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 cisacting unfolded protein-response element (UPRE) which has been thought to be recognized by one or more transcription factor(s).

An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions ("ER stress"). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR in Saccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing of HAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin-proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little or no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stressinduced mRNA splicing, leading to activation of the UPR.

We have identi¢ed cDNA encoding a new member of the adrenomedullin (AM) family, AM2, for the ¢rst time in mammals (mouse, rat and human). The predicted precursor carried mature AM2 in the C-terminus, which had an intramolecular ring formed by an S^S bond and a possibly amidated C-terminus. Phylogenetic analyses clustered AM2 and AM into two distinct but closely related groups. Similarity of exon^intron structure and synteny of neighboring genes showed that mammalian AM2 is an ortholog of pu¡er¢sh AM2 and a paralog of mammalian AM. AM2 mRNA was expressed in submaxillary gland, kidney, stomach, ovary, lymphoid tissues and pancreas of mice, but not in adrenal and testis. Intravenous injection of synthetic mature AM2 decreased arterial pressure more potently than AM, and induced antidiuresis and antinatriuresis in mice. These results show that at least two peptides, AM and AM2, comprise an adrenomedullin family in mammals, and that AM2 may play pivotal roles in cardiovascular and body £uid regulation.

When unfolded proteins are accumulated in the endoplasmic reticulum (ER), an intracellular signaling pathway termed the unfolded protein response (UPR) is activated to induce transcription of ER-localized molecular chaperones and folding enzymes in the nucleus. In Saccharomyces cerevisiae, at least six lumenal proteins including essential Kar2p and Pdi1p are known to be regulated by the UPR. We and others recently demonstrated that the basic-leucine zipper protein Hac1p/ Ern4p functions as a trans-acting factor responsible for the UPR. Hac1p binds directly to the cis-acting unfolded protein response element (UPRE) responsible for Kar2p induction. Moreover, we showed that the KAR2 UPRE contains an E box-like palindrome separated by one nucleotide (CAGCGTG) that is essential for its function. We report here that the promoter regions of each of five target proteins (Kar2p, Pdi1p, Eug1p, Fkb2p, and Lhs1p) contain a single UPRE sequence that is necessary and sufficient for induction and that binds specifically to Hac1p in vitro. All of the five functional UPRE sequences identified contain a palindromic sequence that has, in four cases, a spacer of one C nucleotide. This unique characteristic of UPRE explains why only a specific set of proteins are induced in the UPR to cope with ER stress.

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) according to the needs within the ER. In Saccharomyces cerevisiae, expression of the UPR-specific transcription factor Hac1p is tightly regulated at the level of mRNA splicing that depends on an unconventional system. Thus, HAC1 precursor mRNA is constitutively expressed but not translated. A sensor molecule Ire1p͞Ern1p-mediated signaling from the ER specifically removes an intron of 252 nucleotides from the precursor mRNA, and the resulting mature mRNA is translated to produce Hac1p. Because the 5 splice site is located near the C-terminal end of the Hac1p-coding region, this splicing replaces the last 10 codons of the ORF with an exon encoding 18 aa without affecting the N-terminal 220-aa region which contains the DNA-binding domain. Here, we found that this C-terminal 18-aa segment functions as a potent activation domain. Therefore, the splicing event joins the HAC1 DNA-binding domain to its activation domain, allowing rapid posttranscriptional generation of a potent transcriptional activator (238-aa Hac1p) that activates the UPR efficiently. This suggests that the UPR is hardly activated by Hac1p produced without splicing (230-aa Hac1p) which may occur in the absence of Ire1p͞Ern1p-mediated signaling from the ER. Based on these and other results, we propose that the control of expression and activity of Hac1p meets the requirements of the ER. T he levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) are adjusted by an intracellular signaling pathway from the ER to the nucleus termed the unfolded protein response (UPR). Thus, when unfolded proteins are accumulated in the ER, the UPR is activated to induce transcription of these luminal protein genes in the nucleus, primarily to increase the folding capacity and thus maintain homeostasis of the ER (1-3). Extensive studies of the molecular mechanism of the UPR in Saccharomyces cerevisiae led to the identification of three genes as essential components of the UPR: the IRE1͞ERN1 gene encoding a transmembrane protein kinase Ire1p believed to act as a sensor in the ER (4, 5), the HAC1͞ERN4 gene encoding a basic leucine zipper-type transcription factor Hac1p (6-8) which directly recognizes the cis-acting UPR element (UPRE) present in the promoter regions of target genes (9-11), and the RLG1 gene encoding tRNA ligase Rlg1p (12). In addition, the PTC2 gene was identified; Ptc2p is a protein serine͞threonine phosphatase that appears to regulate Ire1p negatively (13). In S. cerevisiae, the UPR is coupled to inositol biosynthesis; cells lacking Ire1p or Hac1p are not only defective in the UPR but also require inositol for growth (4-8, 12, 14). It was suggested that production of ER proteins and ER membrane is coordinately regulated by the UPR (15).The UPR signal transduction system is distinguished by the use of an unconventional mRNA splicing sys...