Unfolded Protein Response Regulates Yeast Small Gtpase Arl1p Activation

Unfolded protein response regulates yeast small PNAS PLUS GTPase Arl1p activation at late Golgi via phosphorylation of Arf GEF Syt1p

Jia-Wei Hsua,b,1, Pei-Hua Tanga,b,1, I-Hao Wanga,b,1, Chia-Lun Liua,b, Wen-Hui Chena,b, Pei-Chin Tsaia,b, Kuan-Yu Chena,b, Kuan-Jung Chena,b, Chia-Jung Yuc, and Fang-Jen S. Leea,b,2

aInstitute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan; bDepartment of Medical Research, National Taiwan University Hospital, Taipei 100, Taiwan; and cDepartment of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan

Edited by Alan R. Fersht, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved February 17, 2016 (received for review September 14, 2015) ADP ribosylation factor (Arf) GTPases are key regulators of mem- Syt1p to the Golgi (14). It is largely unknown how Syt1p GEF brane traffic at the Golgi complex. In yeast, Arf guanine nucleotide- activity is regulated. exchange factor (GEF) Syt1p activates Arf-like protein Arl1p, which Ire1p is a kinase/endoribonuclease, which is localized in the was accompanied by accumulation of golgin Imh1p at late Golgi, endoplasmic reticulum (ER) membrane and activated via ER but whether and how this function of Syt1p is regulated remains stress (15, 16). The accumulation of unfolded proteins in the ER unclear. Here, we report that the inositol-requiring kinase 1 (Ire1p)- triggers the unfolded protein response (UPR), which selectively mediated unfolded protein response (UPR) modulated Arl1p activa- activates the expression of ER-resident chaperones (17). Signaling tion at late Golgi. Arl1p activation was dependent on both kinase in the UPR is initiated by Ire1p, which has a luminal fragment that and endo-RNase activities of Ire1p. Moreover, constitutively active senses the imbalance of unfolded proteins in the ER and a cyto- transcription factor Hac1p restored the Golgi localization of Arl1p plasmic fragment that induces downstream transcriptional acti- IRE1 – and Imh1p in -deleted cells. Elucidating the mechanism of Ire1p vation (17). The most conserved output of Ire1p signaling is the Hac1p axis actions, we found that it regulated phosphorylation of site-specific cleavage of an mRNA, the product of the yeast HAC1 Syt1p, which enhances Arl1p activation, recruitment of Imh1p to the gene (18). Cleavage occurs at two distinct sites and is followed by Golgi, and Syt1p interaction with Arl1p. Consistent with these findings, the ligation of the 5′ and 3′ fragments to generate an ER stress- the induction of UPR by tunicamycin treatment increases phosphor- dependent spliced mRNA that encodes a potent transcription ylation of Syt1p, resulting in Arl1p activation. Thus, these findings clarify how the UPR influences the roles of Syt1p, Arl1p, and Imh1p factor. The target genes of the spliced Hac1p enhance the ability of in Golgi transport. the ER to cope with the accumulation of unfolded proteins and also act more broadly to up-regulate the secretory capacity (19, 20). – Golgi complex | ADP ribosylation factor | GTPase | UPR | ER stress In this study, we demonstrate that Ire1p Hac1p signaling regulates Arl1p activity via modulating the phosphorylation of Syt1p at S416. The Ire1p–Hac1p signaling pathway regulates the DP ribosylation factor (Arf) and ARF-like (Arl) proteins are interaction between Syt1p and Arl1p to activate Arl1p. Impor- major regulators of membrane trafficking in the exocytotic A tantly, both the S416 phosphorylation of Syt1p and the Arl1p and endocytic pathways (1). Arfs cycle between an inactive GDP- activity are up-regulated during treatment with the UPR inducer bound state and an active GTP-bound state. The activation occurs by the GDP to GTP exchange that is mediated by guanine nucleotide-exchange factors (GEFs) (2, 3). All ArfGEFs described to Significance date are peripheral membrane proteins that contain a region of ∼200 amino acids, which is responsible for their GEF activity and The unfolded protein response (UPR) is a cellular response to is known as the Sec7 domain (3). A database analysis has stress caused by accumulation of unfolded or misfolded pro- revealed that there are 15 Sec7 domain-containing proteins in teins in the endoplasmic reticulum lumen. ADP ribosylation humans, eight in plants, and five in flies, worms, and yeast (Sec7p, factor (Arf) GTPases are key regulators of membrane traffic in Gea1p, Gea2p, Yel1p, and Syt1p) (2). The Sec7 domain interacts the Golgi complex. In yeast, Arf guanine nucleotide-exchange with Arfs and mediates GDP to GTP exchange on Arfs in vitro (4, factor (GEF) Syt1p activates the Arf-like protein Arl1 at late 5). The regions outside the Sec7 domain are largely unrelated; Golgi, thereby recruiting golgin Imh1p, but how Syt1p GEF CELL BIOLOGY however, they have been reported to play important biological activity is regulated is largely unknown. We demonstrate that roles, including membrane localization and substrate specificity UPR signaling regulated phosphorylation of Syt1, which is critical (6). Recently, the domains outside the Sec7 domain have been for Arl1p activation, recruitment of golgin protein Imh1p to shown to regulate ArfGEF activity via both autoinhibition and the Golgi, and Syt1p interaction with Arl1. These findings reveal positive feedback (7, 8). It has been suggested that ArfGEF ac- a previously unsuspected relationship between the UPR and tivity could be regulated through additional mechanisms. Golgi transport. Arl1p, the best-studied Arl protein, localizes to the trans-Golgi and regulates anterograde and retrograde transport in the trans- Author contributions: J.-W.H., P.-H.T., I.-H.W., C.-J.Y., and F.-J.S.L. designed research; – J.-W.H., P.-H.T., I.-H.W., C.-L.L., W.-H.C., P.-C.T., K.