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Signal Motif-Dependent ER Export of the Qc-SNARE BET12 Interacts With

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Signal Motif-Dependent ER Export of the Qc-SNARE BET12 Interacts With © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838 RESEARCH ARTICLE SPECIAL ISSUE: PLANT CELL BIOLOGY Signal motif-dependent ER export of the Qc-SNARE BET12 interacts with MEMB12 and affects PR1 trafficking in Arabidopsis Kin Pan Chung1, Yonglun Zeng1, Yimin Li2, Changyang Ji1, Yiji Xia2 and Liwen Jiang1,3,* ABSTRACT A large number of SNARE proteins are encoded in the plant Soluble N-ethylmaleimide-sensitive fusion protein attachment protein genome (Sanderfoot, 2007; Sanderfoot et al., 2000). Numerous receptors (SNAREs) are well-known for their role in controlling studies have unraveled the important role of SNARE proteins membrane fusion, the final, but crucial step, in vesicular transport in plants, involving various biological processes including in eukaryotes. SNARE proteins contribute to various biological pathogen defense, cytokinesis, abiotic stress, cell expansion, processes including pathogen defense and channel activity regulation, symbiosis, gravitropism, gametophyte and seed development as well as plant growth and development. Precise targeting of SNARE (Ebine et al., 2008; El-Kasmi et al., 2011; Grefen et al., 2010a; proteins to destined compartments is a prerequisite for their proper Hachez et al., 2014; Honsbein et al., 2009; Huisman et al., 2016; functioning. However, the underlying mechanism(s) for SNARE Pan et al., 2016; Reichardt et al., 2007; Uemura et al., 2012b; Yano targeting in plants remains obscure. Here, we investigate the targeting et al., 2003). The precise targeting of SNARE proteins to a distinct – mechanism of the Arabidopsis thaliana Qc-SNARE BET12, which is compartment is essential for mediating the vesicle target- involved in protein trafficking in the early secretory pathway. Two distinct membrane fusions which secures an efficient and accurate protein signal motifs that are required for efficient BET12 ER export were trafficking. Mis-targeting of SNARE proteins results in numerous identified. Pulldown assays and in vivo imaging implicated that both the cellular defects. For instance, the cell plate formation is disrupted in COPI and COPII pathways were required for BET12 targeting. Further a mutant with an impaired trafficking of the syntaxin KNOLLE studies using an ER-export-defective form of BET12 revealed that (Park et al., 2013; Teh et al., 2013). In addition, a recent study has the Golgi-localized Qb-SNARE MEMB12, a negative regulator of revealed the novel role of the endoplasmic reticulum (ER)- pathogenesis-related protein 1 (PR1; At2g14610) secretion, was its associated SNARE SYP73 in maintaining the ER integrity and interacting partner. Ectopic expression of BET12 caused no inhibition in consequently streaming, since these features were altered in a syp73 the general ER-Golgi anterograde transport but caused intracellular mutant (Cao et al., 2016). Therefore, correct localization of SNARE accumulation of PR1, suggesting that BET12 has a regulatory role in proteins seems to be a prerequisite for their proper functioning in PR1 trafficking in A. thaliana. different cellular processes. Most SNARE proteins are tail-anchored (TA) proteins, their KEY WORDS: BET12, ER export, PR1, Protein trafficking, SNARE membrane association being conferred by the C-terminal transmembrane domain (TMD). TA proteins are post-translationally INTRODUCTION inserted into the membrane through the guided entry of TA proteins Compartmentalization of cells in eukaryotes presupposes the (GET) pathway (Schuldiner et al., 2008; Stefanovic and Hegde, development of mechanisms for protein trafficking between 2007). Recent studies have demonstrated the functional GET different membrane-enclosed organelles. Vesicular transport is components for TA protein targeting in Arabidopsis (Xing et al., the predominant pathway for protein trafficking in eukaryotic 2017) for the process whereby the SNARE SYP72 is inserted into the cells. Multiple molecular machineries are required for the ER membrane through the GET pathway (Srivistava et al., 2016). formation, transport, tethering and fusion of the vesicles to Once translocated into the ER, further targeting of membrane proteins the target compartment (Bonifacino and Glick, 2004). Soluble is determined by either specific signal motifs, the length of TMD or a N-ethylmaleimide-sensitive fusion protein attachment protein combination of both (Brandizzi et al., 2002; Hanton et al., 2006, receptors (SNAREs) have been identified as critical components 2005b; Matheson et al., 2006; Rojo and Denecke, 2008; Saint-Jore- involved in the final fusion step for the vesicular transport pathways Dupas et al., 2006). In the early secretory pathway of plants, proteins (Söllner et al., 1993). These facilitate vesicle–target-membrane that are exported from the ER and traffic to the Golgi are mediated by fusion by forming hetero-tetrameric trans-SNARE complexes coat protein complex II (COPII) machinery (Brandizzi and Barlowe, derived from a specific set of SNARE proteins (Jahn and 2013; DaSilva et al., 2004; Hawes et al., 2008; Moreau et al., 2007; Scheller, 2006; McNew et al., 2000). Robinson et al., 2015; Stefano et al., 2014). According to the model in yeast and mammals, the formation of COPII vesicles is initiated by the recruitment of the small GTPase SAR1 to the ER membrane. 