SUMMARY Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.

Natural rubber (NR) is stored in latex as rubber particles (RPs), rubber molecules surrounded by a lipid monolayer. Rubber transferase (RTase), the enzyme responsible for NR biosynthesis, is believed to be a member of the cis-prenyltransferase (cPT) family. However, none of the recombinant cPTs have shown RTase activity independently. We show that HRT1, a cPT from Heveabrasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed HeveaRPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactucasativa also exhibited RTase activity, indicating proper introduction of cPT on RP is the key to reconstitute active RTase. RP proteomics and interaction network analyses revealed the formation of the protein complex consisting of HRT1, rubber elongation factor (REF) and HRT1-REF BRIDGING PROTEIN. The RTase activity enhancement observed for the complex assembled on WRPs indicates the HRT1-containing complex functions as the NR biosynthetic machinery.DOI: http://dx.doi.org/10.7554/eLife.19022.001

A novel gene, IRE1, of Saccharomyces cerevisiae was cloned through genetic complementation of a myoinositol auxotrophic mutant. The predicted amino acid sequence indicated that IRE1 encodes a protein of 126983 Da with two highly hydrophobic regions, probably a signal sequence and a membrane-spanning region. The carboxy-terminal region of IRE1 showed close sequence similarity to the catalytic domains of protein kinases. Disruption of the IRE1 locus caused myo-inositol auxotrophy. The IRE1 product is very likely a protein kinase required for myo-inositol synthesis.

Protein palmitoylation represents an important mechanism governing the dynamic subcellular localization of many signaling proteins. Palmitoylation of endothelial nitric-oxide synthase (eNOS) promotes its targeting to plasmalemmal caveolae; agonist-promoted depalmitoylation leads to eNOS translocation. Depalmitoylation and translocation of eNOS modulate the agonist response, but the pathways that regulate eNOS palmitoylation and depalmitoylation are poorly understood. We now show that the newly characterized acylprotein thioesterase 1 (APT1) regulates eNOS depalmitoylation. Immunoblot analyses indicate that APT1 is expressed in bovine aortic endothelial cells, which express eNOS. APT1 overexpression appears to accelerate the depalmitoylation of eNOS in COS-7 cells cotransfected with eNOS and APT1 cDNAs. (2). N-Myristoylation involves cleavage of the N-terminal Met residue and attachment of the 14-carbon unsaturated fatty acid myristic acid to Gly 2 via an acyl-amide bond. Myristoylation is a cotranslational, usually irreversible process that is catalyzed by a well characterized N-myristoyltransferase (for review, see Ref.3). Palmitoylation represents a distinct type of acylation in which the 16-carbon unsaturated fatty acid palmitic acid is post-translationally attached to the thiol group of a specific Cys residue via an acyl-thioester bond (for review, see Ref. 4). In contrast to the stable acyl-amide bond in N-myristoylation, the chemical lability of the thioester bond allows the existence of regulated cycles of palmitoylation and depalmitoylation that may control a protein's subcellular localization.One critical function of protein palmitoylation appears to be tethering otherwise soluble proteins to the plasmalemmal membrane. Indeed, although N-myristoylated and prenylated signaling proteins are found in the cytoplasm as well as on cellular membranes, palmitoylation of signaling proteins confines them to cellular membranes (for review, see Ref. 4). Moreover, for several signaling proteins (including Src-like tyrosine kinases (5), the heterotrimeric G protein ␣ subunit G␣ i1 (6), and eNOS (7)), palmitoylation targets the protein to specific signal transduction microdomains in the plasmalemmal membrane termed caveolae. Cycles of protein palmitoylation and depalmitoylation may control a protein's distribution between membrane and cytoplasm and/or between subdomains of the plasma membrane (e.g. caveolae versus non-caveolae) and may modulate the coupling of specific signaling proteins to either receptors or intracellular effectors. To date, four examples of agonist-regulated protein depalmitoylation have been reported: the ␤ 2 -adrenergic receptor (8), the m 2 muscarinic acetylcholine receptor (9), the heterotrimeric G protein ␣ subunit G␣ s (10), and eNOS (11). However, the mechanisms by which activation-dependent changes in these proteins' palmitoylation state are achieved remain elusive.Numerous studies have attempted to identify protein palmitoyltransferases that palmitoylate proteins and protein palmitoylthio...

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2′,4,4′,6′-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.