吉田 秀郎

All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasinic reticulum (ER) by signaling an adaptive pathway termed the unfolded protein response (UPR). In yeast, a type-I ER transmembrane protein kinase, Ire1p, is the proximal sensor of unfolded proteins in the ER lumen that initiates an unconventional splicing reaction on HAC1 mRNA. Hac1p is a transcription factor required for induction of UPR genes. In higher eukaryotic cells, the UPR also induces site-2 protease (S2P)-mediated cleavage of ER-localized ATF6 to generate an N-terminal fragment that activates transcription of UPR genes. To elucidate the requirements for IRE1alpha and ATF6 for signaling the mammalian UPR, we identified a UPR reporter gene that was defective for induction in IRE1alpha-null mouse embryonic fibroblasts and S2P-deficient Chinese hamster ovary (CHO) cells. We show that the endoribonuclease activity of IRE1alpha is required to splice XBP1 (X-box binding protein) mRNA to generate a new C terminus, thereby converting it into a potent UPR transcriptional activator. IRE1alpha was not required for ATF6 cleavage, nuclear translocation, or transcriptional activation. However, ATF6 cleavage was required for IRE1alpha-dependent induction of UPR transcription. We propose that nuclear-localized IRE1alpha and cytoplasmic-localized ATF6 signaling pathways merge through regulation of XBP1 activity to induce downstream gene expression. Whereas ATF6 increases the amount of XBP1 mRNA, IRE1alpha removes all unconventional 26-nucleotide intron that increases XBP1 transactivation potential. Both processing of ATF6 and IRE1alpha-mediated splicing of XBP1 mRNA are required for full activation of the UPR.

In yeast, the transmembrane protein kinase/endoribonuclease Ire1p activated by endoplasmic reticulum stress cleaves HAC1 mRNA, leading to production of the transcription factor Hac1p that activates the unfolded protein response (UPR). In mammals, no Hac1p counterpart has yet been discovered despite the presence of Ire1p homologs in the endoplasmic reticulum. Instead, the transcription factor ATF6 specific to the mammalian UPR is regulated by intramembrane proteolysis. Here, we identified the transcription factor XBP1, a target of ATF6, as a mammalian substrate of such an unconventional mRNA splicing system and showed that only the spliced form of XBP1 can activate the UPR efficiently. Our results reveal features of the UPR conserved during evolution and clarify the relationship between IRE1- and ATF6-dependent pathways.

The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). We have used C. elegans as a genetic model system to dissect UPR signaling in a multicellular organism. C. elegans requires ire-l-mediated splicing of xbp-1 mRNA for UPR gene transcription and survival upon ER stress. In addition, ire-1/xbp-1 acts with pek-1, a protein kinase that mediates translation attenuation, in complementary pathways that are essential for worm development and survival. We propose that UPR transcriptional activation by ire-1 as well as translational attenuation by pek-1 maintain ER homeostasis. The results demonstrate that the UPR and ER homeostasis are essential for metazoan development.

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus: their basic regions seem to function as a nuclear localization signal, Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6 alpha acid the G13 gene product ATF6 beta.

Transcription of genes encoding molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) is induced by accumulation of unfolded proteins in the ER. This intracellular signaling, known as the unfolded protein response (UPR), is mediated by the cis-acting ER stress response element (ERSE) in mammals. In addition to ER chaperones, the mammalian transcription factor CHOP (also called GADD153) is induced by ER stress. We report here that the transcription factor XBP-1 (also called TREE5) is also induced by ER stress and that induction of CHOP and XBP-1 is mediated by ERSE. The ERSE consensus sequence is CCAAT-N-9-CCACG. As the general transcription factor NF-Y (also known as CBF) binds to CCAAT, CCACG is considered to provide specificity in the mammalian UPR. We recently found that the basic leucine zipper protein ATF6 isolated as a CCACG-binding protein is synthesized as a transmembrane protein in the ER, and ER stress-induced proteolysis produces a soluble form of ATF6 that translocates into the nucleus. We report here that overexpression of soluble ATF6 activates transcription of the CHOP and XBP-1 genes as well as of ER chaperone genes constitutively, whereas overexpression of a dominant negative mutant of ATF6 blocks the induction by ER stress. Furthermore, we demonstrated that soluble ATF6 binds directly to CCACG only when CCAAT exactly 9 bp upstream of CCACG is bound to NF-Y. Based on these and other findings, we concluded that specific and direct interactions between ATF6 and ERSE are critical for transcriptional induction not only of ER chaperones but also of CHOP and XBP-1.