-Y.C., and K.-J.C. performed research; Golgi network (TGN) (9 13), including Gas1p transport to the C.-L.L., W.-H.C., K.-J.C., and C.-J.Y. contributed new reagents/analytic tools; J.-W.H., P.-H.T., plasma membrane, the recruitment of golgin Imh1p to the Golgi, I.-H.W., C.-L.L., W.-H.C., P.-C.T., K.-Y.C., C.-J.Y., and F.-J.S.L. analyzed data; and J.-W.H., and the association of Gga2p with the late Golgi. Arl1p activation P.-H.T., I.-H.W., and F.-J.S.L. wrote the paper. requires a second Arl GTPase, Arl3p, and recruitment of Arl3p to The authors declare no conflict of interest. the Golgi needs a transmembrane protein Sys1p (10, 11). We have This article is a PNAS Direct Submission. recently demonstrated that the ArfGEF Syt1p activates Arl1p and 1J.-W.H., P.-H.T., and I.-H.W. contributed equally to this work. thus recruits Imh1p to the Golgi (14). Although the Syt1p-medi- 2To whom correspondence should addressed. Email: [email protected]. ated Arl1p activation could be regulated by Arl3p, our previous This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. data indicated that Arl3p does not interact with Syt1p nor recruit 1073/pnas.1518260113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1518260113 PNAS | Published online March 10, 2016 | E1683–E1690 Downloaded by guest on September 24, 2021 tunicamycin. Our findings suggest that the UPR-induced Ire1p– (14). Consistent with the change in Arl1p-mRFP localization in Hac1p signaling activates Arl1p and enhances the recruitment of ire1Δ cells, we found that the GTP-bound form of Arl1p was Imh1p via the phosphorylation of Syt1p at S416. decreased in syt1Δ and ire1Δ cells compared with wild-type cells (Fig. 1C). Our previous study showed that Syt1p prefers to in- Results teract with the GDP-bound forms of Arl1p (14). Notably, we Ire1p Regulates Arl1p Activation and the Golgi Localization of Imh1p. found that the interaction between Syt1p and the constitutively Previous large-scale proteomics screening data have indicated inactive Arl1p (T32N) was reduced in ire1Δ cells (Fig. 1D). These that Syt1p is phosphorylated at seven N-terminal residues (21– data demonstrated that Ire1p specifically regulates Arl1p activa- 25). To identify the kinase signaling that may regulate Syt1p tion and the localization of Imh1p at late Golgi. activity, we first searched for possible candidates in the kinase signaling pathway through physical and genetic interactions with The Kinase and Endoribonuclease Activities of Ire1p Are Required for ARL1 reported in the Saccharomyces Genome Database (SGD; the Golgi Localization of Arl1p and Imh1p. Ire1p is a transmembrane www.yeastgenome.org). Consistent with our previous report, we sensor protein that transmits signals from the lumen of the ER to first observed that mCherry-Imh1p displays a Golgi distribution the cytosol. Ire1p consists of an N-terminal luminal domain, a in wild-type cells but is present in a mostly diffuse pattern in arl3Δ, single-pass transmembrane domain, and a cytosolic region arl1Δ,andsyt1Δ cells (14). Interestingly, we found that mCherry- divided into a serine/threonine protein kinase domain and a Imh1p showed a cytosolic distribution in an ire1Δ mutant strain C-terminal endoribonuclease (RNase) domain (26). Accumulation (Fig. 1A). We also found that Arl1p-mRFP (monomeric red fluo- of misfolded proteins in the ER is thought to induce the oligo- rescent protein) showed a diffuse cytosolic localization in ire1Δ cells merization of Ire1p, leading to the phosphorylation of Ire1p at in trans (Fig. 1B). We further performed quantitative colocalization analysis S840 and S841 by another Ire1p molecule (27). There- by confocal microscopy and showed that mCherry-Imh1p and fore, Ire1p possesses two distinct enzymatic activities, both a Arl1p-mRFP were much less colocalized with GFP-Sec7p in ire1Δ kinase activity and a site-specific endoribonuclease activity, that cells (Figs. S1 and S2, respectively). However, we found that Arl3p- are regulated by its intrinsic kinase module (28). Both the olig- mRFPandHA-Syt1premainedinpunctuatestructuresandcolo- omerization and phosphorylation of Ire1p are required for its calized with the trans-Golgi marker GFP-Sft2p in the ire1Δ cells endoribonuclease activity to conduct the UPR (26). To examine (Fig. S3 A and B). We also observed that Arl3p-mRFP exhibits a which Ire1p activity is required for Arl1p and Imh1p localiza- sys1arl3Δ tion, we generated a series of HA-tagged Ire1p–mutant proteins, diffuse cytosolic localization in cells. Furthermore, in K702A ire1Δ cells, Arf1p-mRFP was colocalized with the cis-Golgi marker including the kinase-dead mutant Ire1p , the phospho- C defective Ire1pS840AS841A, the phospho-mimetic Ire1pS840ES841E, GFP-Sed5p (Fig. S3 ). Together, these data support the notion K1058A that Ire1p specifically affects the Golgi localization of Arl1p and and the endoribonuclease-dead mutant Ire1p .First,we Imh1p. The activation of Arl1p to its GTP-bound form is known to confirmed that the expression of the kinase-dead mutant, the facilitate its association with the late Golgi membranes (14). We phospho-defective, and the endoribonuclease-dead mutant of Ire1p in ire1Δ cells induced a growth defect in rich media con- further examined whether Ire1p is required for Syt1p GEF activity A to promote Arl1p activation in vivo. We used recombinant GST- taining tunicamycin, a UPR inducer (Fig. S4 ). We found that Imh1-C177 to pull down the active GTP-bound forms of Arl1p the kinase-dead mutant, the phospho-defective mutant, and the endo-ribonuclease-dead mutant of Ire1p could not restore the Golgi localization of Imh1p and Arl1p (Fig. 2 A and B). These data indicated that both the kinase and endoribonuclease activity are required for the Golgi localization of Imh1p and Arl1p.

