Eukaryotic cells possess multiple intracellular signaling pathways from the ER to the nucleus, each modulating gene expression in response to changes in or surrounding the ER (reviewed by Pahl and Baeuerle, 1997
). One of these, the UPR, deals with homeostasis of the folding capacity in the ER. Without this system, both yeast cells (Cox et al., 1993
; Mori et al., 1993
; Nikawa et al., 1996
) and mammalian cells (Li and Lee, 1991
; Little and Lee, 1995
; Morris et al., 1997
; Liu et al., 1998
) are unable to survive under ER stress conditions that cause continuous accumulation of unfolded proteins in the ER.
In this study, we analyzed the mechanism of activation of ATF6 by ER stress, which we recently isolated as a candidate for a mammalian UPR-specific transcription factor. We showed that constitutively expressed 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) specifically in ER-stressed cells before the induction of GRP78, a major target protein of the mammalian UPR (Figures –). The results presented here revealed the most important consequence of this conversion, that is, alteration of subcellular localization (see our current model depicted in Figure ). Fractionation (Figure ), differential solubilization (Figure A), topological studies (Figure B), glycosylation analysis (Figure C), and immunoflurescence analysis (Figures A and D) clearly demonstrated that p90ATF6 is a type II transmembrane glycoprotein embedded in the ER membrane. In contrast, p50ATF6 is a soluble nuclear protein (Figures and ). When overexpressed, ATF6 mutants representing p50ATF6 were accumulated in the nucleus (Figure ) and constitutively activated transcription of the GRP78 gene, leading to enhanced levels of GRP78 in the ER (Figure ). We thus propose that p50ATF6 represents an active and mature form of the mammalian transcription factor ATF6, and its production is mediated by ER stress-induced proteolysis of p90ATF6, which is synthesized as a precursor protein embedded in the ER membrane. Upon ER stress, p50ATF6 is released from the ER membrane, allowing it to enter the nucleus. In the nucleus, p50ATF6 containing a bZIP domain activates transcription of ER chaperone genes such as GRP78 through ERSE in collaboration with the general transcription factor NF-Y; we recently found that ATF6 recognizes ERSE and directly interacts with NF-Y (our unpublished observation).
Figure 10 Model for ER stress-induced processing of ATF6. ATF6 is constitutively synthesized as a precursor protein (p90ATF6) that anchors in the ER membrane through the single transmembrane domain near the center of the molecule. ER stress-induced proteolysis (more ...)
Interestingly, p90ATF6 appears to be turned over fairly quickly; its half-life within the cell is ~2 h (Figure ). This rapid turnover rate allowed the cells to restore p90ATF6 at 16 h after thapsigargin treatment (Figure A). Similarly, p90ATF6 was restored at 16 h after tunicamycin treatment, although it was unglycosylated (Figure ). Therefore, p90ATF6 itself might serve as a sensor molecule of ER stress; under the conditions that cause accumulation of unfolded proteins in the ER, p90ATF6 would not be able to fold properly, and this may somehow activate proteolytic processing of p90ATF6, resulting in production of p50ATF6. In this connection, it is noteworthy that overexpression of full-length ATF6, ATF6 (670), constitutively activated transcription of the GRP78 gene, albeit only slightly (Figure ). We reasoned that overproduction of ATF6 (670), a transmembrane protein in the ER, is sensed as ER stress by the cell probably because normal levels of ER chaperones are insufficient for proper folding of exogenous proteins expressed at high levels. As a result, portions of endogenous p90ATF6 and exogenous ATF6 (670) are subjected to proteolytic processing constitutively, resulting in enhanced transcription of ER chaperone genes by constitutively produced p50ATF6 through ERSE in the nucleus. Indeed, p50ATF6-like doublet protein bands were detected in extracts of cells transfected with pCGN-ATF6 (670) (Figure B, lane 2). This also explains why various promoters of ER chaperone genes were constitutively activated when full-length ATF6 was overexpressed in HeLa cells (Yoshida et al., 1998
We showed here that the mammalian UPR uses a system very similar to that previously identified for cholesterol homeostasis, another well-investigated intracellular signaling pathway from the ER to the nucleus (Yokoyama et al., 1993
; Wang et al., 1994
; Sakai et al., 1996
, reviewed by Brown and Goldstein, 1997
). Cholesterol metabolism is primarily regulated at the level of transcription, and sterol regulatory element binding proteins (SREBPs) mediate transcriptional activation of genes involved in cholesterol biosynthesis as well as receptor-mediated endocytosis of cholesterol-containing lipoproteins from plasma. Under normal conditions, SREBPs are bound to membranes of the ER and nuclear envelope through two hydrophobic stretches separated by a spacer of 31 amino acids and located around the center of the molecule. Depletion of sterols from culture medium activates proteolytic cleavage of SREBPs at the two sites in a sequential manner, allowing entrance of the N-terminal fragment into the nucleus. This released N-terminal fragment contains all of the functional domains necessary for active transcription factors such as DNA-binding (basic helix–loop–helix), dimerization (leucine zipper), and transactivation (acid blob) domains and thus enhances transcription of target genes in the nucleus.
