. Chem. 274:63976404.
Ozawa, K., Y. Tsukamoto, O. Hori, Y. Kitao, H. Yanagi, D. Stern, and S. Ogawa. 2000. Regulation of tumor angiogenesis by oxygen-regulated protein 150, an inducible endoplasmic reticulum chaperone. Cancer Res. 61:42064213.
Rep, M., J.M. van Dijl, K. Suda, G. Scahtz, L.A. Grivell, and C.K. Suzuki. 1996. Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon. Science. 274:103106.
Schnaitman, C., and J.W. Greenawalt. 1968. Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J. Cell Biol. 38:158175.
Suzuki, Y., N. Sato, M. Tohyama, A. Wanaka, and T. Takagi. 1996. Efficient isolation of differentially expressed genes by means of a newly established method, "ESD." Nucleic Acids Res. 24:797799.
van Dijl, J.M., E. Kutejova, K. Suda, D. Perecko, G. Schatz, and C.K. Suzuki. 1998. The ATPase and protease domains of yeast mitochondrial Lon: roles in proteolysis and respiration-dependent growth. Proc. Natl. Acad. Sci. USA. 95:1058410589.
van Dyck, L., D.A. Pearce, and F. Sherman. 1994. PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 269:238242.
von Heijne, G., J. Steppuhn, and R.G. Herrmann. 1989. Domain structure of mitochondrial and chloroplast targeting peptides. Eur. J. Biochem. 180:535545.[Abstract]
Wang, N., S. Gottesman, M.C. Willingham, and M.M. Gottesman. 1993. A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease. Proc. Natl. Acad. Sci. USA. 90:1124711251.[Abstract]
Yamaguchi, A., O. Hori, D. Stern, E. Hartmann, S. Ogawa, and M. Tohyama. 1999. Stress-associated endoplasmic reticulum protein 1 (SERP)/ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation. J. Cell Biol. 147:11951204.
Yoshida, H., K. Haze, H. Yanagi, T. Yura, and K. Mori. 1998. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins J. Biol. Chem. 273:3374133749.
Related Article