SummaryBacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelASpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins (~25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.

Arabidopsis thaliana calcineurin B-like protein (AtCBL2)is a member of a recently identified family of calcineurin B-like calcium-binding proteins in A. thaliana. The crystal structure of AtCBL2 has been determined at 2.1 Å resolution. The protein forms a compact ␣-helical structure with two pairs of EF-hand motifs. The structure is similar in overall folding topology to the structures of calcineurin B and neuronal calcium sensor 1, but differs significantly in local conformation. The two calcium ions are coordinated in the first and fourth EF-hand motifs, whereas the second and third EF-hand motifs are maintained in the open form by internal hydrogen bonding without coordination of calcium ions. Both a possible site and a possible mechanism for the target binding to AtCBL2 are discussed based on the three-dimensional structure.

The roles of three membrane proteins, BOR1, DUR3, and FPS1, in boron (B) transport in yeast were examined. The boron concentration in yeast cells lacking BOR1 was elevated upon exposure to 90 mM boric acid, whereas cells lacking DUR3 or FPS1 showed lower boron concentrations. Compared with control cells, cells overexpressing BOR1 or FPS1 had a lower boron concentration, and cells overexpressing DUR3 had a higher boron concentration. These results suggest that, in addition to the efflux boron transporter BOR1, DUR3 and FPS1 play important roles in regulating the cellular boron concentration. Analysis of the yeast transformants for tolerance to a high boric acid concentration revealed an apparent negative correlation between the protoplasmic boron concentration and the degree of tolerance to a high external boron concentration. Thus, BOR1, DUR3, and FPS1 appear to be involved in tolerance to boric acid and the maintenance of the protoplasmic boron concentration.

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions.

The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5'-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH(2) terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca(2+) and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca(2+)-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca(2+) and (p)ppGpp signaling in regulation of the genetic system of these organelles.

We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.

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Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies.