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Summary: The collective function of calcineurin B‐like (CBL) calcium ion (Ca2+) sensors and CBL‐interacting protein kinases (CIPKs) in decoding plasma‐membrane‐initiated Ca2+ signals to convey developmental and adaptive responses to fluctuating nitrate availability remained to be determined.Here, we generated a cbl‐quintuple mutant in Arabidopsis thaliana devoid of these Ca2+ sensors at the plasma membrane and performed comparative phenotyping, nitrate flux determination, phosphoproteome analyses, and studies of membrane domain protein distribution in response to low and high nitrate availability.We observed that CBL proteins exert multifaceted regulation of primary and lateral root growth and nitrate fluxes. Accordingly, we found that loss of plasma membrane Ca2+ sensor function simultaneously affected protein phosphorylation of numerous membrane proteins, including several nitrate transporters, proton pumps, and aquaporins, as well as their distribution within plasma membrane microdomains, and identified a specific phosphorylation and domain distribution pattern during distinct phases of low and high nitrate responses.Collectively, these analyses reveal a central and coordinative function of CBL–CIPK‐mediated signaling in conveying plant adaptation to fluctuating nitrate availability and identify a crucial role of Ca2+ signaling in regulating the composition and dynamics of plasma membrane microdomains. [ABSTRACT FROM AUTHOR]

P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1–2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism. [ABSTRACT FROM AUTHOR]

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth. • Transceptor NRT1.1 is critical for plasma membrane ATPase AHA2 activity under LN • Upon nitrate binding, NRT1.1 promotes coreceptor QSK1 phosphorylation • QSK1 physically links NRT1.1 and AHA2, specifically phosphorylating AHA2 on S899 • NRT1.1-QSK1 transduces LN sensing to AHA2, repressing proton efflux and LR growth Zhu and Krall et al. uncover a mechanism in which NRT1.1, in LN, increases the phosphorylation of QSK1. This forms a complex to transduce LN sensing to the PM H+-ATPase AHA2, regulating the phosphorylation ratio of AHA2 activation and inhibitory phosphorylation sites, resulting in altered apoplast acidification and regulation of LR growth. [ABSTRACT FROM AUTHOR]