ER stress signaling by regulated proteolysis of ATF6

Abstract

ATF6 is an endoplasmic reticulum (ER) membrane-anchored transcription factor activated by intramembrane proteolysis in the ER stress response. Upon ER stress, ATF6 is transported from the ER to the Golgi to be processed by site-1 and site-2 proteases. The trafficking is controlled by the ER chaperone BiP/GRP78. Here, we describe the experimental methods that we have used to study of ATF6 regulation in tissue culture cells. These methods were used to investigate several key steps of ATF6 activation in the ER stress response including binding and dissociation of BiP to ATF6, translocation from the ER to the Golgi and cleavage in the Golgi. In addition, luciferase reporter assays were a sensitive way to monitor ER stress and ATF6 activation. These methods were not only useful for the study ATF6 and the ER stress response, they might also help to elucidate the roles of the ER stress response in a number of human diseases involving misfolded proteins and in the differentiation of secretory tissues which require higher ER folding capacities.

Introduction

The homeostasis of the endoplasmic reticulum (ER) is tightly regulated and accumulation of misfolded proteins in the ER lumen induces a coordinated cellular response known as the ER stress response or unfolded protein response (UPR). The ER stress response is an auto-regulatory program that up-regulates a large number of genes that expand the folding capacity of the ER, such as ER chaperones and membrane trafficking factors [1], [2], [3], [4]. In mammals, the bZIP transcription factor ATF6 serves as a critical proximal signal transducer to transmit the ER stress signal across the ER membrane into the nucleus.

One aspect of the ER stress response is the induction of new gene expression. Mapping of the promoters of a number of ER stress responsive genes, such as the ER chaperones BiP/GRP78 and GRP94, identified an ER stress response element (ERSE) [5], [6]. ATF6 was first identified in a yeast one-hybrid screen for factors bound to the ER stress response element (ERSE). The ERSE is a bipartite sequence with the consensus CCAAT-N₉-CCACG. ATF6 was found to bind this sequence with NF-Y, a CCAAT box binding factor [7]. However, we found by oligonucleotide selection that ATF6 can also bind alone to the sequence TGACGTGG, similar to the opposite strand of the 3′ half of the ERSE [8]. An alternative ERSE sequence, ATTGG-N-CCACG, has also been identified in the human Herp promoter, suggesting that some alternative configurations of NF-Y and ATF6 binding are allowed [9].

An unusual property of the transcription factor ATF6 is that it exists as an ER membrane-tethered precursor regulated by intramembrane proteolysis [6], [10]. ATF6 is a type II transmembrane protein with its C-terminus located in the ER lumen and its N-terminal DNA binding domain facing the cytosol (Fig. 1). In response to ER stress, ATF6 translocates from the ER to the Golgi and within the Golgi it is processed to its active form through sequential cleavage by site-1 and site-2 proteases (S1P and S2P) [11], [12]. S1P and S2P are also involved in the proteolytic processing of sterol response element binding proteins (SREBPs), transcription factors that control cellular sterol levels [13]. SREBP is similarly regulated by translocation from the ER to the Golgi and sequential cleavage by S1P and S2P. The key difference is that SREBP translocation is triggered by low cellular sterol levels while ATF6 is induced by high levels of misfolded proteins in the ER. The ER stress-induced trafficking of ATF6 to the Golgi is controlled by the ER chaperone BiP/GRP78. Dissociation of BiP from ATF6’s lumenal domain unmasks ER export signals, which then target ATF6 to the Golgi [14], [15].

Proteolytic activation of ATF6 in the ER stress response represents a recently recognized mechanism to regulate membrane-bound factors, termed regulated intramembrane proteolysis (RIP) [16]. S1P cleavage removes most of the lumenal domain of ATF6 and the N-terminal fragment is further cut by S2P within the transmembrane domain, releasing the cytosolic fragment of ATF6 from the membrane. The cytoplasmic fragment of ATF6 moves to the nucleus and activates the expression of ER stress target genes by binding to the ERSE within their promoter regions (Fig. 2) [11], [12]. Several lines of evidence support the essential role of ATF6 in the ER stress response. First, ATF6 failed to be processed to its active form and BiP induction by ER stress was completely abolished in cells deficient for S2P or where S1P activity was inhibited [11], [17]. Second, dominant negative forms of ATF6 blocked the induction of BiP reporter genes [8]. Third, knock down of ATF6 levels by RNA interference reduced the induction of many ER stress inducible genes in microarray experiments [18].

Here, we describe the detailed experimental procedures that we have used to analyze ATF6 activation in the ER stress response. First, we will review the use of luciferase reporter genes to measure ER stress and ATF6 activation. We will then cover in reverse order the three key steps in the proteolytic processing of ATF6: BiP binding and ER stress-induced dissociation, translocation to the Golgi, and cleavage by the Golgi proteases. It should be noted that there are two isoforms of ATF6 in mammals: ATF6α and ATF6β. They share significant sequence homology and both are proteolytically processed during ER stress, suggesting they may be regulated in a similar way [19]. However, recent evidence has shown that ATF6β is a poor transcriptional activator and can inhibit activation by ATF6α [20]. The experiments described here are based on ATF6α.