Ire1p–Hac1p Signaling Regulates the Golgi Localization of Arl1p and Imh1p. The well-known downstream substrate of Ire1p is HAC1, a transcriptional activator. During the UPR, activated Ire1p cuts the intron of precursor HAC1 mRNA to generate a spliced mRNA that encodes an activator of UPR target genes (29, 30). To further verify whether Ire1p signaling regulates Arl1p and Imh1p Golgi localization through its downstream HAC1 pro- Δintron cessing, we expressed HAC1 , in which the intron sequences were deleted, under the control of the HAC1 native promoter and compared its phenotype with the wild-type HAC1 gene. We Δintron observed that expression of HAC1 and HAC1 could restore tunicamycin hypersensitivity in hac1Δ cells (Fig. S4B); however, Δintron expression of HAC1 , but not HAC1, suppressed tunicamycin hypersensitivity in ire1Δ cells (Fig. S4B). Consistently, Δintron these data support the notion that the intron-less HAC1 produces constitutively processed HAC1 mRNA, which can be directly translated into the Hac1p transcription factor to constitutively induce the UPR. We next examined the localization of Fig. 1. Ire1p regulates the Golgi localization of Arl1p and Imh1p. (A) mCherry- Imh1p and Arl1p in hac1Δ cells and found that GFP-Imh1p and Imh1p shows a cytosolic distribution in the ire1Δ cells. The indicated yeast cells Arl1p-mRFP showed a cytosolic distribution in hac1Δ and ire1Δ expressing mCherry-Imh1p were observed via fluorescence microscopy. (B)Arl1p- cells (Fig. 3 A and B). In addition, expression of HAC1 in the mRFP Golgi localization is altered in ire1arl1Δ cells. Arl1p-mRFP was transformed hac1Δ cells, but not in the ire1Δ cells, restored the Golgi local- into the indicated yeast cells. The live cells were observed in midlog phase by ization of Imh1p and Arl1p (Fig. 3 A and B). Moreover, the Δintron fluorescence microscopy. (C)DeletionofIRE1 in yeast cells decreases GTP-bound expression of intron-less HAC1 rescued the Golgi locali- Arl1p. The indicated yeast cells expressing Arl1p were lysed and analyzed for – zation of Imh1p and Arl1p in the hac1Δ and ire1Δ cells (Fig. 3 A GST-Imh1-C177 bound Arl1p proteins by Western blot. (D) The association of B HAC1Δintron Syt1p and inactive Arl1p is altered in ire1Δ cells. Yeast lysates were prepared, and ). Notably, the expression of did not restore pulled down with glutathione–Sepharose beads, and analyzed for bound pro- the Golgi localization of Imh1p in arl3Δ, syt1Δ,orarl1Δ cells teins by Western blot. (Scale bar, 5 μm.) (Fig. 3C), suggesting that Ire1p–Hac1p signaling acts as the

