No abstract

Many strains of lactic acid bacteria (LAB) and bifidobacteria have exhibited strain-specific capacity to produce γ-aminobutyric acid (GABA) via their glutamic acid decarboxylase (GAD) system, which is one of amino acid-dependent acid resistance (AR) systems in bacteria. However, the linkage between bacterial AR and GABA production capacity has not been well established. Meanwhile, limited evidence has been provided to the global diversity of GABA-producing LAB and bifidobacteria, and their mechanisms of efficient GABA synthesis. In this study, genomic survey identified common distribution of gad operon-encoded GAD system in Lactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems in Lb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights that Lb. brevis applies GAD system as the main strategy against extracellular and intracellular acidification demonstrating its high capacity of GABA production. In addition, the abundant GadA retained its activity toward near-neutral pH (pH 5.5–6.5) of cytosolic acidity thus contributing to efficient GABA synthesis in Lb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis in Lb. brevis.

Published under the PNAS license.Data deposition: The crystallography, atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 6JIM).

The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated.

Tail-anchored (TA) proteins possess an N-terminal functional domain and a single transmembrane domain (TMD) at the C-terminus followed by a hydrophilic tail.1 Newly synthesized TA proteins are released from free ribosomes with the C-terminal hydrophobic region inserted into various membranes, such as the endoplasmic reticulum, chloroplast outer envelope, mitochondrial outer membrane and the peroxisomal membrane. 2The functional domain of TA proteins orients to the cytosol and the TMD of TA proteins is inserted into membranes posttranslationally. Sorting of proteins by the C-terminal tail (CT) to their specific intracellular destinations is essential for their functions.3 For instance, overexpression of a C-terminal TMDtruncated AtPAP2 in Arabidopsis abolishes its faster plant growth phenotype. 4 Over 500 proteins in Arabidopsis have been predicted to have TA structures, of which, 130 have had their subcellular localization experimentally confirmed based on either GFP targeting or mass spectrometry.2 Most TA proteins were assigned to the ER and secretory membranes, 27 proteins to mitochondria and 32 proteins to plastids.2,5 These include several isoforms of Tom20 and Tom22 (also known as Tom9) of the mitochondrial outer membrane translocon 6,7 and the GTPase receptors of the outer membrane translocon of plastids, including AtToc33, AtToc34. 5,8Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only plant TA protein shown to be dual-targeted to chloroplasts and mitochondria. 4 It was predicted to carry a putative N-terminal signal peptide, a phosphatase domain and a transmembrane domain (TMD) followed by a short hydrophilic C-terminal tail (CT) (a.a. 614-636) by the TMHMM analysis.4 AtPAP2 was to date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (om) of chloroplasts and mitochondria and exposed its n-terminal enzymatic domain to the cytosol. it was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria Keywords: purple acid phosphatase, mitochondria, plastid, dual-targeting, outer membrane detected in the membrane fraction using immunoblotting. 4 An in vivo targeting assay using chimeric GFP vectors showed that the C-terminal TMD motif of AtPAP2, but not the predicted N-terminal signal peptide, can direct GFP to both plastids and mitochondria in Arabidopsis PSB-D protoplasts. 4 In transgenic Arabi...

Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin–Benson–Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

Most high γ-aminobutyric acid (GABA) producers are Lactobacillus brevis of plant origin, which may be not able to ferment milk well due to its poor proteolytic nature as evidenced by the absence of genes encoding extracellular proteinases in its genome. In the present study, two glutamic acid decarboxylase (GAD) genes, gadA and gadB, were found in high GABA-producing L. brevis NPS-QW-145. Co-culturing of this organism with conventional dairy starters was carried out to manufacture GABA-rich fermented milk. It was observed that all the selected strains of Streptococcus thermophilus, but not Lactobacillus delbrueckii subsp. bulgaricus, improved the viability of L. brevis NPS-QW-145 in milk. Only certain strains of S. thermophilus improved the gadA mRNA level in L. brevis NPS-QW-145, thus enhanced GABA biosynthesis by the latter. These results suggest that certain S. thermophilus strains are highly recommended to co-culture with high GABA producer for manufacturing GABA-rich fermented milk.