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Plant stomata sense CO2 via reversible interaction of the Raf-like HT1 protein kinase with non-activity requiring MAP kinase. 

Abstract Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction–modification and CRISPR–Cas systems 1 . In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors 2–4 . However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation. 

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr,Microbiol. Rev.57, 522–542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al.,Chem. Biol.17, 776–785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein–protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart,ChemBioChem16, 528–547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein–protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

 

Increases in CO₂concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO₂] rise cause closing of stomatal pores, thus affecting plant–water relations globally. However, the underlying CO₂/bicarbonate (CO₂/HCO₃⁻) sensing mechanisms remain unknown. [CO₂] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO₂/HCO₃⁻regulation of SLAC1 is relevant for CO₂control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO₂/HCO₃⁻inXenopusoocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO₃⁻regulation of SLAC1. Notably, gas-exchange experiments withslac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO₂regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO₂/HCO₃⁻, but not by ABA, was impaired, indicating the relevance of R256 for CO₂signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO₂/HCO₃⁻sensors in guard cells.