A Rice Transmembrane bZIP Transcription Factor ...

A Rice Transmembrane bZIP Transcription Factor, OsbZIP39, Regulates the Endoplasmic Reticulum Stress Response Hideyuki Takahashi, Taiji Kawakatsu, Yuhya Wakasa, Shimpei Hayashi and Fumio Takaiwa*

Regular Paper

Functional Transgenic Crops Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan *Corresponding author: E-mail, [email protected]; Fax, +81-29-838-8397 (Received September 12, 2011; Accepted November 7, 2011)

Keywords: ER stress Oryza sativa Transcription factor Unfolded protein response. Abbreviations: ATF6, activating transcription factor 6; BiP, binding protein; bZIP, basic leucine zipper; DBP, DNAbinding domain; DMSO, dimethylsulfoxide; DTT, dithiothreitol; ER, endoplasmic reticulum; GFP, green fluorescent protein; GRP, glucose-regulated protein; GUS, b-glucuronidase; IRE1, inositol-requiring protein 1; NOS, nopaline synthase; PDI, protein disulfide isomerase; RIP, regulated

intramembrane proteolysis; RT–PCR, reverse transcription– PCR; SF, Strep/FLAG; S1P, site-1 protease; S2P, site-2 protease; TM, tunicamycin; TMD, transmembrane domain

Introduction The accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen causes ER stress. In plants, it is speculated that biotic and abiotic stresses including pathogen attack, drought, heat stress and salinity disturb protein folding and trigger ER stress. In response, eukaryotic cells activate specific signal transduction pathways that function in the maintenance of ER homeostasis. These pathways are designated the ER stress response or the unfolded protein response (Ron and Walter 2007, Urade 2007, Liu and Howell 2010). ER stress signaling in mammalian cells has three pathways, and each of these pathways transduces different classes of ER stress sensors (Ron and Walter 2007). One pathway is mediated by the activation of transcription factor 6 (ATF6) as a membrane-associated transcription factor, which results in the targeting of specific stress response genes. Another pathway is facilitated by inositol-requiring protein 1 (IRE1)-mediated RNA splicing of a basic leucine zipper (bZIP) transcription factor. This results in the activation of stress sensor targeting stress response genes. The remaining pathway involves a protein kinase-like ER kinase that regulates translation via phosphorylation of translation initiation factor eIF2a. In each of these pathways, a membrane-associated protein, either individually or in association with other factors, senses the accumulation of misfolded proteins and transmits the signal from the ER lumen to the nucleus. ATF6, a type II transmembrane protein, is normally retained in the ER by association with binding protein (BiP)/ glucose-regulated protein (GRP) 78 (Chen et al. 2002, Shen et al. 2002). Upon ER stress, ATF6 dissociates from BiP/GRP78 and is transported to the Golgi for proteolytic processing (Chen et al. 2002, Shen et al. 2002), first by a site-1 protease (S1P) and then by a site-2 protease (S2P) (Haze et al. 1999, Ye et al. 2000). The released N-terminal domain of ATF6, containing a bZIP

Plant Cell Physiol. 53(1): 144–153 (2012) doi:10.1093/pcp/pcr157, available online at www.pcp.oxfordjournals.org ! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

144

Plant Cell Physiol. 53(1): 144–153 (2012) doi:10.1093/pcp/pcr157 ! The Author 2011.

Downloaded from http://pcp.oxfordjournals.org/ at Nogyo Seibutsu Shigen on March 17, 2015

The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways. These pathways are known as the ER stress response or the unfolded protein response. In this study, three rice basic leucine zipper (bZIP) transcription factors (OsbZIP39, OsbZIP50 and OsbZIP60) containing putative transmembrane domains (TMDs) in their C-terminal regions were identified as candidates of the ER stress sensor transducer. One of these proteins, OsbZIP39, was characterized in this study. OsbZIP39 was shown to associate with microsomes as a membrane-integrated protein using the subcellular fractionation method. When the full length and a truncated form of OsbZIP39 without the TMD (OsbZIP39C) was fused to green fluorescent protein (GFP) and transfected into rice protoplasts, the proteins were identified in the cytoplasm and nucleus, respectively. This suggests that OsbZIP39 may be converted into a soluble truncated form by proteolytic cleavage and subsequently translocated to the nucleus. Expression of OsbZIP39DC clearly activated the binding protein 1 (BiP1) promoter in a rice protoplast transient assay. Overexpression of OsbZIP39DC in stable transgenic rice also led to the up-regulation of several ER stress response genes including BiP1 and OsbZIP50 in the absence of ER stress. However, in the OsbZIP39DCoverexpressing line, OsbZIP50 mRNA did not undergo IRE1 (inositol-requiring protein 1)-mediated cytoplasmic splicing that is required for its activation. These data indicate that OsbZIP39 may be directly involved in the regulation of several ER stress response genes.

