IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.

Proteins synthesized in the endoplasmic reticulum (ER) of eukaryotic cells must be folded correctly before translocation out of the ER. Disruption of protein folding results in the induction of genes for ER-resident chaperones, for example, BiP. This phenomenon is known as the ER stress response. We report here that bZIP60, an Arabidopsis thaliana basic leucine zipper (bZIP) transcription factor with a transmembrane domain, is involved in the ER stress response. When compared with wildtype Arabidopsis plants, homozygous bzip60 mutant plants show a markedly weaker induction of many ER stressresponsive genes. The bZIP60 protein resides in the ER membrane under unstressed condition and is cleaved in response to ER stress caused by either tunicamycin or DTT. The N-terminal fragment containing the bZIP domain is then translocated into the nucleus. Cleavage of bZIP60 is independent of the function of Arabidopsis homologs of mammalian S1P and S2P proteases, which mediate the proteolytic cleavage of the mammalian transcription factor ATF6. In Arabidopsis, expression of the bZIP60 gene and cleavage of the bZIP60 protein are observed in anthers in the absence of stress treatment, suggesting that the ER stress response functions in the normal development of active secretory cells.

Analysis of transcripts of 75 genes encoding putative basic leucine zipper (bZIP) transcription factors in the Arabidopsis genome identified AtbZIP60, which was induced by tunicamycin. AtbZIP60 encodes a predicted protein of 295 aa with a putative transmembrane domain near its C terminus after a bZIP domain. A truncated form of AtbZIP60 without a transmembrane domain (AtbZIP60⌬C) fused with GFP localized to the nucleus, suggesting translocation of native protein to the nucleus by release from the membrane. AtbZIP60 was also induced by DTT and azetidine-2-carboxylate, which induce the endoplasmic reticulum (ER) stress response (also called the unfolded protein response). Expression of AtbZIP60⌬C clearly activated any of three BiP and two calnexin promoters in a dual luciferase assay using protoplasts of cultured cells. The induction was considered to be through cis-elements plant-specific unfolded protein response element and ER stress-response element. Interestingly, AtbZIP60⌬C also appeared to induce the expression of AtbZIP60 through an ER stress-response element-like sequence in the promoter of AtbZIP60. These characteristics of AtbZIP60 imply a signal transduction pathway of the ER stress response unique to plants.Arabidopsis thaliana ͉ BiP ͉ tunicamycin ͉ unfolded protein response T he endoplasmic reticulum (ER) consists of a three-dimensional structure in eukaryotic cells where proteins for the secretary pathway are synthesized. Proper folding and assembly of proteins synthesized in the ER are necessary for transport to their final destinations. When folding or assembly of proteins in the ER is disordered, unfolded proteins accumulate in the ER and expression of genes for ER-resident chaperones, such as BiP, and folding enzymes are induced. This phenomenon is conserved among eukaryotic cells and is referred to as the ER stress response or the unfolded protein response (UPR) (1-4). Recent studies conducted in yeast and mammalian cells have shown that the ER stress response plays essential roles not only under specific stresses but also under normal growth conditions (5-8). In plants, the ER stress response has been implicated in plant-specific processes, such as seed development and pathogen response (9).The mechanism of signal transduction for the ER stress response has been extensively characterized in yeast and mammalian cells. In yeast cells, IRE1, an ER membrane-located protein kinase͞ribonuclease, plays a pivotal role for the perception of ER stress (10, 11). Sensing ER stress, IRE1 dimerizes and transautophosphorylates, activating its ribonuclease activity (12, 13). Activated IRE1 catalyzes the spliceosome-independent splicing of Hac1 mRNA, encoding a basic leucine zipper (bZIP) transcription factor. Hac1 protein is efficiently synthesized from spliced Hac1 mRNA and binds to a cis-element, UPR element (UPRE; consensus sequence CAGCGTG), resulting in induction of downstream chaperone genes, such as BiP (14-16).The ER stress response pathways of mammalian cells are multiple, in contrast to that of yeas...

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