E1684 | www.pnas.org/cgi/doi/10.1073/pnas.1518260113 Hsu et al. Downloaded by guest on September 24, 2021 and GFP-Sft2p (late Golgi marker) in syt1arl1Δ cells. As PNAS PLUS expected, Arl1p-mRFP displays a mostly diffuse pattern in the syt1arl1Δ cells, and Syt1p expression restored the Golgi localization of Arl1p-mRFP (14) (Fig. 4D). We observed that expression of Syt1pS416D,butnotSyt1pS416A, restored the punctuate distribution of Arl1p-mRFP in syt1arl1Δ cells, and the proteins were colocalized with GFP-Sft2p in the late Golgi (Fig. 4D). We further examined whether Syt1p phosphorylation at S416 is required for its GEF activity to promote Arl1p activation in vivo. We found that more Arl1p was bound to GST-Imh1-C177 in syt1Δ cells expressing wild-type Syt1p and Syt1pS416D compared with those expressing Syt1S416A (Fig. 4E). This result indicates that phosphorylation at S416 is required for Syt1p GEF activity. We also demonstrated that Syt1pS416D, but not Syt1pS416A, interacted significantly with the constitutively inactive GDP-bound form of Arl1p (T32N) (Fig. 4F). These data demonstrated that S416 phosphorylation of Syt1p facilitated the interaction with Arl1p, thereby promoting the activation and Golgi localization of Arl1p. We next addressed whether Ire1p–Hac1p signaling may regulate the phosphorylation at S416 of Syt1p. We observed that expression of the phospho-mimetic mutant Syt1pS416D, but not wild-type Syt1p or phospho-defective Syt1pS416A, maintained the Fig. 2. The kinase and endoribonuclease activity of Ire1p are required for Golgi localization of Arl1p and Imh1p in ire1Δ and hac1Δ cells the Golgi localization of Arl1p and Imh1p. (A) The kinase and endoribonuclease (Fig. 5 A and B). These data suggested that Ire1p–Hac1p sig- activity of Ire1p disrupt the Golgi localization of Imh1p. Different forms of naling mediates Syt1p phosphorylation at S416 to regulate Arl1p Ire1p were coexpressed with GFP-Imh1p in ire1Δ yeast cells. Live cells were and Imh1p Golgi localization. observed in midlog phase using fluorescence microscopy. (B) The localization Δ of Arl1p-mRFP in ire1arl1 cells expressing different forms of Ire1p is shown. UPR Up-Regulates Syt1p S416 Phosphorylation to Promote Arl1p (Scale bar, 5 μm.) Activation. We finally questioned whether the activation of Arl1p and phosphorylation of Syt1p are indeed enhanced by the UPR. N upstream determinant to regulate the recruitment of Arl1p and Tunicamycin is known to trigger ER stress by inhibiting -glyco- Imh1p to the Golgi. Taken together, these data indicated that Ire1p– sylation of newly synthesized proteins in the ER, thus up-regulating Hac1p signaling regulates Arl1p and Imh1p Golgi localization. Ire1p activity (31). We next examined whether tunicamycin-induced ER stress contributes to the phosphorylation of Syt1p and Phosphorylation at S416 of Syt1p Modulates the Syt1p GEF Activity to Arl1p activity. First, we confirmed the ER morphology using Promote Arl1p Activation and the Golgi Localization of Imh1p. Con- the ER marker GFP-Sec63p, an abundant ER transmembrane sidering that deletion of IRE1 in yeast cells did not alter the Golgi localization of Syt1p, we were curious about whether Ire1p– Hac1p signaling can modulate Syt1p phosphorylation. We first examined the Syt1p phosphorylation sites by mass spectrometry. In addition to the residues identified from the prior large-scale screening data (21–25), we found several previously unidentified phosphorylation sites on Syt1p, including S214, S408, S410, S416, and S417 (Fig. 4A and Table S1). Interestingly, all of these phosphorylation sites are enriched at the N terminus of Syt1p. Our previous report showed that the N-terminal region of Syt1p is necessary for its function in maintaining the Golgi localization of Imh1p by promoting Arl1p activation (14). We next performed mass spectrometry to analyze the phosphorylated levels of Syt1p in ire1Δ, hac1Δ, and wild-type cells and found that phospho-S416, but not phospho-S417 or other phosphorylated residues of Syt1p, CELL BIOLOGY was decreased in both ire1Δ and hac1Δ cells (Fig. 4B and Fig. S5), suggesting that Ire1p–Hac1p signaling specifically affects the phosphorylation at S416 of Syt1p. To examine whether Syt1p phosphorylation may regulate its function, we next generated phospho-mimetic (change of Ser to Asp) and phospho-deficient (change of Ser to Ala) Syt1p mutants and coexpressed them with GFP-Imh1p in syt1Δ cells. Consistent with a previous report, we observed that GFP-Imh1p displays a Fig. 3. Ire1p–Hac1p signaling regulates the Golgi localization of Arl1p and Δ Golgi distribution in wild-type cells but is present in a mostly Imh1p. (A) Expression of HAC1 intron suppressed the mislocalization of Imh1p Δ diffuse pattern in syt1Δ cells (14). Importantly, we found that in ire1Δ cells. Unspliced HAC1 and spliced HAC1 intron were coexpressed with expressions of the phospho-mimetic Syt1pS416D and wild-type GFP-Imh1p in hac1Δ and ire1Δ cells. Live cells were observed in midlog S416A phase using fluorescence microscopy. (B) The localization of Arl1p-mRFP Syt1p, but not Syt1p , could rescue the mislocalization of Δ in hac1arl1Δ and ire1arl1Δ cells expressing HAC1 or HAC1 intron is shown. syt1Δ C Δ GFP-Imh1p in the cells (Fig. 4 ). To examine whether (C) Expression of HAC1 intron did not suppress the mislocalization of Imh1p in Δ Syt1p phosphorylation is required for promoting Arl1p activation arl3Δ, syt1Δ,orarl1Δ cells. Spliced HAC1 intron was coexpressed with GFP- and the late Golgi localization of Arl1p, we coexpressed Syt1p Imh1p in ire1Δ, arl3Δ, syt1Δ, and arl1Δ cells. Live cells were observed in wild-type protein and phosphorylation mutants with Arl1p-mRFP midlog phase using fluorescence microscopy. (Scale bar, 5 μm.)