A transcription factor in rice ER stress signaling

Results

Fig. 1 Mapping of the transactivation domain of OsbZIP39. Left: schematic structure of OsbZIP39 and the fusion proteins constructed from the GAL4 DNA-binding domain (GAL4DBD) and various OsbZIP39 fragments. The positions of the bZIP domain (bZIP) and transmembrane domain (TMD) are indicated. The arrowhead indicates the position of a putative canonical S1P site. An amino acid region for each OsbZIP39 fragment is also shown. Right: transactivation activity of various fusion proteins. Protoplasts isolated from rice suspension cells were transiently transformed with effector plasmids carrying each OsbZIP39 fragment fused with the GAL4DBD and a reporter plasmid expressing the GUS gene driven by a synthetic promoter containing the GAL4 upstream activator sequence. A plasmid carrying the firefly luciferase gene driven by the maize ubiquitin promoter was co-transformed for normalization. Transactivation levels were represented as the fold induction relative to the basal activity obtained from the plasmid expressing OsbZIP39C (amino acids 1-311) fused with the GAL4DBD. Data represent means with SD of three independent experiments.


DNA-binding domain, is translocated to the nucleus where it is involved in the transcriptional activation of ER stress response genes including BiP/GRP78 (Wang et al. 2000). Two membrane-associated bZIP transcription factors (AtbZIP28 and AtbZIP60) play key roles in transducing ER stress signals in Arabidopsis (Iwata and Koizumi 2005, Liu et al. 2007). AtbZIP28 is similar to ATF6 in both structure and mode of action (Liu et al. 2007, Tajima et al. 2008). Specifically, AtbZIP28 is a type II membrane protein, containing an N-terminal bZIP DNA-binding domain that faces the cytosol and a C-terminal regulatory domain that faces the ER lumen. Under normal conditions, AtbZIP28 is localized to the ER. When misfolded proteins accumulate in response to treatment with ER stress agents, such as tunicamycin (TM) and dithiothreitol (DTT), AtbZIP28 undergoes proteolytic processing of its transmembrane domain (TMD). This results in the release of the N-terminal, cytoplasm-facing bZIP domain and its translocation into the nucleus (Liu et al. 2007, Tajima et al. 2008). Among the 89 predicted bZIP genes in the rice genome (Nijhawan et al. 2008), three bZIP transcription factors were previously shown to possess a putative TMD (OsbZIP39, OsbZIP50 and OsbZIP60) (Oono et al. 2010, Wakasa et al. 2011). Among these three bZIPs, expression of OsbZIP50 was induced by DTT and TM (Wakasa et al. 2011). Transient expression of a truncated OsbZIP50 lacking the putative TMD resulted in the activation of the promoters of several ER stress response genes in rice protoplasts (Wakasa et al. 2011). These results suggested that OsbZIP50 plays a critical role in the ER stress response. Recently, mRNA encoding AtbZIP60, an ortholog of OsbZIP50, was identified to be a target of IRE1-mediated splicing (Deng et al. 2011, Nagashima et al. 2011). IRE1-mediated splicing of AtbZIP60 mRNA associated with a frameshift in the region coding for its C-terminal region resulted in activation of ER stress response genes (Deng et al. 2011, Nagashima et al. 2011). OsbZIP39, a bZIP transcription factor that possesses a putative TMD, is characterized in this study. Truncated OsbZIP39, lacking a putative TMD, is localized to the nucleus and is involved in activating the BiP1 promoter in rice protoplasts. Furthermore, in stable transgenic rice, overexpression of the truncated OsbZIP39 resulted in the up-regulation of several ER stress response genes in the absence of ER stress. This included OsbZIP50. Based on the results of this study, OsbZIP39 is involved in the regulation of many ER stressresponsive genes.

transcription factor and has transactivation activity, the transcriptional activation domain of OsbZIP39 was identified. A series of plasmids, encoding C-terminal deletion mutants of OsbZIP39 fused to the GAL4 DNA-binding domain (GAL4DBD), were prepared and used as effectors. Protoplasts were isolated from rice suspension cells and transiently co-transformed with an effector plasmid and a reporter plasmid expressing the b-glucuronidase (GUS) gene driven by a synthetic promoter containing the GAL4 upstream activator sequence. As shown in Fig. 1, the N-terminal region of OsbZIP39 (amino acids 1–98) increased the expression of the GUS reporter gene by approximately 24-fold, in comparison with the control containing the GAL4DBD alone. When amino acids 51–98 were deleted, transactivation decreased by approximately 4-fold when compared with the GAL4DBD-only control. These results indicate that OsbZIP39 functions as a transcriptional activator. Furthermore, the critical transcriptional activation domain is located between amino acids 51–98.

