Armin Huber

Light-regulated subcellular translocation of Drosophila TRPL channels induces long-term adaptation and modifies the light-induced current

Drosophila phototransduction results in the opening of two classes of cation channels, composed of the channel subunits transient receptor potential (TRP), TRP-like (TRPL), and TRPgamma. Here, we report that one of these subunits, TRPL, is translocated back and forth between the signaling membrane and an intracellular compartment by a light-regulated mechanism. A high level of rhabdomeral TRPL, characteristic of dark-raised flies, is functionally manifested in the properties of the light-induced current. These flies are more sensitive than flies with no or reduced TRPL level to dim background lights, and they respond to a wider range of light intensities, which fit them to function better in darkness or dim background illumination. Thus, TRPL translocation represents a novel mechanism to fine tune visual responses.

Phosphorylation of the InaD Gene Product, a Photoreceptor Membrane Protein Required for Recovery of Visual Excitation

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.

The TRP Ca2+ channel assembled in a signaling complex by the PDZ domain protein INAD is phosphorylated through the interaction with protein kinase C (ePKC)

Photoreceptors which use a phospholipase C‐mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cβ (PLCβ), the light‐activated Ca2+ channel TRP, and an eye‐specific protein kinase C (ePKC) are clustered by the PDZ domain protein INAD. Here we investigated the function of ePKC by cloning the Calliphora homolog of Drosophila ePKC, by precipitating the TRP signaling complex with anti‐ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids (MW=78 036) and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti‐ePKC antibodies led to the co‐precipitation of PLCβ, TRP, INAD and ePKC but not of rhodopsin. Phorbolester‐ and Ca2+‐dependent protein phosphorylation revealed that, apart from the PDZ domain protein INAD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC‐mediated phosphorylation of TRP is part of a negative feedback loop which regulates Ca2+ influx through the TRP channel.

Molecular cloning of Drosophila Rh6 rhodopsin: the visual pigment of a subset of R8 photoreceptor cells 1

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 the Drosophila compound eye. Compared to the other visual pigments of Drosophila, this rhodopsin is equally homologous to Rh1 and Rh2 (51% amino acid identity) but shows only 32% and 33% amino acid identity with Rh3 and Rh4, respectively. The open reading frame codes for a protein of 369 amino acids (MW=41 691). The primary structure of Rh6 displays sites typical for rhodopsin molecules in general, for example, a chromophore binding site in transmembrane domain VII, sequence motifs in the intracellular loops 2 and 3 required for the binding of a heterotrimeric G‐protein, and a glycosylation site near the N‐terminus which seems to be important for protein transport and maturation. Since R8 cells are founder cells in the developing compound eye, the isolation of a rhodopsin gene expressed in these cells may aid the understanding of terminal differentiation of photoreceptor cells.

Scaffolding proteins organize multimolecular protein complexes for sensory signal transduction

Scaffolding proteins composed of protein–protein interaction domains have emerged as organizers of multiprotein complexes in diverse cellular compartments, including neuronal synapses, cell–cell junctions of epithelial cells, and the stimulus perceiving structures of sensory neurons. This review focuses on the INAD‐assembled signalling complex of Drosophila photoreceptors, which organizes key components of the phototransduction cascade into a multiprotein signal transduction unit. The structure, the physiological consequences, and the assembly and targeting of the members of the INAD signalling complex will be described. In addition, the existence of signalling complexes in vertebrate photoreceptors, olfactory neurons and mechanosensitive hair cells will be discussed.