Hsu et al. PNAS | Published online March 10, 2016 | E1685 Downloaded by guest on September 24, 2021 Fig. 4. Syt1p phosphorylation at S416 is required for Arl1p activation and the Golgi localization of Imh1p. (A) Diagram of the phosphorylation residues identified in Syt1p by MS. The phospho-residues identified in previous reports are labeled in black (S211, S275, T277, S356, S369, S385, and S409); the previously unidentified phospho-residues identified in this study are labeled in red (S214, S408, S410, S416, and S417). (B) Quantification of Syt1p phosphorylation at S416 and S417 in wild-type, ire1Δ, and hac1Δ cells by mass spectrometric analysis. (C) The phospho-mimetic Syt1pS416D and unphosphorylated mimetic Syt1pS416A were expressed in syt1Δ cells to examine the localization of GFP-Imh1p. (D) Expression of Syt1pS416A could not restore the Golgi localization of Arl1p. Different forms of Syt1p were coexpressed with Arl1p-mRFP in syt1arl1Δ cells. The live cells were observed in midlog phase by fluorescence microscopy. (Scale bar, 5 μm.) (E) Expression of Syt1pS416A decreased the amount of active Arl1p. Yeast lysates from cells expressing Arl1p and either Syt1p, Syt1pS416A, or Syt1pS416D were prepared and analyzed for bound proteins as described in Materials and Methods.(F) The association of Syt1p and Arl1p in vivo. Indicated cells were lysed with glass beads. After centrifugation, the clarified lysates were immunoprecipitated with anti-HA antibodies at 4 °C for 2 h and then washed three times with binding buffer before the bound proteins were analyzed by Western blot. (Scale bar, 5 μm.)

protein. Consistent with previous studies (32), vast extensions of in response to tunicamycin treatment (Fig. 6C). Notably, deletion the peripheral ER into the cell interior were observed when yeast of ARL3, SYT1, ARL1,orIMH1 also induced tunicamycin hy- cells were exposed to ER stress with tunicamycin treatment (Fig. persensitivity (Fig. S4C), suggesting that the Arl3p–Syt1p–Arl1p– S4D). We also observed that tunicamycin treatment led to a Imh1p cascade in the Golgi has an important role in the UPR. marked increase of Golgi-like Arl1p-mRFP puncta in wild-type Furthermore, we observed an increase of Golgi-like mCherry- cells but not in ire1Δ cells (Fig. 6A). Consistently, we also detected Imh1p puncta after tunicamycin treatment (Fig. 6B). In tunicamycin- elevated GTP-bound Arl1p in wild-type cells, but not syt1Δ cells, treated yeast, mCherry-Imh1p puncta were highly colocalized with

E1686 | www.pnas.org/cgi/doi/10.1073/pnas.1518260113 Hsu et al. Downloaded by guest on September 24, 2021 PNAS PLUS

Fig. 5. Ire1p–Hac1p signaling regulates Syt1p phosphorylation at S416 to induce Arl1p activation. (A and B) Expression of SYT1S416D suppressed the mislocalization of Imh1p and Arl1p in ire1Δ and hac1Δ cells. SYT1, SYT1S416A, and SYT1S416D were coexpressed with GFP-Imh1p or Arl1p-mRFP in the indicated yeast cells. Cells containing punctate mCherry-Imh1p and Arl1p-mRFP signals were quantified using ImageJ software (n = 50). Live cells were observed in midlog phase using fluorescence microscopy. (Scale bar, 5 μm.)

