Handbook of Photosensory Receptors (BRIGGS:PHOTORECEPTORS O-BK) || Discovery and Characterization of Photoactivated Adenylyl Cyclase (PAC), a Novel Blue-Light Receptor Flavoprotein,

22Discovery and Characterization of Photoactivated AdenylylCyclase (PAC), a Novel Blue-Light Receptor Flavoprotein,from Euglena gracilis

Masakatsu Watanabe and Mineo Iseki

22.1Introduction

Photoactivated adenylyl cylase (PAC), a novel blue-light receptor f lavoprotein with anintrinsic effector function, was first isolated from the photosensory organelle(paraf lagellar body, PFB) of Euglena gracilis, a unicellular eukaryotic photosyntheticunif lagellate alga. This f lavoprotein was originally shown to mediate one Euglena be-havioral response namely photoavoidance (the step-up photophobic response) butnot a second behavioral response, photoaccumulation (the step-down photophobicresponse) (Iseki et al., 2002). Here we summarize (1) the story of discovery and char-acterization of this unique photoreceptor molecule and (2) its additional characteri-zation made thereafter, as well as (3) its future research prospects. All of the above re-search findings are the result of combined multidisciplinary efforts by investigatorswith independent minds, different capabilities, and mutual respect.

22.2Action Spectroscopy

In the late 1980s, Sugai and Watanabe began a collaboration to extend the reportedUVA/blue action spectra of Euglena photomovements (Checcucci et al., 1976) to theUV-B/C region of the spectrum in order to examine the possible f lavoprotein natureof the putative UV-A/blue light receptor(s) in this organism. Individual-cell-levelmethods for assay of photomovement had been developed by the joint efforts ofWatanabe, Aono, Kondo, Takahashi and Kubota (Kondo et al., 1988; Matsunaga et al.,1998). At the beginning of the study we made the embarrassing finding that replace-ment of an organic culture medium by a simple inorganic medium (Diehn’s restingmedium) (Diehn, 1969) before the photomovement assay caused the Euglena cells to

Handbook of Photosensory Receptors. Edited by W. R. Briggs, J. L. SpudichCopyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN 3-527-31019-3

448

respond only to step-up signals in a photophobic response. Matsunaga solved thisproblem by addition of small amount of Hyponex, a commercial plant fertilizer,which caused the cells to respond only to step-down signals in a photophobic re-sponse. Subsequent careful examination revealed that ammonium ion is the respon-sible factor that converts the Euglena cells from responding photophobically only to astep-up light signal to cells responding photophobically only to a step-down light sig-nal (Figure 22.1; Matsunaga et al., 1999). On the basis of this methodological findingit became possible to determine reliable action spectra for both of these photophobicresponses at the Okazaki Large Spectrograph (Figure 22.2; Matsunaga et al., 1998;Watanabe, 2004).

In the UV-A and visible regions of the spectrum, the shapes of the action spectrawere of the so-called UV-A/blue type, originally named the cryptochrome type byGressel (1979). In the newly studied UV-B/C region, an action peak was found at270 nm for the step-down response and at 280 nm for the step-up response. The ab-sorption spectrum of f lavin adenine dinucleotite (FAD) appeared to fit the action

22 Discovery and Characterization of Photoactivated Adenylyl Cyclase (PAC)

Figure 22.1 Fluence rate-response curves forstep-up and step-down photophobic respons-es at 440 nm (Matsunaga et al. 1999). Step-up(Su, open circle) and step-down (Sd, closedcircle) photophobic responses were measuredfor (a) the cells exponentially grown in Koren-Hutner’s medium for 3 days, or, (b) the cells

transferred on the 5th day to Diehn’s restingmedium with addition of 1 mM NH4Cl andkept for 2 d until measurement of the re-sponses. The technique for measuring theseresponses is described elsewhere (Matsunagaet al., 1998).

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spectrum for the step-up response (Figure 22.2a), whereas the shape of the step-downaction spectrum, which has a UV-A peak (at 370 nm) that is higher than the blue peak(at 450 nm), appeared to be mimicked by the absorption spectrum of a mixed solu-tion of 6-biopterin and FAD (Figure 22.2b). These observations could also account forthe fact that the UV-B/C peak wavelength at 270 nm of the action spectrum for thestep-down response is shorter by 10 nm than the homologous peak in the action spec-trum for the step-up response at 280 nm.