1School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, Activated SAR1 then recruits the inner-coat dimeric complex New Territories, Hong Kong, China. 2Department of Biology, Hong Kong Baptist SEC23–SEC24 which captures protein cargos. Subsequent University, Hong Kong, China. 3The Chinese University of Hong Kong Shenzhen recruitment of the outer-coat complex SEC13–SEC31 stimulates Research Institute, Shenzhen 518057, China. the GTP hydrolysis of activated SAR1, and eventually leads to *Author for correspondence ([email protected]) the formation of COPII carriers containing protein cargos to be exported (Bassham et al., 2008; Chung et al., 2016; Hwang and K.P.C., 0000-0003-2786-3095; Y.Z., 0000-0002-9512-6487; C.J., 0000-0003- 4399-8272; L.J., 0000-0002-7829-1472 Robinson, 2009; Marti et al., 2010). Among the Golgi-localized SNARE proteins (Uemura et al., 2004), SYP31 and MEMB11 have Received 24 February 2017; Accepted 23 May 2017 been reported to have a critical role in mediating ER-to-Golgi Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202838. doi:10.1242/jcs.202838 anterograde transport (Bubeck et al., 2008; Chatre et al., 2005). Similarly, the fluorescence pattern of YFP–BET12 changed from Although the roles of SNARE proteins in the early secretory pathway punctate to aggregates upon BFA treatment (Fig. 1D). However, have been investigated, the mechanism for SNARE targeting to the unlike ST–YFP and VHAa1–GFP, the YFP–BET12 aggregate was Golgi membrane remains elusive. Until now, only a single study has not only found in the dense-core FM4-64 aggregate but also at its demonstrated that ER export and Golgi targeting of SYP31 depends periphery (Fig. 1D). Fluorescence intensity line plots along the on the di-acidic motif in its N-terminus (Chatre et al., 2009). YFP–BET12 aggregate showed a different distribution pattern from In the present study, we aimed to elucidate the targeting ST–YFP and VHAa1–GFP in that the YFP–BET12 signal intensity mechanism and the functional role of the Qc-SNARE BET12 remained high in both the periphery and the core region of the (also termed Bs14b) in the early secretory pathway. Previous studies FM4-64 peak (Fig. 1E), indicating that YFP–BET12 is being suggested that BET12 is involved in plant fertility and displayed a trapped in both the Golgi-derived and TGN-derived aggregates. Golgi localization in Arabidopsis protoplasts (Bolanos-Villegas To further confirm the localization of YFP–BET12 from the et al., 2015). BET11 (also termed Bs14a), shares a 78% amino acid CLSM analysis, we performed immunogold electron microscopy similarity to BET12, and was also suggested to localize on Golgi (EM) with anti-GFP antibodies (recognizing YFP) on ultrathin membranes (Uemura et al., 2004). Unlike SYP31 and MEMB11, sections prepared from high-pressure frozen and freeze-substituted BET11 overexpression did not severely affect protein ER-to-Golgi root cells of transgenic Arabidopsis seedlings expressing YFP– anterograde transport (Bubeck et al., 2008; Chatre et al., 2005). BET12. Consistent with our confocal findings, EM observations Strikingly, an in vitro study in yeast has suggested that BET11 and showed that gold particles (on secondary antibodies recognizing BET12 tend to form a distinct quaternary SNARE complex with anti-GFP) were present on both the Golgi stacks as well as the different yeast Golgi SNAREs, as BET11 had a SNARE-binding associated TGN (Fig. 1F). Quantification of immunogold labeling profile that resembled that of Sft1p, whereas the binding profile of indicated that ∼37% and ∼52% of the gold particles were associated BET12 resembled that of Bet1p (Tai and Banfield, 2001). In order with the Golgi (including on both the cis- and trans-side) and TGN, to characterize the role of BET12 in ER-to-Golgi protein trafficking, respectively (Fig. 1G). Taken together, CLSM and EM studies we first determined the subcellular localization of BET12 in demonstrated that YFP–BET12 localized to both the Golgi and transgenic plants and uncovered a signal motif-dependent targeting TGN in transgenic
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