the TGN markers GFP-Sec7p and GFP-Sft2p but not the ER covered a previously unsuspected relationship between the UPR marker GFP-Sec63p or the cis-Golgi marker GFP-Sed5p (Fig. S6), and Golgi transport. indicating UPR-induced additional GFP-Imh1 structures that are Phosphorylation of ArfGEFs plays important roles in regulating associated with amplification of the TGN. Overexpression of IRE1, their function (22, 33). For example, protein kinase A-catalyzed Δintron HAC1,orHAC1 in wild-type cells also increased Golgi-like phosphorylation of BIG2 decreases its nucleotide exchange ac- mCherry-Imh1p puncta (Fig. S7). Collectively, these data demon- tivity, which is restored by protein phosphatase 1γ, to coordinate strated that tunicamycin-induced UPR promotes Arl1p activation the cAMP and Arf regulatory pathways (34). In addition, AMP- and function in the late Golgi. activated protein kinase has been reported to phosphorylate To further assess the effect of Syt1p S416 phosphorylation on GBF1 at T1337 to attenuate the function of GBF1 in the disas- tunicamycin-induced Arl1p activation, we generated and affinity- sembly of the Golgi structure in response to glucose blockade (35). purified rabbit polyclonal antibodies that were specific for S416 Furthermore, phosphorylation of GBF1 at T1337 by activated phosphorylation on Syt1p. The affinity-purified antibodies de- AMP-activated protein kinase also dissociates GBF1 from the tected Syt1pS416D in cell lysates, although Syt1pS416A was also Golgi membrane and inhibits its GEF catalytic activity, leading to detected at low levels (Fig. 6D). We found that S416 phosphory- the inactivation of Arf during mitosis (36). In yeast, Sec7p and lation on Syt1p was elevated in tunicamycin-treated wild-type cells Gea2p have also been reported to be phosphoproteins in large- (Fig. 6D). Increased phosphorylation of Syt1p at S416 was ob- scale proteomics analyses (22, 23, 37); however, there are no ire1Δ served in tunicamycin-treated wild-type cells but not in cells known conserved phosphorylation sites in yeast ArfGEFs that are CELL BIOLOGY (Fig. 6E), indicating that tunicamycin-induced S416 phosphoryla- involved in the regulation of GEF activity and membrane associ- tion on Syt1p is Ire1p-dependent. We further found a marked ation. Thus, our study demonstrates for the first time, to our increase in Golgi-like mCherry-Imh1p puncta in syt1Δ cells knowledge, that phosphorylation of an Arf GEF in yeast has S416D expressing the phospho-mimetic SYT1 , but not the phospho- functional relevance. S416A defective SYT1 , in the absence of tunicamycin treatment (Fig. The mammalian homologs of yeast Arl3p and Arl1p are 6F). Taken together, these data strongly support the idea that ARFRP1 and ARL1, respectively. Mammalian ARL1 has been tunicamycin-induced ER stress triggers the phosphorylation of shown to recruit different GRIP domain-containing golgins, Syt1p at S416 through Ire1 signaling to activate Arl1p and recruit golgin-97 and golgin-245/p230, to different subdomains of the Imh1p to the late Golgi (Fig. 7). TGN, which act on retrograde and anterograde cargo transport (38, 39). Silencing of the ARFRP1 gene dissociates ARL1, gol- Discussion gin-97, and Golgin-245 from the TGN (40). It is therefore pro- In this study, we showed that the Ire1p–Hac1p axis regulates posed that the Arl3p–Arl1p–Imh1p cascade in yeast also occurs Syt1p phosphorylation, thereby promoting Arl1p activation and in mammalian cells. However, the GEF for mammalian ARL1 the Golgi recruitment of Imh1p. Moreover, upon the induction activation has not been identified, and phylogenetic analysis of UPR by tunicamycin, phosphorylation of Syt1p at S416 and showed that Syt1p GEF for Arl1p seems unique for the fungi (6). Arl1p activation are augmented. Thus, these findings have un- The GEFs for mammalian ARL1 activation and the regulation

Hsu et al. PNAS | Published online March 10, 2016 | E1687 Downloaded by guest on September 24, 2021 Fig. 6. ER stress up-regulates the phosphorylation of Syt1p at S416 to induce Arl1p activation. (A) The Golgi localization of Arl1p was augmented in yeast cells treated with tunicamycin. Yeast cells expressing Arl1p-mRFP were treated with DMSO or 1 μg/mL tunicamycin for 2.5 h, and then the mRFP signals were observed using fluorescence microscopy. (B) The localization of mCherry-Imh1p in yeast cells treated with DMSO or tunicamycin is shown. (C) Tunicamycin treatment induces the up-regulation of Arl1p activity. Wild-type and syt1Δ cells expressing Arl1p were treated with tunicamycin. Total cell lysates were prepared and the bound proteins were analyzed as described in Materials and Methods.(D) Detection of Syt1p phosphorylation at S416 in yeast cells expressing Syt1p, Syt1pS416A, or Syt1pS416D.(E) Detection of Syt1p phosphorylation at S416 in tunicamycin-treated wild-type and ire1Δ cells. (F) The localization of mCherry-Imh1p in tunicamycin-treated yeast cells expressing Syt1p, Syt1pS416A, or Syt1pS416D is shown. Cells containing punctate mCherry-Imh1p signals were quantified (n = 100). The data are reported as the mean ± SD of three independent experiments. **P < 0.01. (Scale bar, 5 μm.)

