Article |
Address correspondence to David Ron, Skirball Institute of Biomolecular Medicine, Room 3-10, 540 First Avenue, New York, NY 10016. Tel.: (212) 263-7786. Fax: (212) 263-8951. E-mail: ron{at}saturn.med.nyu.edu
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Abstract |
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Key Words: chaperone; protein folding; protein degradation; gene expression; functional genomics
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Introduction |
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In yeast, ER stress activates the ER resident transmembrane protein kinase and endonuclease Ire1p, leading to the posttranscriptional processing of a downstream mRNA encoding the transcription factor Hac1p. Hac1p directly induces target genes of the UPR. These target genes encode proteins involved in processing, trafficking, and degradation of ER client proteins (for reviews see Mori, 2000; Patil and Walter, 2001). The UPR has diversified considerably in metazoans. In addition to a conserved signaling pathway of IRE1 homologues and a downstream HAC1-like transcription factor, XBP-1 (Yoshida et al., 2001; Calfon et al., 2002), two new pathways, not present in yeast, have evolved. Pancreatic-enriched ER kinase (PERK) phosphorylates eukaryotic translation initiation factor 2 to attenuate protein synthesis and activate specific gene expression during ER stress (for review see Ron and Harding, 2000), and the ER stressactivated transcription factor ATF6 directly activates UPR target genes (Haze et al., 1999; Ye et al., 2000; Yoshida et al., 2001).
The coordination of these three pathways and their specific contribution to the metazoan ER stress response are unclear, though in Caenorhabditis elegans, the IRE1 and PERK pathways provide redundant protection against ER stress (Shen et al., 2001). We used cDNA microarrays to characterize the transcriptional response to ER stress in C. elegans, a simple metazoan that has counterparts of all three known components of the mammalian UPR (Shen et al., 2001; Calfon et al., 2002). We find that in C. elegans, the ire-1 and xbp-1 pathway has retained its essential role in upregulating expression of many UPR target genes that are similarly upregulated by the homologous pathway in yeast. We also discovered a novel family of highly related genes that protect against ER stress when the ire-1 and xbp-1 signaling pathway is defective.
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Results |
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Genes active in degradation of malfolded ER proteins (ER-associated degradation [ERAD] genes) are not essential for yeast viability under normal growth conditions but are essential during ER stress or when the UPR is blocked. Furthermore, disruption of ERAD genes in yeast activates the UPR (Casagrande et al., 2000; Friedlander et al., 2000; Ng et al., 2000; Travers et al., 2000). We considered the possibility that abu genes might interact with the process of malfolded protein disposal from the C. elegans ER. First we confirmed that in C. elegans, like yeast, inactivation of ERAD genes causes ER stress. RNAi of sel-1, the C. elegans homologue of the yeast ERAD gene HRD3 (Hampton et al., 1996), resulted in marked upregulation of the ER stress indicator hsp-4::gfp (Fig. 5 B). The induction of hsp-4::gfp by sel-1(RNAi) and by RNAi of a homologue of another yeast ERAD gene, HRD1 (encoded in C. elegans by F55A11.3), were both xbp-1 dependent (unpublished data).
Inactivation of sel-1 by RNAi had no impact on the viability of wild-type animals and only modestly reduced the viability of xbp-1 mutants. However, when combined with inactivation of abu-1, sel-1(RNAi) increased lethality of xbp-1 mutant animals (Fig. 5 C). Combined inactivation of sel-1 and abu-1 also affected the appearance of xbp-1 mutant animals. Viewed under Nomarski optics, the intestine of wild-type, xbp-1 mutant, abu-1(RNAi), or sel-1(RNAi) young adults had a similar, fine, granular appearance. Animals with compound xbp-1;abu-1(RNAi) or xbp-1;sel-1(RNAi) genotypes had more dark staining large intestinal granules. The xbp-1;abu-1(RNAi);sel-1(RNAi) animal had a marked increase in such granules (Fig. 5 D). These observations suggest that abu-1 (and possibly other abu genes) and sel-1 perform partially redundant functions in animals with a blocked UPR.
