S‐acylation of CBL10/SCaBP8 by PAT10 is crucial for its tonoplast association and function in salt tolerance

• DOI: 10.1111/jipb.12864
• Publication Date: 2019-08-23T08:49:44Z
• Authors: Sen Chai; Fu‐Rong Ge; Yan Zhang; Sha Li
• Keywords: Palmitoylation; Ankyrin repeat; Lipid-anchored protein; Ankyrin

Crop yield is sensitive to salt stresses, for which Calcineurin B-like proteins (CBLs) are major response factors. This study shows that Arabidopsis CBL10, through protein S-acylation by S-acyl transferase10, targets the vacuolar membrane confer tolerance. Protein a reversible posttranslational modification involves addition of 16-carbon saturated lipid group, usually palmitate cysteine residues substrate (Hemsley and Grierson 2008; Hemsley et al. 2013; Running 2014). plays important roles in targeting, interaction, stability In plants, most potentially S-acylated involved signaling 2013), implying crucial plant development environmental responses. A class transferases (PATs) have enzymatic domains containing DHHC-signature motif Plant genomes encode large number these DHHC-type PATs; however, only four PATs thaliana, TIP GROWTH DEFECTIVE1 (TIP1/PAT24), PAT10, PAT14, PAT4, been functionally characterized 2005; Qi Zhou Lai 2015; Zhao 2016; Wan 2017). No substrates biochemically verified (Running 2014), although cellular genetic evidence PAT10 mediates several calcineurin (CBL) (CBL2, CBL3, CBL6) (Zhou 2013). CBLs Ca2+ sensors play essential responses (Gong 2004; Yang Guo 2018). interact with CBL-interacting kinases (CIPKs) initiate CBL-CIPK module contributes spatial control information carried specific subcellular targeting Batistic 2010; Adams Shin 2014; Among 10 encoded Arabidopsis, CBL1, CBL4, CBL5, CBL9 associate plasma (PM) N-terminal myristylation S-acylation, whereas tonoplast association CBL2, CBL6 mediated (Batistic 2008). Consistent this being dependent on mutations CBL2 CBL3 result developmental defects similar those (Tang 2012b; However, pat10 mutants hypersensitive stresses 2013) cbl2;cbl3 double mutant not 2012b). difference raises question whether there other whose activity stress. We hypothesized another member CBL family, CBL10 (also referred as SCaBP8 [SOS3-like Ca2+-binding 8]), might be potential compromised function loss-of-function could account its hypersensitivity. CBL10/SCaBP8 critical tolerance (Kim 2007; Quan 2018), associates 2018) PM vesicles (Quan 2007). Furthermore, predicted sites using prediction software CSS-Palm (Ren To test hypothesis, we first compared sensitivity pat10-2, cbl10/scabp8, cbl2;cbl3, all reported null Tang 2012a; pat10-2 cbl10/scabp8 were NaCl, showed comparable MgCl2 hypersensitivity (Figure S1) 2015). These results suggested different contribute distinctly mutant. then analyzed localization confocal laser scanning fluorescence microscopy (CLSM). The GFP-translational fusion generated signals clear 1A, B). Because same transgene fully suppressed NaCl 2C, D), possibility GFP fusions interfered native pattern was excluded. By contrast, expressed or after treatment inhibitor 2-bromopalmitate (2-BP) mostly cytoplasmic Coexpressing GFP-CBL10 together fluorescent marker RFP-INOSITOL TRANSPORTER1 (INT1) (Wolfenstetter 2012) further demonstrated SCaBP8/CBL10 requires 1A). Finally, cell fractionation assays an anti-GFP antibody confirmed switch from cytoplasm both 2-BP 1C). suggest PAT10-mediated S-acylation. (A) Confocal (CLSM) leaf protoplasts plants transformed Pro35S:GFP-CBL10 Pro35S:GFP-CBL10;pat10-2, Pro35S:GFP-CBL10C38S, treated 50 µM 2 h. label tonoplast, RFP-INT1 transiently expressed. G, R, B represent GFP, RFP, transmission channels, respectively. Magenta green lines at rightmost panels show intensity RFP defined left (E, enlarged view). Arrowheads indicate membrane. V, vacuole. (B) CLSM root epidermal cells Pro35S:GFP-CBL10C38S (C) Cell antibody. Immunoblot anti-ACTIN performed demonstrate efficiency fractionation. CBB, Coomassie brilliant blue staining input samples. S, soluble fraction; P, pellet. Results shown representatives three biological replicates. For treatment, seedlings d germination incubated 12 h before extraction, DMSO used control. Bars = 5 µm (A); (B). C38 stress state assayed Acyl-RAC assay method. EX: experimental (S-acylation state); LC: loading control; Hyd+, hydroxylamine present (selectively cleaves groups); Hyd¯, absent (S-acyl groups cleaved). Proteins considered if signal observed Hyd+ lane Hyd¯ EX site SCaBP8/CBL10. transmembrane domain indicated black line, (C38) red. Relative fresh weight above genotypes. means ± standard errors (SE). Three replicates, each 30 seedlings, analyzed. Means letters significantly (One-way ANOVA, Tukey's multiple comparison test, P < 0.05). (D) Seedlings wild type (WT) scabp8/cbl10 empty vector (EV), Pro35S:CBL10, Pro35S:CBL10C38S (C38S) growing 1/6 Murashige Skoog (MS) medium MS supplemental 60 mM NaCl. Images taken challenge. Next, determine PAT10. Indeed, when Hyd (Hyd+), detected, indicating 2A). decreased background level (Hyd¯) depends One residue N-terminus, C38, 2008) proximal (TM) 2010), making it likely candidate 2B). C38S CBL10C38S, largely cytoplasmic, wild-type 1A–C). also mutation abolished support notion PAT10-dependent C38. explore and, thus function, expressing either (GFP-CBL10) point-mutated version (GFP-CBL10C38S). expression such complemented growth 2C–E). GFP-CBL10C38S completely rescue F, G), supporting key role function. That CBL10C38S enhanced partially under G) consistent fact severely affected upon challenge S1A, B), suggesting enacted locations than tonoplast. Taken together, our responsible S-acylation-dependent association. Our highlight conclusion mediate various distinct substrates. should guide future studies CBL-mediated well functional plants. thank Dr. Yan ABRC European Stock Center materials described article. work supported Natural Science Foundation Shandong Province (ZR2014CM027 S. L.), China Postdoctoral (2015M570605 National (31771558 S.L., 31271578, 31625003, 31871422 Y.Z.). Y.Z.'s laboratory Tai-Shan Scholar Program Provincial Government. conflict interest declared. Additional Supporting Information may found online tab article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.12864/suppinfo Please note: publisher content functionality any supplied authors. Any queries (other missing content) directed corresponding author