Robert Renthal

The major antennal chemosensory protein of red imported fire ant workers

Some chemosensory proteins (CSPs) are expressed in insect sensory appendages and are thought to be involved in chemical signalling by ants. We identified 14 unique CSP sequences in expressed sequence tag (EST) libraries of the red imported fire ant, Solenopsis invicta. One member of this group (Si‐CSP1) is highly expressed in worker antennae, suggesting an olfactory function. A shotgun proteomic analysis of antennal proteins confirmed the high level of Si‐CSP1 expression, and also showed expression of another CSP and two odorant‐binding proteins (OBPs). We cloned and expressed the coding sequence for Si‐CSP1. We used cyclodextrins as solubilizers to investigate ligand binding. Fire ant cuticular lipids strongly inhibited Si‐CSP1 binding to the fluorescent dye N‐phenyl‐naphthylamine, suggesting cuticular substances are ligands for Si‐CSP1. Analysis of the cuticular lipids showed that the endogenous ligands of Si‐CSP1 are not cuticular hydrocarbons.

Structure and distribution of antennal sensilla of the red imported fire ant.

The morphology of the antenna of the red imported fire ant, Solenopsis invicta, was examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. The antennae are sexually dimorphic: the worker antenna has porous sensilla on the two distal segments (the antennal club), whereas the clubless male antenna has porous sensilla on all segments past the pedicel. The major type of porous sensilla on both male and female is sensilla tricodea curvata. However, the male s. tricodea curvata are rather uniform in size, whereas the female s. tricodea curvata vary considerably in thickness. The number of sensilla on the distal segment of the worker antenna increases with segment length. This suggests a possible mechanism by which task assignments in S. invicta could be determined by the presence or absence of sensilla sensitive to specific task-related odor or pheromone cues. The sensilla basiconica have an invariant spatial pattern on worker and queen antennae.

Helix insertion into bilayers and the evolution of membrane proteins

Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed.

Influenza A (H3N2) Outbreak, Nepal

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain.

Reaction of the purple membrane with a carbodimide

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (delta h) and decay of photointermediate M) show changes indicative of slowed proton uptake. The delta h is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH2OH. However, the reaction is inhibited by millimolar concentrations of CaCl2. The altered delta h is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropylcarbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a diffent type of site and appears to inhibit the proton pump.

Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence

Purple membrane was covalently labeled with 5-(dimethylamino) naphthalene-1-sulfonyl hydrazine (dansyl hydrazine) by carbodiimide coupling to the cytoplasmic surface (carboxyl-terminal tail: 0.7 mol/mol bacteriorhodopsin) or by periodate oxidation and dimethylaminoborane reduction at the extracellular surface (glycolipids: 1 mol/mol). In 2 mM acetate buffer, pH 5.6, micromolar concentrations of UO2 +(2) were found to quench the dansyl groups on the cytoplasmic surface (maximum = 26%), while little quenching was observed at the extracellular surface (maximum = 4%). Uranyl ion quenched dansyl hydrazine in free solution at much higher concentrations. Uranyl also bound tightly to unmodified purple membrane, (apparent dissociation constant = 0.8 microM) as measured by a centrifugation assay. The maximum stoichiometry was 10 mol/mol of bacteriorhodopsin, which is close to the amount of phospholipid phosphorus in purple membrane. The results were analyzed on the assumptions that UO2 +(2) binds in a 1:1 complex with phospholipid phosphate and that the dansyl distribution and quenching mechanisms are the same at both surfaces. This indicates a 9:1 ratio of phosphate between the cytoplasmic and extracellular surfaces. Thus, the surface change density of the cytoplasmic side of the membrane is more negative than -0.010 charges/A2.

Control of bacteriorhodopsin color by chloride at low pH: Significance for the proton pump mechanism

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).

Proteomic Insights into the Protective Mechanisms of an In Vitro Oxidative Stress Model of Early Stage Parkinson’s Disease

Previous studies in Parkinsons disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease. We now report results from proteomic analysis of a rotenone-induced oxidative stress model of PD in the human neuroblastoma cell line, SH-SY5Y. Cells were exposed to sub-micromolar concentrations of rotenone for 48h prior to whole cell protein extraction and shotgun proteomic analysis. Evidence for activation of the UPR comes from our observation of up-regulated binding immunoglobulin protein (BiP), heat shock proteins, and foldases. We also observed up-regulation of proteins that contribute to the degradation of misfolded or unfolded proteins controlled by the UPS and ERAD pathways. Activation of the UPR may allow neurons to maintain protein homeostasis in the cytosol and ER despite an increase in reactive oxygen species due to oxidative stress, and activation of the UPS and ERAD may further augment clean-up and quality control in the cell.

Dansylation of bacteriorhodopsin near the retinal attachment site.

Abstract The purple membrane of Halobacterium halobium was reacted with 5-dimethylaminonaphthalene-l-sulfonyl chloride (dansyl chloride) at pH 8.0. Chromophoric and functional properties of the product appear unaltered. Approximately 2 moles of dansyl group were incorporated per mole of bacteriorhodopsin, part bound to bacteriorhodopsin and part bound to lipids. Purification and fragmentation of the protein showed most of the dansyl modification in a fragment containing residues 33 to 56. Amino acid analysis indicates that the major dansylated site is lysine 40. We conclude that, contrary to published models, 1) bacteriorhodopsin folds in a way that exposes lysine 40 at the membrane surface, and 2) this side chain is not involved in the proton pump mechanism.

Carbodiimides inhibit the acid-induced purple-to-blue transition of bacteriorhodopsin

Reaction of purple membrane with water soluble carbodiimides inhibits the spectral transition from purple to blue observed at acid pH. The pK and Hill constant for this transition are shifted from 3.4 to 2.6 and from 1.8 to 0.85, respectively. The results suggest a connection between the uptake side of the proton pump and the purple-to-blue transition.