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Discussion |
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Our genome-wide survey also identified 19 genes that were activated by ER stress in xbp-1 mutant animals but not in wild-type animals. Among these were nine genes predicted to encode a family of highly related ABU proteins. These abu genes are coregulated across different physiological and developmental perturbation; clustering together in a single, small, isolated and well-defined "expression mountain," mount 29, in the recently established gene expression map of C. elegans (Kim et al., 2001). GFP driven by the promoter of a representative family member, abu-1::gfp, was expressed strongly in the pharynx and head region of transgenic animals from larval stages L3L4 to young adult (Fig. 5 A). A similar expression pattern was also observed in animals transgenic for a transcriptional fusion of gfp to a different abu gene F19G12.7 (unpublished data). Furthermore, the basal expression pattern of the abu-1 transcriptional reporters correlated nicely with the in situ hybridization pattern of another abu family member, C03A7.7. Together, these observations suggest that the abu-1::gfp reporter reflects the activity of the endogenous gene. In older animals, we noted low levels of basal expression of abu-1::gfp in the intestine that was markedly upregulated by ER stress in xbp-1 mutant animals (Fig. 5 A). This last observation is consistent with the Northern blot analysis of abu-1 expression (Fig. 1 D). Although the significance of basal expression of abu genes in the pharynx is currently not understood, the intestinal expression correlates with the phenotype of animals in which abu-1 had been inactivated by RNAi. We do not know if abu-1 induction by ER stress in xbp-1 mutant animals is dependent on PERK (pek-1) or ATF6 (atf-6) signaling, as compound xbp-1;pek-1 and xbp-1;atf-6(RNAi) mutant animals arrest at early larval stages before we could reliably examine abu-1::gfp expression in the gut (Shen et al., 2001; unpublished data).
Inactivation of abu-1 by RNAi led to marked induction of the ER stress indicator hsp-4::gfp in the intestine (Fig. 5 B). This induction was completely blocked by the xbp-1 mutation (unpublished data), indicating the development of ER stress in the gut of abu-1(RNAi) animals and implying that abu-1 plays a role in an ER function, which, when inactivated, causes ER stress. This ER stress appeared to be well tolerated, as the abu-1(RNAi) animals developed at a normal rate and survived exposure to Cd2+, a toxin that induced further ER stress. However, in xbp-1 mutant animals, abu-1(RNAi) had serious consequences, significantly reducing their viability after exposure to additional ER stress (Fig. 4 B). The abu genes have a very high level of sequence identity at the nucleotide level, and the double-stranded RNA used to inactivate abu-1 could also initiate RNA inactivation of other family members. It is therefore possible that the strong phenotype observed in the abu-1(RNAi) animals reflects the simultaneous inactivation of other abu family members. Collectively, these results point to a role for abu-1, and possibly other abu genes, in some aspect of ER function that is also active under basal conditions. Reduced levels of this abu-dependent activity can be compensated by a normal UPR. However, when the UPR is compromised, loss of this abu-dependent activity becomes limiting, killing the affected animals.
In yeast, the ability to dispose of misfolded proteins in the secretory pathway (ERAD) plays an important role in adapting to high levels of ER stress, but is dispensable for viability under normal conditions (Casagrande et al., 2000; Friedlander et al., 2000; Ng et al., 2000; Travers et al., 2000). Given that abu-1(RNAi) is well tolerated under normal conditions, but not in animals experiencing high levels of ER stress, we considered the possibility that the abu genes may interact genetically with components of the C. elegans ERAD apparatus. An interaction was uncovered between sel-1, a C. elegans gene encoding an ER-localized component of the ERAD apparatus, and abu-1. Animals with diminished xbp-1, sel-1, and abu-1 activity had reduced viability and accumulated unusually large vesicles with dark content in their intestinal cells (Fig. 5 D). sel-1 and abu-1 are unlikely to function in the exact same pathway, as abu-1(RNAi) did not affect the phenotype of a hypomorphic allele of lin-12, whereas sel-1(RNAi) suppressed the egg-laying defect of lin-12(n676; n930)III (unpublished data). However, the observation that inactivating either gene leads to ER stress and the synthetic interactions between them suggest that sel-1 and abu-1 function in interacting cellular processes.