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in PR-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), transcription of glucose-regulated proteins (GRPs) representing ER-resident molecular chaperones is markedly induced via the unfolded protein response (UPR) pathway. In contrast to recent progress in the analysis of yeast UPR, both cis-acting elements and transactivators responsible for mammalian UPR have remained obscure, Here, we analyzed the promoter regions of human GRP78, GRP94, and calreticulin genes and identified a novel element designated the ER stress response element (ERSE), ERSE, with a consensus of CCAATN(9)CCACG, was shown to be necessary and sufficient for induction of these GRPs, Using yeast one-hybrid screening, we isolated a human cDNA encoding a basic leucine zipper (bZIP) protein, ATF6, as a putative ERSE-binding protein. When overexpressed in HeLa cells, ATF6 enhanced transcription of GRP genes in an ERSE-dependent manner, whereas CREB-RP, another bZIP protein closely related ito ATF6, specifically inhibited GRP induction. Endogenous ATF6 constitutively expressed as a 90-kDa protein was converted to a 50-kDa protein in ER-stressed cells, which appeared to be important for the cellular response to ER stress. These results suggest that, as in yeast, bZIP proteins are involved in mammalian UPR, acting through newly defined ERSE.

The untranslatable, RNA polymerase II-dependent gene (dutA) of Dictyostelium discoideum is induced early in development. However, unlike other early genes, dutA induction was not affected by cAMP pulses and occurred normally in various cAMP-related mutant cells, the results indicating that this induction depended solely on factors other than cAMP. In the knockout strain of the catalytic subunit of protein kinase A, dutA expression was severely blocked and not recovered by cAMP pulses. This demonstrates that even the cAMP-independent gene, dutA, requires protein kinase A for its expression.

Background: Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the transcriptional induction of molecular chaperones and folding enzymes localized in the ER. Thus, eukaryotic cells possess an intracellular signalling pathway from the ER to the nucleus, called the unfolded protein-response (UPR) pathway. In Saccharomyces cerevisiae, such induction is mediated by the cis-acting unfolded protein-response element (UPRE) which has been thought to be recognized by one or more transcription factor(s). Results: Extensive mutational analysis revealed that UPRE contains a partial palindrome with a spacer of one nucleotide (CAGCGTG) that is essential for its function. We then cloned the ERN4 (presumably identical with HAC1) gene using yeast one-hybrid screening, in which the GAL4-ERN4 fusion gene constitutively activates the UPR pathway. The ERN4 gene encodes a basic-leucine zipper protein (Ern4p) that specifically binds to UPRE in vitro and activates transcription in vivo. Cells lacking Ern4p are unable to induce transcription of any of the five target genes tested and exhibit sensitivity to ER stress and inositol requirement for growth. Conclusion: We concluded that Ern4p represents a major component of the putative transcription factor (UPRF) responsible for the UPR leading to the induction of ER-localized stress proteins. © Blackwell Science Limited.

We report the isolation and characterization of a gene (designated mcp60) encoding the mitochondrial (mt) 60-kDa heat-shock protein (HSP60) in the fission yeast Schizosaccharomyces pombe. The deduced amino-acid sequence (582 aa) of this gene is highly similar to the known mt HSP60 from diverse organisms. When its sequence was related to the known functional domains of bacterial HSP60 (GroEL), the similarity was particularly high for the intermediate domains that connect the apical domain with the equatorial domain. The mRNA level of mcp60 increased several-fold upon temperature upshift (from 25 to 35 degrees C), while gradually decreased during sporulation. Gene disruption experiments revealed that mcp60 is essential for cell viability at all temperatures.

In Dictyostelium discoideum, a novel type of RNA (dutA RNA), which is untranslatable, cytosolic and 1.3 kb in size, appears specifically after the aggregation stage [Yoshida, H. el. al. (1994) Nucleic Acids Res. 22, 41-46]. We here show that the dutA gene is transcribed by RNA polymerase II, based on its alpha-amanitin sensitive nature of in vitro transcriptional activity. We also show that the stage-specific accumulation of dutA RNA is primarily due to the stage-specific enhancement of the transcriptional activity of the gene. (C) 1995 Academic Press, Inc.

dutA is a gene specifically expressed during the development of Dictyostelium discoideum. Toward understanding its possible role in development, we isolated and characterized the gene and its complete cDNA. We found that dutA is encoded by the nuclear genome as a single copy gene without introns. In addition, the following unique and interesting features of dutA RNA (1322 nt) emerged: (1) it has no sustained ORFs(MAX = 126 nt) (2) it is extremely AU-rich (83%) (3) it contains peculiar sequence motifs (large palindromes, long AU-stretches and CC-clusters) (4) it is localized in the cytoplasm but completely absent from ribosomes. These features suggest that dutA RNA functions without being translated into protein. Disruption of the dutA gene did not cause phenotypic changes, suggesting that the function of dutA is redundant.

We have isolated a gene, DC6, which is induced in the early aggregative stages of development in Dictyostelium discoideum. The increase in DC6 expression is dependent on high cell density, indicating that cellular interactions are required for DC6 induction. In low-cell-density cultures, the induction of DC6 occurs is supplied with conditioned medium of developing cells, suggesting that secreted factors are involved in DC6 induction. The expression of DC6 is not affected (1) in the presence of caffeine or adenosine, which block the production or the action of cAMP pulses, (2) in the presence of high concentrations of cAMP, or (3) in mutant strains (Synag7 and FrigidA), which are defective in transduction pathways of cAMP pulse signals. These results indicate that the induction of DC6 does not require extracellular cAMP pulse signals, which are known to regulate the expression of many genes in the early development. Independence of cAMP signals and dependence on other unknown cellular interactions are prominent characteristics of DC6.