(2005

ABSTRACT. When increased production of secretory proteins overwhelms the capacity of the endoplasmic reticulum (ER) and the Golgi apparatus, eukaryotic cells expand their capacity to sustain secretory function. The capacity of the ER is enhanced by the mechanism called the ER stress response, but the mechanism regulating Golgi capacity (the Golgi stress response) has remained unclear. Here, we found that transcription of Golgirelated genes, including glycosylation enzymes as well as factors involved in post-Golgi vesicular transport and maintenance of Golgi structure, was upregulated upon treatment with monensin, an ionophore that disrupts the function of acidic organelles, including the Golgi apparatus and lysosomes by neutralizing their lumen. This transcriptional induction was found to be commonly regulated by a novel cis-acting element called the Golgi apparatus stress response element (GASE), whose consensus sequence is ACGTGgc. When the function of the Golgi apparatus was specifically disturbed by overexpression of GCP60, a Golgi-localized protein that binds to giantin, transcription from GASE was significantly induced. These results suggest that mammalian cells have the Golgi stress response, and that GASE regulates transcriptional induction involved in the Golgi stress response.

ABSTRACT. Cells from yeast to humans activate unconventional mRNA splicing when unfolded proteins accumulate in the endoplasmic reticulum (ER) under ER stress conditions. The substrate of this splicing in mammalian cells is XBP1 mRNA, which encodes the unfolded protein response (UPR)-specific transcription factor XBP1. The C-terminal region of XBP1 is switched as a result of the splicing. Thus, unspliced and spliced mRNAs produce pXBP1(U) of 261 aa and pXBP1(S) of 376 aa, respectively, with the N-terminal region containing the DNA-binding domain shared. As the pXBP1(S)-specific C-terminal region functions as an activation domain, pXBP1(S) can activate transcription efficiently. We recently found that pXBP1(U) shuttles between the nucleus and cytoplasm, owing to the presence of a nuclear exclusion signal in the pXBP1(U)-specific C-terminal region, in marked contrast to the exclusively nuclear localization of pXBP1(S). pXBP1(U) can associate with pXBP1(S), and pXBP1(U)-pXBP1(S) complex is rapidly degraded by the proteasome. Two other transcription factors are activated in response to ER stress, namely ATF6 and ATF4. ATF6 is a UPR-specific transcription factor, whereas ATF4 is activated by not only ER stress but also various other stimuli. In this study, we show that pXBP1(U) targets the active form of ATF6 but not ATF4 for destruction by the proteasome via direct association. This enhanced degradation is mediated by the degradation domain located at the pXBP1(U)-specific C-terminal end. We conclude that pXBP1(U) functions as a negative regulator of the UPR-specific transcription factors ATF6 and pXBP1(S).

ABSTRACT. The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism against the accumulation of unfolded proteins in the ER (ER stress) that consists of three response pathways (the ATF6, IRE1 and PERK pathways) in mammals. These pathways regulate the transcription of ER-related genes through specific cis-acting elements, ERSE, UPRE and AARE, respectively. Because the mammalian ER stress response is markedly activated in professional secretory cells, its main function was thought to be to upregulate the capacity of protein folding in the ER in accordance with the increased synthesis of secretory proteins. Here, we found that ultraviolet A (UVA) irradiation induced the conversion of an ER-localized sensor pATF6α(P) to an active transcription factor pATF6α(N) in normal human dermal fibroblasts (NHDFs). UVA also induced IRE1-mediated splicing of XBP1 mRNA as well as PERK-mediated phosphorylation of an α subunit of eukaryotic initiation factor 2. Consistent with these observations, we found that UVA increased transcription from ERSE, UPRE and AARE elements. From these results, we concluded that UVA irradiation activates all branches of the mammalian ER stress response in NHDFs. This suggests that the mammalian ER stress response is activated by not only intrinsic stress but also environmental stress.

ABSTRACT. XBP1 is a key transcription factor regulating the mammalian endoplasmic reticulum (ER) stress response, which is a cytoprotective mechanism for dealing with an accumulation of unfolded proteins in the ER (ER stress). The expression of XBP1 is regulated by two different mechanisms: mRNA splicing and protein stability. When ER stress occurs, unspliced XBP1 mRNA is converted to mature mRNA, from which an active transcription factor, pXBP1(S), is translated and activates the transcription of ER-related genes to dispose of unfolded proteins. In the absence of ER stress, pXBP1(U) is translated from unspliced XBP1 mRNA and enhances the degradation of pXBP1(S). Here, we analyzed the regulatory mechanism of pXBP1(S) stability, and found that a SUMO-conjugase, UBC9, specifically bound to the leucine zipper motif of pXBP1(S) and increased the stability of pXBP1(S). Suppression of UBC9 expression by RNA interference reduced both the expression of pXBP1(S) and ER stress-induced transcription by pXBP1(S). Interestingly, overexpression of a UBC9 mutant deficient in SUMO-conjugating activity was able to increase pXBP1(S) expression as well as wild-type UBC9, indicating that UBC9 stabilizes pXBP1(S) without conjugating SUMO moieties. From these observations, we concluded that UBC9 is a novel regulator of the mammalian ER stress response.