In contrast to SREBPs, the precise cleavage site of ATF6 is as yet unknown. The region from amino acid 378 to 398 functions as a transmembrane domain because it is the only hydrophobic segment found in ATF6 (Figure B). The calculated molecular weight of the N-terminal region (1–377) is 41,161 and is thus significantly smaller than the size of p50ATF6 estimated from the mobility on SDS-PAGE. However, yeast Hac1p behaves as a 41-kDa protein on SDS-PAGE despite its calculated molecular weight of 26,903, presumably because of high contents of basic amino acids (Kawahara et al., 1997
). Similarly, full-length ATF6 behaves as a 90-kDa protein on SDS-PAGE despite its calculated molecular weight of 74,597 (Zhu et al., 1997
; Yoshida et al., 1998
). Comparison of the mobilities of various C-terminal deletion mutants with that of p50ATF6 on SDS-PAGE (Figure B) suggested that ATF6 is cleaved between the bZIP and transmembrane domains, although we cannot exclude the possibility that ATF6 is cleaved within the transmembrane domain, similarly to cleavage at site 2 in SREBP2 (Sakai et al., 1996
), or cleaved around the boundary between the cytoplasmic and transmembrane domains, similarly to Notch-1 (Chan and Jan, 1998
; Schroeter et al., 1998
). We are currently carrying out mutational analysis to determine the precise cleavage site. Such information will be useful for identification of the protease(s) responsible for proteolysis of ATF6.
We have identified a key regulatory step that connects events in the ER with those in the nucleus in mammalian UPR, and our observations have raised an important question: i.e., whether ER stress-induced proteolysis of ATF6 is regulated by mammalian Ire1p, a putative sensor molecule of ER stress identified recently (Tirasophon et al., 1998
; Wang et al., 1998
). The detection of endonuclease activity in the C-terminal tail region of human Ire1p suggests the presence of a mammalian mRNA splicing system similar to that for yeast HAC1
mRNA (Tirasophon et al., 1998
), but ATF6 mRNA is not spliced (Yoshida et al., 1998
). This raises the question of whether there are any substrates of such a mammalian splicing system, and if so, what is the consequence of such mRNA splicing. In yeast, transcriptional induction of ER chaperones is coupled to phospholipid biosynthesis; yeast cells lacking either Ire1p or Hac1p require exogenous inositol for growth (Nikawa and Yamashita, 1992
; Cox et al., 1993
; Mori et al., 1993
; Nikawa et al., 1996
; Sidrauski et al., 1996
). It has been proposed that yeast UPR coordinates the synthesis of ER chaperones and ER membranes via the Ire1p–Hac1p pathway (Cox et al., 1997
). This provides a basis for one intriguing speculation that in mammalian cells ER membrane biosynthesis may be controlled differently from transcriptional regulation of ER chaperone genes. Thus, the mammalian mRNA splicing system may be specialized to adjust the production of phospholipids according to the requirements within the ER. On the other hand, mammalian Ire1p may regulate the synthesis of ER chaperones by phosphorylating a putative protease(s), which activates ATF6. Answers to these important questions will further extend our understanding of the molecular mechanism of the mammalian UPR.