ABU-1 is a transmembrane protein that likely functions within the endomembrane system (Figs. 2 and 3). The lumenal domain of some ABU proteins resembles the extracellular domains of a mammalian scavenger receptor and C. elegans CED-1. Both the scavenger receptors and CED-1 are believed to serve as cell surface receptors for chemically modified macromolecules, oxidized lipoproteins, and other altered plasma proteins in the case of the scavenger receptors (Van Berkel et al., 2000) and corpses of apoptotic cells in the case of CED-1 (Zhou et al., 2001). Endocytosis and lysosomal degradation follow binding of these abnormal ligands to their receptors. Scavenger receptors and CED-1 are therefore believed to play a role in recycling the building blocks of damaged macromolecules that are present outside the cell. It is possible therefore that the ABU proteins may be playing a similar role within the endomembrane system, perhaps by binding to altered ER client proteins and modulating their intracellular fate.
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Materials and methods |
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Transgenic C. elegans
A 1.3-kb fragment of C. elegans genomic DNA immediately 5' of the predicted initiation ATG codon of abu-1 (AC3.3) was amplified by PCR using the oligonucleotides AC3.1S (5'-GGCATTGTGGCACGCATTGAACTG-3') and AC3Bam.2AS (5'-GATAGGATCCATTGTTAATATGCTTGAAGAGCTGC-3') and ligated in frame with the GFP coding region in the plasmid of pPD95.75 (gift of Andy Fire, Carnegie Institute of Washington, Baltimore, MD). The abu-1::gfp(zcEx8) strain was created by coinjecting the ac3.3.pPD95.75 plasmid (25 µg/ml) with a lin-15 rescuing plasmid, pSK1 (25 µg/ml), into lin-15(n765ts) strain. The extrachromosomal array was integrated into the chromosome with ultraviolet/trimethylpsoraren treatment, yielding the abu-1::gfp(zcIs8)X reporter strain.
To produce the ges-1::gfp(zcEx6) strain, the ges-1 promoter was amplified by PCR from C. elegans genomic DNA using the primers ges-1.1S (5'-CTCATACATCATTGTCAAGTGACG-3') and ges-1.Pst.2S (5'-AGTACTGCAGAGACAAGGAATATCCGCATCT-3') and ligated into the HindIII-PstI sites of pPD95.81 (gift of Andy Fire). The abu-1 coding region fragment encoding ABU-1 amino acids 7425 was ligated in frame with GFP into PstI-BamHI sites of this plasmid to produce the ges-1::abu-1::gfp(zxEx7) strain. The promoter and coding region of abu-1 was ligated in frame with GFP to produce abu1::abu-1::gfp(ZcEx8) strain. The animals containing transmissible extrachromosomal arrays were analyzed using a 510 laser scanning confocal microscope (ZEISS). Digital images were processed with Adobe Photoshop® 5.0.
RNAi feeding and activation of ER stress by pharmacological means
Interference with gene function by RNAi followed an established protocol (Timmons et al., 2001). In brief, double-stranded RNA was produced in HT115 strain of Escherichia coli transformed with pPD129 plasmids containing cDNA fragments of genes being studied (nucleotides 10321278 of abu-1 cDNA and nucleotides 4781631 of sel-1 cDNA). IPTG (1mM) was added to the bacterial growth media to induce transcription of the double-stranded RNA, and L4-staged animals of defined genotype were added to plates individually and produced their brood. RNAi phenotypes were evaluated in these progeny. Where indicated, 30 L4-stage progeny from the RNAi plate or from a control plate were transferred to a new plate seeded with fresh E. coli where they were exposed to tunicamycin (5 µg/ml) for 5 h or CdCl2 (10 mM) for the indicated period. Animals were scored as dead when pharyngeal pumping had ceased and they failed to move after being tapped on the nose with a platinum wire.
