All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic 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 IRE1␣ and ATF6 for signaling the mammalian UPR, we identified a UPR reporter gene that was defective for induction in IRE1␣-null mouse embryonic fibroblasts and S2P-deficient Chinese hamster ovary (CHO) cells. We show that the endoribonuclease activity of IRE1␣ 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. IRE1␣ was not required for ATF6 cleavage, nuclear translocation, or transcriptional activation. However, ATF6 cleavage was required for IRE1␣-dependent induction of UPR transcription. We propose that nuclear-localized IRE1␣ 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, IRE1␣ removes an unconventional 26-nucleotide intron that increases XBP1 transactivation potential. Both processing of ATF6 and IRE1␣-mediated splicing of XBP1 mRNA are required for full activation of the UPR.

Eukaryotes respond to the presence of unfolded protein in the endoplasmic reticulum (ER) by up-regulating the transcription of genes encoding ER protein chaperones, such as BiP. We have isolated a novel human cDNA encoding a homolog to Saccharomyces cerevisiae Ire1p, a proximal sensor for this signal transduction pathway in yeast. The gene product hIre1p is a type 1 transmembrane protein containing a cytoplasmic domain that is highly conserved to the yeast counterpart having a Ser/Thr protein kinase domain and a domain homologous to RNase L. However, the luminal domain has extensively diverged from the yeast gene product. hIre1p expressed in mammalian cells displayed intrinsic autophosphorylation activity and an endoribonuclease activity that cleaved the 5 splice site of yeast HAC1 mRNA, a substrate for the endoribonuclease activity of yeast Ire1p. Overexpressed hIre1p was localized to the ER with particular concentration around the nuclear envelope and some colocalization with the nuclear pore complex. Expression of Ire1p mRNA was autoregulated through a process that required a functional hIre1p kinase activity. Finally, overexpression of wild-type hIre1p constitutively activated a reporter gene under transcriptional control of the rat BiP promoter, whereas expression of a catalytically inactive hIre1p acted in a trans-dominant-negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress induced by inhibition of N-linked glycosylation. These results demonstrate that hIre1p is an essential proximal sensor of the unfolded protein response pathway in mammalian cells.

The unfolded protein response (UPR) is a signal transduction pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER). In Saccharomyces cerevisiae the ER transmembrane receptor, Ire1p, transmits the signal to the nucleus culminating in the transcriptional activation of genes encoding an adaptive response. Yeast Ire1p requires both protein kinase and site-specific endoribonuclease (RNase) activities to signal the UPR. In mammalian cells, two homologs, Ire1␣ and Ire1␤, are implicated in signaling the UPR. To elucidate the RNase requirement for mammalian Ire1 function, we have identified five amino acid residues within IRE1␣ that are essential for RNase activity but not kinase activity. These mutants were used to demonstrate that the RNase activity is required for UPR activation by IRE1␣ and IRE1␤. In addition, the data support that IRE1 RNase is activated by dimerization-induced trans-autophosphorylation and requires a homodimer of catalytically functional RNase domains. Finally, the RNase activity of wild-type IRE1␣ down-regulates hIre1␣ mRNA expression by a novel mechanism involving cis-mediated IRE1␣-dependent cleavage at three specific sites within the 5 end of Ire1␣ mRNA.

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. Escherichia coli and related bacteria regulate the activity of glutamine synthetase (GS) in at least three ways (reviewed in references 24, 33, 36, and 42-44). GS is subjected to concerted feedback inhibition by several metabolic intermediates (27). Also, the activity of GS is regulated by reversible adenylylation, catalyzed by adenylyltransferase (ATase), with the adenylylated form of GS being less active. Finally, the biosynthesis of GS is regulated by the control of the initiation of transcription of its structural gene, glnA. These regulatory mechanisms enable the cell to rapidly adjust GS activity in response to changes in the availability of the preferred nitrogen source, ammonia. When nitrogen is in excess, cells contain little GS and the enzyme is mostly adenylylated; under conditions of nitrogen limitation, the intracellular concentration of GS is 7-to 10-fold higher and the enzyme is mostly unadenylylated. Under the latter conditions, the reaction catalyzed by GS constitutes the major route for the assimilation of ammonia.DNitrogen regulation of the transcription of glnA and certain other genes results from the action of the two-component regulatory system consisting of the bifunctional histidine protein kinase/phosphoprotein phosphatase nitrogen regulator II (NRII or NtrB, the product of glnL or ntrB) and the response regulator nitrogen regulator I (NRI or NtrC, the product of glnG or ntrC) (reviewed in references 24, 33, and 36). NRII controls the intracellular concentration of the phosphorylated form of NRI, NRIϳP; NRIϳP is an enhancer-binding transcriptional activator of RNA polymerase containing the minor sigma factor 54 . This activator stimulates transcription of glnA and certain other nitrogen-regulated genes and also acts to repress gene transcription (40,41).The regulation of ATase and the nitrogen regulation of gene transcription by NRI and NRII utilize a common sensory and signal transduction system consisting of the uridylyltransferase/ uridylyl-removing enzyme (UTase/UR, the product of glnD) and the PII protein (the product of glnB) (Fig. 1) (1, 3, 7-10,...

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