Requirement of Calcium Binding, Myristoylation, and Protein-Protein Interaction for the Copine BON1 Function in Arabidopsis

Abstract

Copines are highly conserved proteins with lipid-binding activities found in animals, plants, and protists. They contain two calcium-dependent phospholipid binding C2 domains at the amino terminus and a VWA domain at the carboxyl terminus. The biological roles of most copines are not understood and the biochemical properties required for their functions are largely unknown. The Arabidopsis copine gene BON1/CPN1 is a negative regulator of cell death and defense responses. Here we probed the potential biochemical activities of BON1 through mutagenic studies. We found that mutations of aspartates in the C2 domains did not alter plasma membrane localization but compromised BON1 activity. Mutation at putative myristoylation residue glycine 2 altered plasma membrane localization of BON1 and rendered BON1 inactive. Mass spectrometry analysis of BON1 further suggests that the N-peptide of BON1 is modified. Furthermore, mutations that affect the interaction between BON1 and its functional partner BAP1 abolished BON1 function. This analysis reveals an unanticipated regulation of copine protein localization and function by calcium and lipid modification and suggests an important role in proteinprotein interaction for the VWA domain of copines.

Opposing effects on two phases of defense responses from concerted actions of HSC70 and BON1 in Arabidopsis

“…The regulation of SNC1 by BON1 may mimic the regulation of RESISTANCE TO P. SYRINGAE PV MACULICOLA1 and RPS2 by RPM1 INTERACTING PROTEIN4 (RIN4), a classical example of guard hypothesis (Chisholm et al, 2006;Jones and Dangl, 2006). Coincidently, both RIN4 and BON1 are plasma membrane-localized proteins (Hua et al, 2001;Kim et al, 2005;Takemoto and Jones, 2005;Li et al, 2010a), and RIN4 is also involved in stomatal closure (Liu et al, 2009). It remains to be tested if BON1 is targeted by potential effectors for modification or degradation.…”

“…7), and therefore BON1 may not directly regulate the activity of PAMP receptors. As BON1 is a calcium-binding protein and calcium-binding activity is critically essential for its function (Hua et al, 2001;Li et al, 2010a), it could be involved in calcium signal perception and, in turn, modulates signaling pathways or channels/transporters. Similarly, HSC70 and SGT1b were thought to function not in early signal perception but in later signaling and execution events (Clément et al, 2011).…”

“…The BON1 cDNA was cloned from the entry vector into the pDEST-GBKT7 to generate a BD-BON1 construct, while HSC70 genes and SGT1b or truncated SGT1b were cloned into pDEST-GADT7 to generate AD-HSC70 or AD-SGT1b constructs, respectively. The yeast two-hybrid assay was performed as previously described (Li et al, 2010a).…”

“…The BON1 protein is localized to the plasma membrane through N-terminal myristoylation; and its calcium-binding property, though not responsible for its localization, is essential for its function (Li et al, 2010a). How BON1 at the plasma membrane regulates the transcript level of the NB-LRR-coding gene SNC1 is not fully understood.…”

“…It encodes a calcium-dependent phospholipidbinding copine protein that is conserved in protozoa, plants, nematodes, and mammals (Hua et al, 2001). Copine proteins in diverse species studied so far are involved in signaling, although no common processes can be readily deduced (Li et al, 2010a). In Arabidopsis, the loss-of-function (LOF) mutant bon1-1 (hereafter, bon1) has a dwarf phenotype due to constitutive defense responses triggered by the up-regulation of the NB-LRR gene SUPPRESSOR OF NPR1-1, CONSTITUTIVE1 (SNC1; Hua et al, 2001;Yang and Hua, 2004).…”