Selective activation of the transcription factor ATF6 mediates endoplasmic reticulum proliferation triggered by a membrane protein

Abstract

It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an Nterminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5)tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three-to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFPb(5)tail expression; peak ATF6α activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6α abolished the ER proliferation response, whereas knockdown of Ire1 was without effect. Because GFP-b(5)tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6.phospholipid biosynthesis | tail-anchored proteins | membrane biogenesis C ells are capable of adjusting the dimensions, architecture, and molecular composition of their organelles to changing functional needs. The endoplasmic reticulum (ER) offers a well-studied example of such organelle plasticity. When exposed to altered conditions, it initiates signaling cascades that result in the adjustment of its size and composition to the new situation. Among these signaling pathways, the one triggered by increased demand on the lumenal folding machinery, known as the unfolded protein response (UPR), plays a prominent role and has been extensively investigated (1).In yeast, the UPR is mediated by the ER transmembrane kinase/ endonuclease Ire1p, whereas in mammalian cells, the pathway has evolved to a higher degree of complexity, with the participation of two additional transmembrane sensors: the basic leucine zipper (bZIP) transcriptional regulator activating transcription factor 6α/ β (ATF6α/β) and the PKR-like ER kinase (PERK) (1). In the presence of unfolded proteins in the ER lumen, the three sensors induce changes in the activity of the transcriptional and translational apparatus that alleviate ER stress by (i) increasing the capacity of the lumenal folding machinery; (ii) enhancing ER-associated degradation; (iii) diminishing delivery of newly synthesized proteins to the ER lumen; and (iv) increasing phospholipid synthesis, with a resulting expans...

Pharmacological Chaperoning of Nicotinic Acetylcholine Receptors Reduces the Endoplasmic Reticulum Stress Response

“…Table 1 summarizes data obtained for the four manipulations [three exemplar nicotinic ligands and nAChRs containing ␤2 enhanced-ER-export mutant subunits]. All four manipulations suppressed ATF6 translocation, which serves as a key sensor for ER stress and the UPR (Ron and Walter, 2007;Hetz and Glimcher, 2009;Maiuolo et al, 2011), but the only other consistent phenomenon was the increased total Sec24D fluorescence in ERES. Therefore, we suggest that increased ER exit of nAChRs underlies the suppression of ATF6 translocation.…”

“…at ASPET Journals on May 12, 2018 molpharm.aspetjournals.org ever, the attenuation of ATF6 translocation is sufficient to suppress ER proliferation when expression of a tail-anchored protein is sensed within the lipid bilayer (Maiuolo et al, 2011). Events downstream from Golgi exit are not thought to influence the UPR markedly, and these events varied among the manipulations.…”

“…During the UPR, ATF6 is translocated from the ER to the Golgi and is cleaved by site 1 and site 2 proteases to release an N-terminal peptide. The N-terminal ATF6 peptide is translocated to the nucleus and initiates transcription of several target UPR genes that enhance ER function (Matsushita et al, 2002;Bommiasamy et al, 2009;Maiuolo et al, 2011).…”

Calcium-independent Phospholipase A2γ Enhances Activation of the ATF6 Transcription Factor during Endoplasmic Reticulum Stress

“…Alternatively, direct sensing of lipid perturbation may contribute to activation of ATF6. For example, perturbation of the lipid bilayer of the ER by the expression of a tail-anchored ER membrane protein resulted in the selective activation of ATF6, in the absence of IRE1␣ and PERK activation (and without ER Ca 2ϩ depletion) (42). Conversely, a recent study has demonstrated that saturation of membrane lipids promoted selective IRE1␣ and PERK activation by enhancing dimerization via their transmembrane domains (43).…”

Vascular Endothelial Cell Growth–Activated XBP1 Splicing in Endothelial Cells Is Crucial for Angiogenesis

“…21,22 To explore whether VEGF could also activate other ER stress responses, the PERK phosphorylation and ATF6 cleavage were detected. 23,24 Thapsigargin, a well-known ER stress inducer via ER calcium release, was included as a positive control triggering XBP1 splicing, PERK phopshorylation, and ATF6 cleavage in Hela cell ( Figure 1D). Interestingly, VEGF treatment only activated XBP1 splicing but had no effect on PERK phosphorylation or ATF6 cleavage ( Figure 1D).…”