OsbZIP39 is a transcriptional activator

Truncated OsbZIP39 lacking a putative TMD localizes to the nucleus

OsbZIP39 contains an open reading frame encoding a putative polypeptide of 646 amino acids. The predicted protein contains a bZIP DNA-binding domain followed by a predicted TMD (Fig. 1). To investigate whether OsbZIP39 functions as a

As the transcriptional activation and bZIP domains are located upstream of the predicted TMD, localization experiments were conducted to investigate whether, following removal of the putative TMD by proteolysis, OsbZIP39 might be converted


145

H. Takahashi et al.

to a functional truncated form and localized to the nucleus. To establish the subcellular localization of a truncated OsbZIP39 containing amino acids 1–311 (OsbZIP39C), green fluorescent protein (GFP) was fused to the N-terminus of OsbZIP39C and transiently expressed in rice protoplasts. Upon observation of the GFP signals by confocal microscopy, OsbZIP39C was detected exclusively in the nucleus (Fig. 2A). In contrast, GFP–OsbZIP39 was detected in both the cytoplasm and the nucleus (Fig. 2B). The signals in the cytoplasm completely merged with a mCherry-fluorescent ER marker (SP-mCherry-HDEL), indicating the presence of OsbZIP39 in the ER (Fig. 2B).

OsbZIP39 is a type II integral membrane protein on the ER To investigate whether OsbZIP39 is associated with the membrane, subcellular fractionation experiments were employed to determine its intracellular localization. The Strep/FLAG (SF) epitope tag was attached to the full-length OsbZIP39 N-terminus (SF–OsbZIP39) and to a truncated form missing the C-terminal putative TMD (SF-OsbZIP39C), and the proteins were transiently expressed in rice protoplasts. When protoplast total cellular extracts were subjected to immunoblot analysis, two major bands were detected for the SF–OsbZIP39 products (Fig. 3A). The upper band, predicted to be the full-length OsbZIP39 protein, was detected exclusively in the microsomal fraction (Fig. 3B). The lower band was close

146

to the size of SF–OsbZIP39C (Fig. 3A). These results suggest that full-length OsbZIP39 may be associated with membranes. Additionally, some portion of OsbZIP39 may be processed to a truncated form similar to OsbZIP39C. Chen et al. (2002) reported that higher expression of ATF6 gave rise to a low level of cleavage product, which was sufficient to activate the reporter gene in uninduced cells. Therefore, since OsbZIP39 was highly expressed under the control of a strong ubiqutin promoter, part of OsbZIP39 may undergo proteolytic cleavage even in the absence of an ER stress-inducing regent. To characterize the manner in which OsbZIP39 is associated with membranes, SF–OsbZIP39 was extracted from the microsomal fraction under various conditions (Fig. 3B). SF–OsbZIP39 was extracted from the microsomal fraction using detergents (Triton X-100 and SDS) but not by treatment with high salt (NaCl) or alkaline solution (at pH 11) (Fig. 3B). This extraction profile matched that of the integral membrane protein calnexin, implying that OsbZIP39 may be localized in the membrane as an integral membrane protein. To determine the membrane topology of OsbZIP39, the microsomal fraction was further digested with proteinase K. While SF–OsbZIP39 was susceptible to proteinase K digestion in the absence of detergent, calnexin was not (Fig. 3C). These results indicate that OsbZIP39 is oriented so that its N-terminal domain is in the cytosol, a characteristic of type II membrane proteins. In conjunction with the subcellular localization of GFP–OsbZIP39 in Fig. 2B, these findings suggest that full-length OsbZIP39 resides in the ER membrane.



Fig. 2 Subcellular localization of GFP–OsbZIP39C and GFP–OsbZIP39 fusion proteins. An ER-localized mCherry (SP-mCherry-HDEL) fusion construct was co-expressed with GFP–OsbZIP39C (A) and GFP–OsbZIP39 (B and C) transiently in rice protoplasts. (C) The cells expressing GFP–OsbZIP39 were treated with 2 mM DTT for 1 h. The cells were inspected with a confocal laser scanning microscope and a phase contrast (PC) microscope. Bars = 5 mm.

