Targeting of Drosophila Rhodopsin Requires Helix 8 but Not the Distal C-Terminus

BACKGROUND: The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs. CONCLUSIONS/SIGNIFICANCE: Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres

Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels.

BACKGROUND: Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. PRINCIPAL FINDINGS: We describe the homologous and heterologous expression of 10 different targets from the three major MP classes--G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2-0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter--both involved in synaptic transmission--showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. SIGNIFICANCE: We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies

Low molecular weight oligo-ß-hydroxybutyric acids and a monomeric amide thereof – new products from micro-organisms, Polymer Bull

A new group of low-molecular weight channel-forming oligo(hydroxybutyric acids) (cPHBs, 1 with n = 8-30; main component MW ≈ 1300 dalton) was isolated from microorganisms of different origin. Inclusion bodies were electron-microscopically visible in cells in the state of autolysis, not in cells in the exponential phase of growth. cPHB and high-molecular poly(ß-hydroxybutyric acid) (sPHB) is cleaved by phenylethylamine and forms the corresponding monomeric hydroxybutyramide and -under drastic conditions, the crotylamide. One of these compounds, the 3-hydroxy-N-phenethyl-butyramide (5), was isolated as a new natural product now

Pseudoanguillosporin A and B: two New Isochromans Isolated from the Endophytic Fungus Pseudoanguillospora sp.

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Enhanced Biogeochemical Cycling and Subsequent Reduction of Hydraulic Conductivity Associated with Soil-Layer Interfaces in the Vadose Zone

Biogeochemical dynamics in the vadose zone are poorly understood due to the transient nature of chemical and hydrologic conditions, but are nonetheless critical to understanding chemical fate and transport. This study explored the effects of a soil layer on linked geochemical, hydrological, and microbiological processes. Three laboratory soil columns were constructed: a homogenized medium-grained sand, a homogenized organic-rich loam, and a sand-over-loam layered column. Upward and downward infiltration of water was evaluated during experiments to simulate rising water table and rainfall events respectively. In-situ collocated probes measured soil water content, matric potential, and Eh while water samples collected from the same locations were analyzed for Br(−), Cl(−), NO(3)(−), SO(4)(2−), NH(4)(+), Fe(2+), and total sulfide. Compared to homogenous columns, the presence of a soil layer altered the biogeochemistry and water flow of the system considerably. Enhanced biogeochemical cycling was observed in the layered column over the texturally homogeneous soil columns. Enumerations of iron and sulfate reducing bacteria showed 1-2 orders of magnitude greater community numbers in the layered column. Mineral and soil aggregate composites were most abundant near the soil-layer interface; the presence of which, likely contributed to an observed order-of-magnitude decrease in hydraulic conductivity. These findings show that quantifying coupled hydrologic-biogeochemical processes occurring at small-scale soil interfaces is critical to accurately describing and predicting chemical changes at the larger system scale. Findings also provide justification for considering soil layering in contaminant fate and transport models because of its potential to increase biodegradation and/or slow the rate of transport of contaminants