Cell culture, transfection, immunocytochemistry, and pulse-chase analysis
COS-1 cells were obtained from American Type Culture Collection. All cells were grown in DME containing 10% FBS (Cellgro®) at 5% CO2 and 37°C.
ABU-1 was tagged with a Flag epitope at its COOH terminus by overlapping PCR using the primers AC3.Bam.7S (5'-GCGCGGATCCGCAATGCGCTTTATCGCAATTGCAG-3'), AC3.Flag.16AS (5'-ATCGTCGTCCTTGTAGTCCTTTCTCTTGCAACACTG-3'), and Flag3'U.Not (5'-CTAG-CGGCCGCTCACTTGTCATCGTCGTCCTTGTAGTC-3'). The full-length coding region of abu-1 with the COOH-terminal Flag tag was cloned as a BamHI-NotI fragment into pcDNA3 (Invitrogen). COS-1 cells were transfected with this plasmid and stained with anti-Flag antibody (Sigma-Aldrich). Rabbit antiribophorin I antibody (a gift from Dr. Gert Kreibich, New York University [NYU] School of Medicine) was used to visualize the ER staining pattern.
The abu-1 coding region fragment encoding ABU-1 amino acids 22425 was amplified by PCR using primers AC3.EcoRI.5S (5'-GGCAACAAGAATTCGCGATAAACG-3') and AC3.XbaI.4AS (5'-GCGCTCTAGATGTCTACTTTCTCTTGCAAC-3'). The abu-1 coding region fragment coding ABU-1 amino acids 22331 was amplified by PCR using AC3.EcoRI.5S and AC3.XbaI.6AS (5'-GGCATCTAGAGCTTGGCATGCAGATTGGC-3'), deleting the transmembrane domain and COOH terminus of ABU-1. These fragments were cloned into pcDNA3 containing a Flag epitope with signal peptides. COS-1 cells were transfected with wild-type ABU-1 or ABU-1 lacking the transmembrane domain with a Flag tag. Newly synthesized proteins were labeled in vivo with 35S-methionine and 35S-cysteine (500 µCi/ml; ICN Biochemicals). After removal of the labeling media, cells were washed and incubated in complete media for cold chase for the indicated times. Cells were washed and lysed with 1% Triton buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 4 mg/ml aprotinin, and 2 mg/ml pepstatin A). Samples were clarified by centrifugation at 14,000 g for 10 min and by preincubation for 1 h with 10 µl protein ASepharose. Soluble proteins from the cells and secreted proteins in the media were immunoprecipitated by anti-Flag antibody bound to 10 µl protein ASepharose and washed three times with RIPA buffer. Bound proteins were resolved by 10% SDS-PAGE under reducing conditions and revealed by autoradiography.
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Footnotes |
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Acknowledgments |
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T. Yoneda was supported by a Postdoctoral Fellowship for Research Abroad for 2001 from the Japan Society for the Promotion of Science. This research was supported by National Institutes of Health grants ES08681 and DK47119. D. Ron is a Scholar of the Ellison Medical Foundation.
Note added in proof. It has recently been noted that ABU genes are predicted to contain glutamine- and asparagine-rich ("prion") domains (Wormbase web site, http://www.wormbase.org, release WS77, April 12, 2002). Such domains are predicted to form polar zipper proteinprotein interactions with similar domains on the same protein or on other proteins. This raises the interesting possibility that ABU proteins may recognize similar domains exposed on abnormally folded ER client proteins through such polar zipper interactions and thereby target them for destruction.
Submitted: 19 March 2002
Revised: 26 June 2002
Accepted: 26 June 2002
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