Subcellular translocation of the eGFP-tagged TRPL channel in Drosophila photoreceptors requires activation of the phototransduction cascade

Signal-mediated translocation of transient receptor potential (TRP) channels is a novel mechanism to fine tune a variety of signaling pathways including neuronal path finding and Drosophila photoreception. In Drosophila phototransduction the cation channels TRP and TRP-like (TRPL) are the targets of a prototypical G protein-coupled signaling pathway. We have recently found that the TRPL channel translocates between the rhabdomere and the cell body in a light-dependent manner. This translocation modifies the ion channel composition of the signaling membrane and induces long-term adaptation. However, the molecular mechanism underlying TRPL translocation remains unclear. Here we report that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, whereas TRPL-eGFP translocation could be directly visualized in intact eyes. TRPL-eGFP failed to translocate to the cell body in flies carrying severe mutations in essential phototransduction proteins, including rhodopsin, Gαq, phospholipase Cβ and the TRP ion channel, or in proteins required for TRP function. Our data, furthermore, show that the activation of a small fraction of rhodopsin and of residual amounts of the Gq protein is sufficient to trigger TRPL-eGFP internalization. In addition, we found that endocytosis of TRPL-eGFP occurs independently of dynamin, whereas a mutation of the unconventional myosin III, NINAC, hinders complete translocation of TRPL-eGFP to the cell body. Altogether, this study revealed that activation of the phototransduction cascade is mandatory for TRPL internalization, suggesting a critical role for the light induced conductance increase and the ensuing Ca2+-influx in the translocation process. The critical role of Ca2+ influx was directly demonstrated when the light-induced TRPL-eGFP translocation was blocked by removing extracellular Ca2+.

Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors

Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptors signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin–membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

The Visual G Protein of Fly Photoreceptors Interacts with the PDZ Domain Assembled INAD Signaling Complex via Direct Binding of Activated Gαq to Phospholipase Cβ

Visual transduction in the compound eye of flies is a well-established model system for the study of G protein-coupled transduction pathways. Pivotal components of this signaling pathway, including the principal light-activated Ca2+ channel transient receptor potential, an eye-specific protein kinase C, and thenorpA-encoded phospholipase Cβ, are assembled into a supramolecular signaling complex by the modular PDZ domain protein INAD. We have used immunoprecipitation assays to study the interaction of the heterotrimeric visual G protein with this INAD signaling complex. Light-activated Gαq- guanosine 5′-O-(thiotriphosphate) andAlF 4 − -activated Gαq, but not Gβγ, form a stable complex with the INAD signaling complex. This interaction requires the presence ofnorpA-encoded phospholipase Cβ, indicating that phospholipase Cβ is the target of activated Gαq. Our data establish that the INAD signaling complex is a light-activated target of the phototransduction pathway, with Gαq forming a molecular on-off switch that shuttles the visual signal from activated rhodopsin to INAD-linked phospholipase Cβ.

Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin

UNLABELLED Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440. BIOLOGICAL SIGNIFICANCE The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a proteome wide scale. The high proteome coverage of our proteomics survey allowed us to analyze the regulation of whole protein networks instead of single proteins. We were able to reconstruct the complete degradation pathway of vanillin and to monitor the changes in the energy metabolism of KT2440 induced by vanillin as sole carbon source. Vanillin dehydrogenase (Vdh) was not mandatory for vanillin degradation in KT2440 and may be substituted by other aldehyde dehydrogenases that were up-regulated in a wild-type as well as in a Vdh-deficient strain in the presence of vanillin. Aldehyde dehydrogenases, vanillin specific porins and efflux pump systems identified in study will be interesting targets for optimization of vanillin production in Pseudomonas bacteria. Furthermore, several mechanisms of solvent tolerance were induced by vanillin in KT2440. These include increased abundance of several efflux pump systems, chaperones as well as enzymes involved in cyclopropane fatty acid synthesis and trehalose formation. The present work will deepen the understanding of metabolism of aromatic compounds in P. putida and may lead to a more comprehensive understanding of solvent tolerance mechanisms in Gram-negative bacteria in general. Moreover, it will serve as a basis for further strain developments for a biotechnological production of vanillin in P. putida KT2440 or other Pseudomonas strains, highlighting the role of proteomics surveys as a powerful screening technology.

NinaB Is Essential for Drosophila Vision but Induces Retinal Degeneration in Opsin-deficient Photoreceptors

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.