Abstract
SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation sites, e.g., 116th and 119th threonines, were detected in ectopically expressed CPN20 in mammalian cells and in Arabidopsis. The O-fucosylation at both threonine residues was confirmed by in vitro peptide O-fucosylation assay. We further show that CPN20 accumulates in the chloroplast of spy mutants, suggesting that SPY negatively regulates CPN20 localization in the chloroplast. In vivo protein degradation assay along with CPN20 localization behavior suggest that import of CPN20 into the chloroplast is negatively regulated by SPY. Genetic analysis shows that ABA insensitive phenotypes of spy-3 in terms of seed germination and early seedling development are partially suppressed by the cpn20 mutation, suggesting that CPN20 acts downstream of SPY in this ABA signaling pathway and that there may exist other pathways in parallel with CPN20. Collectively, the above data support the notion that the O-fucosylation of CPN20 by SPY fine-tunes ABA signaling in Arabidopsis.
Highlights
Glycosylation is a highly elaborate protein post-translational modification that occurs in eukaryotes and procaryotes (Wacker et al, 2002; Young et al, 2002)
In order to identify substrates of SPY and to elucidate the mechanisms by which SPY takes part in developmental and hormone signaling processes, we carried out a yeast two-hybrid screen, using BD-SPY as bait to search for its interacting proteins on the premise that there was no auto-activation of BD-SPY and AD (Supplementary Figure 1)
SPY interacted with CPN20 through the Nterminal TPR domain (Figure 1A)
Summary
Glycosylation is a highly elaborate protein post-translational modification that occurs in eukaryotes and procaryotes (Wacker et al, 2002; Young et al, 2002). Other than the more studied N-linked and mucin-type O-linked modifications, O-linked monosaccharide glycosylations such as O-GlcNAc and O-fucosylation have been studied for the past 20 years (Hofsteenge et al, 2001; Okajima and Irvine, 2002; Panin et al, 2002; Luther and Haltiwanger, 2009; Lira-Navarrete et al, 2011; Bond and Hanover, 2015; Zentella et al, 2017). Ofucosylation occurs in the endoplasmic reticulum (ER), and proteins with EGF-like repeats (EGFs), such as Notch protein or proteins with Thrombospondin Repeats (TSRs), are Ofucose modified (Hofsteenge et al, 2001; Okajima and Irvine, 2002; Luther and Haltiwanger, 2009; Lira-Navarrete et al, 2011; Chen et al, 2012). How O-fucosylation functions in planta are less well-known
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