of GEF activity by UPR signaling in mammals remain to be Materials and Methods further investigated. Strains, Media, Plasmids, and Chemicals. Table S2 lists the yeast strains used in When the synthesis of secretory proteins overloads the capacity this study. The yeast cells were transformed using the lithium acetate of the ER chaperones, many secretory proteins are improperly method (45). The plasmids used in this study are listed in Table S3. The HAC1 Δ folded and accumulate in the ER. To cope with the unfolded and HAC1 intron expression plasmids were kindly provided by Kazutoshi protein-induced ER stress, cells activate ER stress response Mori, Kyoto University, Kyoto, Japan. A stock solution of 2 mg/mL tunica- – – mycin (Sigma) was prepared in DMSO and stored at –20 °C. Activation of the pathways, such as the Ire1p Hac1p and IRE1 XBP1 axes in yeast μ and mammalian systems, respectively (41). ER stress-induced re- UPR was monitored in rich medium or synthetic medium containing 1 g/mL tunicamycin for 2.5 h at 30 °C. sponse involves the transcriptional activation of UPR target genes to facilitate protein folding and ER-associated protein degrada- Microscopy. Images of live cells containing GFP-tagged or mRFP-tagged pro- tion (42). However, the role of the Golgi in this stress response has teins were obtained after growth in synthetic medium to the midlog phase. All been less defined. A recent study showed that cells have sequential fluorescent protein-tagged chimeras were cultured in selection medium with 2% quality control systems for integral membrane proteins in the ER, (wt/vol) glucose. Yeast cells expressing GFP-Imh1p were cultured in the selection Golgi, and plasma membrane and demonstrated that all three medium with 2% (wt/vol) galactose to induce protein expression. After over- integral membrane quality control systems cooperate to limit the night culture or induction, the midlog phase cells were examined and images accumulation of misfolded proteins at the plasma membrane (43). were captured using a Zeiss Axioskop microscope equipped with a Cool Snap FX Golgi quality control, although still poorly understood, has been camera. For all microscopic examinations, the exposure times and image pro- suggested to protect cells from proteotoxic stress by capturing cessing procedures were identical for each sample within an experiment. The light levels were scaled equivalently among all samples within an experiment. misfolded proteins that emanate from the ER and diverting them Only the light level minimum/maximum settings were adjusted for clarity. for degradation in the lysosome (44). Thus, our elucidation of how – – the UPR influences the roles of Syt1p Arl1p Imh1p in Golgi Indirect Immunofluorescence. The cells were grown and prepared for indirect transport shed important previously unidentified mechanistic in- immunofluorescence of HA-Syt1p and GFP-Sft2p (14). The antibodies used sights into how the Golgi contributes to mitigating proteotoxic included monoclonal anti-HA (Covance) and polyclonal anti-GFP antibody stress emanating from the ER. (Sigma). The secondary antibodies included Alexa-Fluor-488–conjugated

E1688 | www.pnas.org/cgi/doi/10.1073/pnas.1518260113 Hsu et al. Downloaded by guest on September 24, 2021 for 30 min. After reduction and alkylation, the proteins were digested with PNAS PLUS sequencing-grade modified porcine trypsin (20 μg/mL; Promega) overnight at 37 °C. The peptide mixtures were extracted with 1% trifluoroacetic acid/ 40% (vol/vol) acetonitrile and dried in a SpeedVac.

Enrichment and Identification of Phosphopeptides Using Online TiO2 Liquid Chromatography Tandem Mass Spectrometry. Each peptide mixture was resuspended and acidified in 10 μL of equilibration buffer (2% formic acid/ 40% acetonitrile). The peptide mixtures were loaded onto an in-house

packed TiO2 trap column (GL Sciences, 10 μm, 0.3 × 5 mm), washed with 10 μL of wash buffer containing 2% formic acid/40% acetonitrile, and followed by 10 μL of wash buffer containing 2% formic acid/80% acetonitrile. The

online TiO2 liquid chromatography tandem mass spectrometry (MS/MS) experiments were performed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher). Briefly, the phosphopeptides were eluted with 10 μL of elution

