Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling

Abstract

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca 2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca 2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca 2+ -dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca 2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca 2+ -dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.signaling | ion transport | membrane biology | abiotic stress M any of the plant-adaptive responses to environmental stresses occur at the cell membrane. In particular, those related to the regulation of plant ion transporters as stress unbalance cell ion homeostasis (1, 2). The phytohormone abcisic acid (ABA) and the second messenger Ca 2+ have central roles in regulating plant stress tolerance through the control of the activity of various families of protein kinases and phosphatases that regulate the activation of different ion channels or transporters (3-7). Given the presence of the channel substrates at the cell membranes and the transient nature of their activation, the functioning of these systems relies on the regulated localization of different molecular entities in the vicinity of the channels via protein-protein (8, 9) and/or protein-membrane interactions (10, 11).The pyrabactin resistance 1/PYR1-like (PYR/PYL)/regulatory components of ABA receptors (RCAR) receptors perceive intracellular ABA levels and, as a result, form ternary complexes with clade A protein phosphatases type 2C (PP2C), thereby inactivating them (12)(13)(14)(15). This prevents the PP2C-mediated dephosphorylation of ABA-activated sucrose nonfermenting 1-related protein kinases (SnRKs) subfamily 2 (SnRK2s), which results in the activation of an SnRK2-dependent phosphorylation cascade affecting a high number of targets in the plant cell (16,17). As a result, ABA-activated SnRK2s are key players in regulating...

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

“…Of the models tested against, the PfCERLI1 C2 domain was most structurally similar to the C2 domain of human itchy homolog E3 ubiquitin protein ligase (ITCH) but the ligand(s) of this protein have not been elucidated 36 . The protein modelled to the C2 domain of PfCERLI1 with highest confidence was that of C2-domain ABA-related protein 1 (CAR1) from Arabidopsis thaliana, which has been shown to bind phospholipids in a calcium ion dependent manner 37,38 . The PH domain of PfCERLI1 was most structurally similar to the PH domain of Protein kinase B/Akt, and like Akt, was predicted to bind to inositol 1,3,4,5-tetrakisphosphate (IP4) ( Supplementary Fig.…”

Advances and current challenges in calcium signaling

“…Recruitment of ABA receptors to the membrane has been found to be controlled by Ca 2+ via the function of CAR proteins (C2domain ABA related) a family of small proteins harboring a Ca 2+binding C2 domain (Rodriguez et al, 2014;Diaz et al, 2016). Also, CDPKs and CBL/CIPKs provide a well-established link between Ca 2+ signaling and ABA responses .…”

“…In this context, it is also increasingly appreciated that the formation and decoding of these Ca 2+ signals occur in defined regions of membranes often designated as nano-or microdomains. However, we are currently only beginning to understand this emerging aspect of Ca 2+ signaling in plants (Rizzuto & Pozzan, 2006;Demir et al, 2013;Steinhorst & Kudla, 2013;Diaz et al, 2016). Since the initial reports on stimulus-specific alterations of cytoplasmic [Ca 2+ ] in the 1990s, there have been not only a growing number of processes described that involve Ca 2+ signaling, but also a steadily increasing number of researchers interested in these phenomena (Fricker et al, 1991;Knight et al, 1991;Shacklock et al, 1992).…”

Control of rice pre‐harvest sprouting by glutaredoxin‐mediated abscisic acid signaling

“…To understand the molecular function of PHS9 in regulating rice PHS, we performed a yeast two‐hybrid (Y2H) screen using PHS9 as the bait. About 38 interacting proteins were obtained (Table S2), and a putative GTPase activating protein (http://rice.plantbiology.msu.edu) (named OsGAP) attracted most of our attention, as the OsGAP homologs in Arabidopsis , known as CAR proteins (C2‐domain abscisic acid‐related proteins), play an important role in ABA signal transduction (Rodriguez et al ., ; Diaz et al ., ; Podia et al ., ). Expression analysis demonstrated that OsGAP could be detected in seeds (Figure a), but was not induced by ABA (Figure S5).…”

“…In Arabidopsis , CAR proteins could interact with the PYR/PYL ABA receptors in an ABA‐independent manner and mediate their approach to the plasma membrane in a Ca 2+ ‐dependent manner. Overexpression of CAR1 leads to an enhanced ABA‐mediated inhibition of seedling establishment and shoot growth, whereas triple car mutants reduced the sensitivity to the ABA‐mediated inhibition of seedling establishment and root growth (Rodriguez et al ., ; Diaz et al ., ). These results suggest that CAR positively regulates ABA signaling in Arabidopsis .…”