We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtgammaCAL1 and AtgammaCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtgammaCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtgammaCA and AtgammaCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtgammaCAs. All plant species analyzed contain both AtgammaCA and AtgammaCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtgammaCAL sequences show a deviation of functionally important active site residues with respect to gammaCAs but could form active interfaces in the interaction with AtgammaCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.

SUMMARYThe myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol that protects cells from osmotic stress. Using proteomics, the enzymes that constitute the MIB pathway, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1), are identified in tilapia (Oreochromis mossambicus) gill epithelium. Targeted, quantitative, label-free proteomics reveals that they are both upregulated during salinity stress. Upregulation is stronger when fish are exposed to severe (34 ppt acute and 90 ppt gradual) relative to moderate (70 ppt gradual) salinity stress. IMPA1 always responds more strongly than MIPS, suggesting that MIPS is more stable during salinity stress. MIPS is N-terminally acetylated and the corresponding peptide increases proportionally to MIPS protein, while non-acetylated N-terminal peptide is not detectable, indicating that MIPS acetylation is constitutive and may serve to stabilize the protein. Hyperosmotic induction of MIPS and IMPA1 is confirmed using western blot and real-time qPCR and is much higher at the mRNA than at the protein level. Two distinct MIPS mRNA variants are expressed in the gill, but one is more strongly regulated by salinity than the other. A single MIPS gene is encoded in the tilapia genome whereas the zebrafish genome lacks MIPS entirely. The genome of euryhaline tilapia contains four IMPA genes, two of which are expressed, but only one is salinity regulated in gill epithelium. The genome of stenohaline zebrafish contains a single IMPA gene. We conclude that the MIB pathway represents a major salinity stress coping mechanism that is regulated at multiple levels in euryhaline fish but absent in stenohaline zebrafish.Supplementary material available online at http://jeb.biologists.org/lookup/suppl

Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.

SUMMARYThe NADH-ubiquinone oxidoreductase complex (complex I) (EC 1.6.5.3) is the main entrance site of electrons into the respiratory chain. In a variety of eukaryotic organisms, except animals and fungi (Opisthokonta), it contains an extra domain comprising trimers of putative c-carbonic anhydrases, named the CA domain, which has been proposed to be essential for assembly of complex I. However, its physiological role in plants is not fully understood. Here, we report that Arabidopsis mutants defective in two CA subunits show an altered photorespiratory phenotype. Mutants grown in ambient air show growth retardation compared to wild-type plants, a feature that is reversed by cultivating plants in a high-CO 2 atmosphere. Moreover, under photorespiratory conditions, carbon assimilation is diminished and glycine accumulates, suggesting an imbalance with respect to photorespiration. Additionally, transcript levels of specific CA subunits are reduced in plants grown under non-photorespiratory conditions. Taken together, these results suggest that the CA domain of plant complex I contributes to sustaining efficient photosynthesis under ambient (photorespiratory) conditions.

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.

MINT‐7266036: gamma CA2 (uniprotkb:Q9C6B3) and gamma CA2 (uniprotkb:Q9C6B3) physically interact (MI:0914) by molecular sieving (MI:0071)

Plant mitochondria include gamma-type carbonic anhydrases (gammaCAs) of unknown function. In Arabidopsis, the gammaCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2-3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H(2)O(2) dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility.

The increasing use of manufactured nanoparticles (NP) in different applications has triggered the need to understand their putative ecotoxicological effects in the environment. Copper oxide nanoparticles (CuO NP) are toxic, and induce oxidative stress and other pathophysiological conditions. The unique properties of NP can change depending on the characteristics of the media they are suspended in, altering the impact on their toxicity to aquatic organisms in different environments. Here, Mozambique tilapia (O. mossambicus) were exposed to flame synthesized CuO NP (0.5 and 5 mg·L−1) in two environmental contexts: (a) constant freshwater (FW) and (b) stepwise increase in environmental salinity (SW). Sublethal effects of CuO NP were monitored and used to dermine exposure endpoints. Fish exposed to 5 mg·L−1 CuO in SW showed an opercular ventilation rate increase, whereas fish exposed to 5 mg·L−1 in FW showed a milder response. Different effects of CuO NP on antioxidant enzyme activities, accumulation of transcripts for metal-responsive genes, GSH∶GSSG ratio, and Cu content in fish gill and liver also demonstrate that additive osmotic stress modulates CuO NP toxicity. We conclude that the toxicity of CuO NP depends on the particular environmental context and that salinity is an important factor for modulating NP toxicity in fish.