buffer (0.1 M Na2CO3, 30% acetonitrile) at a flow rate of 0.5 μL/min and diluted online with 0.1% formic acid at a flow rate of 40 μL/min before reaching the reverse phase trap column (Zorbax 300SB-C18, 0.3 × 5 mm; Agilent Technologies). The trapping process was continued for 30 min, and the analysts were separated on an in-house packed analytical C18 column Fig. 7. A working model of the activation of the Syt1p–Arl1p–Imh1p cas- (Synergi Hydro-RP, 2.5 μm, 0.075 × 150 mm; Phenomenex) with a 15-μm tip cade by the Ire1p–Hac1p pathway. During ER stress, activated Ire1p causes (New Objective). The peptides were separated using a 50-min gradient from the splicing of HAC1 mRNA to turn on the transcriptional activation of UPR 5% to 40% solvent B (0.05% formic acid/99.9% acetonitrile) at a flow rate of target genes, which encode proteins that may phosphorylate Syt1p at Ser- 0.25 μL/min across the analytical column. The LTQ-Orbitrap mass spectrom- 416, resulting in Arl1p activation and recruitment of Imh1p to the late Golgi. eter was operated in the data-dependent mode using the TOP 6 strategy. Briefly, a scan cycle was initiated with a full scan of high mass accuracy (m/z 350–1,700) in the Orbitrap, which was followed by MS/MS scans in the linear – goat anti-rabbit IgG and Alexa-Fluor-594 conjugated goat anti-mouse IgG ion trap on the six most abundant precursor ions. The m/z values selected for (Molecular Probes). The preparations were visualized with a Zeiss Axioskop MS/MS were dynamically excluded for 180 s. The electrospray voltage ap- microscope, and the images were processed using Image Pro Plus software. plied was 1.8 kV. Both the MS and MS/MS spectra were acquired using one Signals were quantified using ImageJ software. microscan with a maximum fill time of 1,000 and 100 ms for MS and MS/MS analysis, respectively. Automatic gain control was used to prevent overfilling Plating Assay for Tunicamycin Hypersensitivity. Midlog phase yeast cells were of the ion trap, and 5 × 103 ions were accumulated in the ion trap to gen- μ spotted at 10-fold serial dilutions onto rich media plates with or without 1 g/mL erate the MS/MS spectra. For the MS scans, the m/z scan range was 350– tunicamycin. The plates were incubated for 3 d before imaging. 2,000 Da. Neutral loss scanning (98 for +1, 49 for +2, and 32.7 for +3 charged ions) was used to detect phosphopeptide, and a third stage MS was used to Active Arl1p Pull-Down Assay. To detect Arl1p-GTP levels in yeast (14), yeast improve backbone fragmentation. cells expressing Arl1p were lysed with glass beads at 4 °C in lysis buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and protease Database Search. All MS and MS/MS data were analyzed and processed in inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 μM MaxQuant v.1.0.13.8, with Mascot searches (46). The top six fragment ions benzamidine, and 1 mM phenylmethylsulfonyl fluoride). The lysates were per 100 Da of each MS/MS spectrum were extracted for a protein database clarified by centrifugation at 10,000 × g for 5 min. The clarified cell lysates search using the Mascot search engine (version 2.2.1, Matrix Science) against were incubated for 2 h at 4 °C with 50 μg GST-Imh1 containing the C-ter- the Swiss-Prot v.56 Saccharomyces cerevisiae forward and reverse protein minal 177 amino acids of yeast Imh1p bound to glutathione–Sepharose sequence datasets, with a set of common contaminant proteins (total 6,090 beads (GE Healthcare). After washing three times with lysis buffer, the entries). The searches were performed with the following parameters: full bound proteins were assayed for the presence of Arl1p by Western blotting. trypsin specificity, a fragment ion mass tolerance of 0.50 Da, a parent ion tolerance of 10.0 ppm, fixed modifications of oxidized M (+15.9949), car- Protein Interaction Analysis. To analyze the in vivo interaction of Arl1p and bamidomethyl C (+57.0215), and variable modifications of phosphorylated Syt1p in yeast, yeast cells expressing HA-Syt1p and GST-Arl1pQ72LdN17 or S, T, and Y (+79.9663). The random sequence database was used to estimate GST-Arl1pT32NdN17 were harvested and lysed with glass beads in binding the false-positive rates for the peptide matches, and the false-positive rate buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 5 mM NaF, for the peptide sequence matches using the criteria described above was and protease inhibitors at 4 °C. The clarified cell lysates were incubated with estimated to be <1% in this study. The posttranslational modification score glutathione–Sepharose beads at 4 °C overnight, and the beads were washed three times with binding buffer. The bound proteins were analyzed by was used to assign the phosphorylation site(s), in which class I phosphory- Western blotting using specific antibodies against Syt1p and Arl1p. lation sites are defined by a localization probability of 0.75 and a probability localization score difference ≥5 (47). CELL BIOLOGY SDS/PAGE Separation and In-Gel Proteolysis. Fifty micrograms of yeast protein extract were separated on a 10% SDS/PAGE gel and stained with Coomassie ACKNOWLEDGMENTS. We acknowledge Kun-Yi Chien for mass spectrometric analysis. We thank Hugh R. B. Pelham, Kai Simons, and Kazutoshi Mori for Brilliant Blue G-250. The protein bands of interest were excised into ∼2-mm providing the expression plasmids and yeast strains. We also thank Ya-Wen cubes and subjected to in-gel tryptic digestion. Briefly, the gel pieces were Liu, Joel Moss, and Randy Haun for their critical review of this manuscript. destained using 50% (vol/vol) acetonitrile/50 mM ammonia bicarbonate. The This work was supported by National Science Council of Taiwan Grant NSC- proteins were reduced with 25 mM NH4HCO3 containing 10 mM DTT at 60 °C 104-2320-B-002-048-MY3 and Yung-Shin Biomedical Research Fund YSP-86-019 for 30 min and alkylated with 55 mM iodoacetamide at room temperature (awarded to F.-J.S.L.).

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