Reference #: WAT-1018-341289
Submit Date: 04/09/2002 01:37:50-0500

Photoactivated adenylyl cyclase (PAC): a novel blue-light receptor flavoenzyme mediating Euglena photomovement

ABSTRACT:
Euglena gracilis, a unicellular flagellate, shows abrupt changes in its swimming direction in response to a sudden increase or decrease in incident light fluence-rate (i. e. step-up or step-down photophobic responses), resulting in photoavoidance or photoaccumullation, respectively. On the basis of obvious similarity of the UVB/UVA/blue-light -type action spectra for photophobic responses to the absorption spectra of flavins, the latter molecule have been candidates for the chromophores of the photoreceptor molecules mediateing the former responses. The Euglena cell has a paraflagellar body (PFB), a small ellipsoidal structure near the base of its flagellum, which is considered as a photosensing organelle for its photomovements (photophobic responses and phototaxis). The PFB exhibits a bright green autofluorescence, consistent with the hypothesis that flavoproteins localized in the PFB might act as the photoreceptor molecules. To examine this hypothesis, we isolated PFBs and purified the flavoproteins from them. The chromatographically purified flavoprotein (about 400 kDa), which noncovalently bound FAD, seemed to be a heterotetramer of - and -subunits ( _{2 2} ). Predicted amino acid sequences of each of the subunits were similar to each other and contained two FAD-binding domains each followed by an adenylyl cyclase catalytic domain. The purified flavoprotein showed an adenylyl cyclase activity, which was elevated near-hundred-fold by blue-light irradiation. Thus, the flavoprotein can directly transduce a light signal into a change in the intracellular cyclic AMP level without any other signal transduction proteins. Based on this unique feature of the flavoprotein, we named it Photoactivated Adenylyl Cyclase (PAC). Suppression of gene expression of PAC by RNA-mediated interference (RNAi) caused dissapearance of both PFB and the step-up photophobic response, demondtrating that PAC actually mediates photoavoidance of Euglena . Presence of PAC in several behavioral mutant strains will also be reported. Reference: Iseki, M. et al. (2002) Nature 415: 1047-1051

Keywords: Photoactivated adenylyl cyclase (PAC), blue-light receptor flavoenzyme , photomovement, Euglena gracilis

Reference #: LOS-1016-535949
Submit Date: 03/19/2002 04:46:53-0500

Photophysics, photochemistry and structural modeling of phototropin-related prokaryotic proteins: a new family of bacterial blue-light receptors

ABSTRACT:
A bacterial protein, YtvA from Bacillus subtilis, containing a photoactive LOV-domain with strong similarity to that of higher plant phototropins, has been identified by sequence homology search and by computer based modeling. Heterologous expression and purification demonstrated the suggested properties (1). The full length YtvA protein exhibits the same spectroscopic and photochemical properties as phototropin-LOV domains. YtvA contains flavin mononucleotide (FMN) as a chromophore and upon light excitation a covalent thiol adduct with a conserved cysteine is formed. This photoproduct decays back to the parent state within two hours in the dark. Time-resolved absorption and optoacoustic experiments demonstrate that some differences with LOV domains do exist, due to the presence of an additional domain that highly stabilize the photoproduct. In plants, phototropins are responsible for blue-light induced phototropism and chloroplasts relocation. The role of proteins having phototropin-like photochemistry in bacteria is presently unknown, but their presence appears to be widely spread, both in non photosynthetic organisms and in cyanobacteria (2). These phototropin-related proteins may represent a novel type of bacterial blue-light photoreceptors and theri role is presently being investigated. 1.Losi, A., E. Polverini, B. Quest, and W. Gaertner (2002) Biophys. J. in press. 2.Losi, A. in preparation

Keywords: phototropin-related , YtvA, optoacoustics

Reference #: 035444
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Reference #: WAD-1016-622496
Submit Date: 03/20/2002 02:48:20-0500

Phototropin function in fern and Arabidopsis

ABSTRACT:
Phototropin is a blue light receptor found in seed plants, fern, moss and even in Chlamydomonas. Phototropin contains two LOV domains at the N-terminus and a serin-threonin kinase domain at C-terminus. FMN in a LOV domain is a chromophore to absorb blue light. Arabidopsis and fern Adiantum have two phototropins, phot1 and phot2. phot1 was first identified as a blue light receptor for phototropic response under low light intensity by the analyses of Arabidopsis mutant defective in tropic response. Then, phot2 was found as a photoreceptor of chloroplast avoidance response under strong light again by mutant analysis. Experiments using Arabidopsis double mutant of phot1 and phot2 revealed that phot1 and phot2 were photoreceptors working redundantly in phototropic response under strong light, chloroplast photo-accumulation movement under weak blue light and in stomatal opening in leaves. Phot2 of Adiantum was also found to be a photoreceptor for chloropalst avoidance response by mutant analysis and its rescue experiment. Adiantum has a chimeric photoreceptor called phytochrome3 (phy3) that has a phytochrome sequence at N-terminus and a phototropin sequence at C-terminus. Analytical study of rap ( red light aphototropic) mutants revealed that phy3 was the photoreceptor of red light-induced tropic response as well as chloroplast movement in Adiantum. It is not yet known whether phy3 is also a blue light receptor, at the moment.

Keywords: Arabidopsis , fern, phototropin, blue light receptor

Reference #: BAT-1017-306350
Submit Date: 03/28/2002 01:47:52-0500

Cryptochromes - not cryptic any longer

ABSTRACT:
Cryptochromes (CRYs) are blue-light photoreceptors, discovered first in plants and later also in animals and human [1]. They are related to the DNA-repair enzyme photolyase, have the same cofactors as photolyase but no photolyase activity [2,3]. In plants, CRYs serve to regulate several important growth and developmental processes in blue- and UV-light, including inhibition of hypocotyl growth, anthocyanin formation and induction of genes involved in this pathway, and flowering time [4,5]. In the model plant species Arabidopsis thaliana, two CRYs (CRY1, CRY2) were identified and studied in detail, although neither their structure nor their photochemistry is known yet. However, several partners of CRY have been identified already which will help to elucidate the signal transduction chain of CRYs. Here we describe a novel protein (CIF1), which is localized in the cytosol, and whose interaction with CRY2 is blue-light regulated. The function of this protein in CRY2 signaling will be discussed in detail. CRY2 shows instability in blue light under certain photoperiodic conditions, a feature that is likely to be very important for CRY2 function. We have further investigated the molecular processes involved in the blue-light mediated degradation of CRY2 and will present recent results from these studies. 1. Sancar A. (2000) Ann. Rev. Biochem. 69:31-67. 2. Malhotra K. et al. (1995) Biochemistry 34 : 6892-6899. 3. Lin C. et al. (1995) Science 269 : 968-970. 4. Cashmore et al. (1999) Science 284 : 760-765. 5. Christie J.M. et al. (2001) J. Biol. Chem. 276:11457-11460.

Keywords: cryptochrome, blue-light photoreceptor, light-signaling