Color discrimination requires the input of different photoreceptor cells that are sensitive to different wavelengths of light. TheDrosophilavisual system contains multiple classes of photoreceptor cells that differ in anatomical location, synaptic connections, and spectral sensitivity. The Rh5 and Rh6 opsins are expressed in nonoverlapping sets of R8 cells and are the onlyDrosophilavisual pigments that remain uncharacterized. In this study, we ectopically expressed Rh5 and Rh6 in the major class of photoreceptor cells (R1–R6) and show them to be biologically active in their new environment. The expression of either Rh5 or Rh6 in “blind”ninaE¹⁷mutant flies, which lack the gene encoding the visual pigment of the R1–R6 cells, fully rescues the light response. Electrophysiological analysis showed that the maximal spectral sensitivity of the R1–R6 cells is shifted to 437 or 508 nm when Rh5 or Rh6, respectively, is expressed in these cells. These spectral sensitivities are in excellent agreement with intracellular recordings of the R8p and R8y cells measured inCalliphoraandMusca. Spectrophotometric analyses of Rh5 and Rh6in vivoby microspectrophotometry, and of detergent-extracted pigmentsin vitro, showed that Rh5 is reversibly photoconverted to a stable metarhodopsin (λ_max= 494 nm), whereas Rh6 appears to be photoconverted to a metarhodopsin (λ_max= 468 nm) that is less thermally stable. Phylogenetically, Rh5 belongs to a group of short-wavelength-absorbing invertebrate visual pigments, whereas Rh6 is related to a group of long-wavelength-absorbing pigments and is the first member of this class to be functionally characterized.

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida . Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.

By screening retinal cDNA libraries for photoreceptor‐specifically expressed genes we have isolated and sequenced a cDNA clone encoding the rhodopsin (Rh6) of a subset of R8 photoreceptor cells of th...

In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase β-carotenoid-15,15′-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.

In an approach directed to isolate and characterize key proteins of the transduction cascade in photoreceptors using the phosphoinositide signaling pathway, we have isolated the Calliphora homolog of the Drosophila InaD gene product, which in Drosophila InaD mutants causes slow deactivation of the light response. By screening a retinal cDNA library with antibodies directed against photoreceptor membrane proteins, we have isolated a cDNA coding for an amino acid sequence of 665 residues (M = 73,349). The sequence displays 65.3% identity (77.3% similarity) with the Drosophila InaD gene product. Probing Western blots with monospecific antibodies directed against peptides comprising amino acids 272-542 (anti-InaD-(272-542)) or amino acids 643-655 (anti-InaD-(643-655)) of the InaD gene product revealed that the Calliphora InaD protein is specifically associated with the signal-transducing rhabdomeral photoreceptor membrane from which it can be extracted by high salt buffer containing 1.5 M NaCl. As five out of eight consensus sequences for protein kinase C phosphorylation reside within stretches of 10-16 amino acids that are identical in the Drosophila and Calliphora InaD protein, the InaD gene product is likely to be a target of protein kinase C. Phosphorylation studies with isolated rhabdomeral photoreceptor membranes followed by InaD immunoprecipitation revealed that the InaD protein is a phosphoprotein. In vitro phosphorylation is, at least to some extent, Ca-dependent and activated by phorbol 12-myristate 13-acetate. The inaC-encoded eye-specific form of a protein kinase C (eye-PKC) is co-precipitated by antibodies specific for the InaD protein from detergent extracts of rhabdomeral photoreceptor membranes, suggesting that the InaD protein and eye-PKC are interacting in these membranes. Co-precipitating with the InaD protein and eye-PKC are two other key components of the transduction pathway, namely the trp protein, which is proposed to form a Ca channel, and the norpA-encoded phospholipase C, the primary target enzyme of the transduction pathway. It is proposed that the rise of the intracellular Ca concentration upon visual excitation initiates the phosphorylation of the InaD protein by eye-PKC and thereby modulates its function in the control of the light response.