A transcription factor in rice ER stress signaling

If OsbZIP39 functions as an ER membrane-associated transcription factor involved in the ER stress response, full-length OsbZIP39 should relocate to the nucleus in response to ER stress. To test this hypothesis, cells expressing GFP–OsbZIP39 were treated with DTT. When observed by confocal microscopy, the GFP signals were predominantly detected in the nucleus (Fig. 2C). In contrast, GFP signals in untreated cells were localized to the ER (Fig. 2B). In response to ER stress, we propose that OsbZIP39 is converted to a soluble truncated form by proteolytic cleavage and then localized to the nucleus.


Fig. 3 OsbZIP39 is a type II membrane protein. (A) Immunoblot analysis of the total extracts of rice protoplasts expressing SF–OsbZIP39 and SF–OsbZIP39C with anti-FLAG antibody. A Strep/FLAG epitope was fused to the N-terminus of the full-length OsbZIP39 (SF–OsbZIP39) and the truncated form lacking the C-terminal region containing the putative TMD (SF–OsbZIP39C), and transiently expressed in rice protoplasts. The molecular masses are given on the right in kiloDaltons. (B) The microsomal fraction from the rice protoplasts expressing SF–OsbZIP39 was suspended in control buffer (100 mM Tris–HCl, pH 7.5), high salt buffer (1 M NaCl and 100 mM Tris–HCl, pH 7.5), alkaline solution (100 mM Na2CO3, pH 11), Triton X-100 buffer [1% (v/v) Triton X-100 and 100 mM Tris–HCl, pH 7.5] and SDS buffer [1% (w/v) SDS and 100 mM Tris–HCl, pH 7.5]. These suspensions were ultracentrifuged to obtain pellet (P) and supernatant (S) fractions. Each fraction was subjected to immunoblot analysis with anti-FLAG antibody. (C) The microsomal fraction from rice protoplasts expressing SF–OsbZIP39 was incubated in the presence (+) or absence () of SDS and proteinase K. The fractions were subjected to immunoblot analysis with anti-FLAG antibody.

Fig. 4 OsbZIP39 effector activity on the expression of ER stress response genes. (A) Effect of DTT treatment on the induction of the 1.2 kb BiP1 promoter. Protoplasts were transfected with a reporter plasmid and treated with 2 mM DTT for 16 h. Transient assays were carried out as described in Fig. 1. Transactivation levels are represented as fold induction relative to the expression of a GUS reporter gene from the BiP1 promoter under untreated conditions. Data represent means with SD of three independent experiments. (B) Effect of OsbZIP39 and OsbZIP39C on the BiP1 promoter. Transient assays were carried out as described above. Instead of DTT treatment, effector plasmids carrying OsbZIP39 or OsbZIP39C driven by the maize ubiquitin promoter were co-transformed. Transactivation levels are represented as fold induction relative to the expression of a GUS reporter gene from the BiP1 promoter without effector plasmids. Data represent means with SD of three independent experiments.

OsbZIP39 activated the BiP1 promoter through cis-elements responsible for the ER stress response The ability of OsbZIP39 to activate ER stress response genes was investigated using transient assays with rice protoplasts. In these assays, cDNAs encoding the full-length OsbZIP39 and its truncated form were expressed as effectors. Previously, BiP1 expression was shown to be induced by DTT in rice (Wakasa et al. 2011). TM and DTT are known to induce the ER stress response in plants by causing unfolded proteins to accumulate in the ER lumen. As a reporter of ER stress, the GUS gene was linked to the BiP1 promoter (1.2 kb). In this study, treatment with DTT clearly enhanced GUS activity, indicating that the 1.2 kb BiP1 promoter was activated by ER stress treatment (Fig. 4A). Furthermore, OsbZIP39C co-expression with the reporter gene significantly enhanced GUS activity in comparison with full-length OsbZIP39 (Fig. 4B). These results also support the notion that the N-terminal cytoplasmic region is the active region of OsbZIP39. The three copies of Motif II (CC-N12-CCACG) (Martinez and Chrispeels 2003) and the one copy of the ERSE motif (CCAATN9-CCACG) (Yoshida et al. 1998) -like motif (ERSE-L) were considered as candidate cis-elements in the BiP1 promoter for its activation in response to ER stress, caused by DTT or co-expression of OsbZIP39C (Fig. 5A). To examine the functional role of these cis-elements, individual mutations were introduced into Motif II and ERSE-L of the BiP1 promoter


147

H. Takahashi et al.

as shown in Fig. 5A. When compared with the wild type, mutagenesis of these cis-elements (m1, m2, m3 and m4) clearly suppressed the activation of the BiP1 promoter following treatment with DTT (Fig. 5B). Notably, disruption of all four cis-elements (m5) severely depressed BiP1 promoter activation to