SUMMARYAcidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H + -ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2 and PSY1R observed might provide a general paradigm for regulation of plasma membrane proton transport by receptor kinases.

Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. The biosynthetic route to the nucleotide dUMP, the precursor of dTTP, as shown in Fig. 1, differs between organisms and sets it apart from the rest of nucleotide metabolism (1). This is in agreement with the general belief that uracil was the original DNA base-pairing partner for adenine and that the biosynthesis of dTTP from dUMP arose later in evolution (2). The deoxyribonucleotides dGTP, dATP, and dCTP are all produced in a pathway that goes through the multisubstrate enzyme ribonucleotide reductase. This enzyme is highly regulated in order to maintain the balance of deoxyribonucleotide pools (3-5). dUTP is also formed by ribonucleotide reductase, but because of the toxicity of this nucleotide (6, 7), all organisms express an enzyme with dUTPase activity that hydrolyzes dUTP to dUMP (8).The direct formation of dUMP via ribonucleotide reductase (Fig. 1) is not an efficient process, as the ribonucleotide reductase has a reduced affinity for UDP compared to the affinities of three other canonical ribonucleotides (9-12). Instead, most of the dUMP arises from deamination of cytosine deoxyribonucleotides (13-15). Enteric bacteria synthesize an enzyme with dCTP deaminase activity that converts dCTP into dUTP (EC 3.5.4.13), which accounts for 70 to 80% of the dUMP production (15). The dCTP deaminase is a homotrimeric enzyme structurally related to the trimeric dUTPase and part of the dCTP deaminase/dUTPase superfamily (16-18). The genomes of many Gram-positive bacteria and all eukaryotes encode a dCMP deaminase (EC 3.5.4.12) that shares no resemblance to the enteric dCTP de...

PSY1R is a leucine-rich repeat (LRR) receptor-like kinase (RLK) previously shown to act as receptor for the plant peptide hormone PSY1 (peptide containing sulfated tyrosine 1) and to regulate cell expansion. PSY1R phosphorylates and thereby regulates the activity of plasma membrane-localized H+-ATPases. While this mechanism has been studied in detail, little is known about how PSY1R itself is activated. Here we studied the activation mechanism of PSY1R. We show that full-length PSY1R interacts with members of the SERK co-receptor family in planta. We identified seven in vitro autophosphorylation sites on serine and threonine residues within the kinase domain of PSY1R using mass spectrometry. We furthermore show that PSY1R autophosphorylation occurs in trans and that the initial transphosphorylation takes place within the activation loop at residues Ser951, Thr959, and Thr963. While Thr959 and Thr963 are conserved among other related plant LRR RLKs, Ser951 is unique to PSY1R. Based on homology modeling we propose that phosphorylation of Ser951 stabilize the inactive conformation of PSY1R.

Model compounds of the green fluorescent protein (GFP) phenolate chromophore are synthesized and investigated for their intrinsic optical properties by state-of-the-art gas-phase action spectroscopy.