22.3PAC Discovery and its Identi>cation as the Blue-light Receptor for Photoavoidance

In searching for protocols to isolate the PFB, we compared two methods: (1) detach-ment of the f lagellum together with PFB, by low temperature, high Ca2+, or UVAtreatment (e. g. Brodhun and Häder, 1990, Schmidt et al., 1990; Lebert, 2001) and (2)non-selective cell disruption by French press, Parr press, or sonication as suggested

22.3 PAC Discovery and its Identif ication as the Blue-light Receptor for Photoavoidance

Figure 22.2 Action spectra for step-down andstep-up photophobic responses in Euglenaand absorption spectra of aqueous solutionsof FAD, 6-biopterin, and their mixture (Mat-sunaga et al. 1998). (a) Action spectrum forthe step-up photophobic response (closed cir-cle), (b) action spectrum for the step-downphotophobic response (closed triangle). Ab-

sorption spectra of aqueous solutions (in50 mM potassium phosphate buffer, pH 8.0)of 1 × 10–5 M FAD [dotted line in (a) and (b)],of (2 × 10–5 M) 6-biopterin [broken line in (a)],mixed aqueous solution of FAD and 6-biop-terin with molar ratio of 1:2 [solid line in (a)]are shown.

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by Akio Murakami (personal communication). The cell disruption method workedwell in liberating the PFB from the Euglena cells with a very short fragment of the f la-gellum attached, whereas the former seemed to retain the PFB within the cell bodyas indicated from examination by f luorescence microscopy (Matsunaga, pers.comm.). This simple but effective technique provided us with a sound basis for fur-ther steps of analysis.

The isolated PFBs (Figure 22.3a) were lysed by sonication in an appropriate bufferand a f lavoprotein of about 400 kDa was purified chromatographically as follows:The crude extract (CE) was subjected to anion-exchange chromatography (AE) fol-lowed by gel filtration (GF). The f luorescence of the 400-kDa f lavoprotein was faintbut showed significant peaks at 370 nm and 450 nm in the excitation spectrum, con-sistent with the action spectra for photophobic responses of Euglena (Matsunaga et al.1998). The f luorescence became greater after boiling, indicating that the protein non-covalently binds f lavins (Figure 22.3b). The f luorescence intensity showed an obvi-ous pH dependency that is characteristic of f lavin adenine dinucleotide (FAD), sug-gesting that the chromophore of the protein is most probably FAD (Figure 22.3c).

From the results of SDS-polyacrylamide gel electrophoresis, the 400-kDa f lavopro-tein appeared to be a heterotetramer composed of two 105-kDa- and two 90-kDa sub-units (Figure 22.3d). We determined N-terminal amino acid sequences of the sub-units and amplified cDNAs encoding the subunits by PCR. Nucleotide sequences ofthe cDNAs were determined by direct sequencing of the PCR products and extendedby 5’- and 3’ rapid amplification of cDNA ends (RACE) methods. The deduced aminoacid sequences of the 105-kDa and 90-kDa polypeptides indicated that they are com-posed of 1019 and 859 amino acids, respectively, and are homologous over the regionwhere they overlap: the sequence identity over the 800 amino acids of the N-terminalportion is 75% (Figure 22.4a, b). In both sequences we found four characteristic re-gions: a pair of homologous regions (F1 and F2) that are separated by one (C1) of theother homologous pairs (C1 and C2) (Figure 22.4a). F1 and F2 show similarity to thef lavin-binding domain of AppA (activation of photopigment and puc expression) inRhodobacter sphaeroides (Gomelsky and Kaplan, 1998; Masuda and Bauer, 2002) andseveral hypothetical proteins found in genome sequences of other prokaryotes(Gomelsky and Klug, 2002) (see Chapter 21, Masuda and Bauer). The apparent molarratio of FAD to the 400-kDa protein of Euglena, estimated from f lavin f luorescenceand Bradford assay of the protein, varied from 2.5:1 (FAD:protein) to 7:1 in severalexperiments. Taking into account the sequence similarity and the limited accuracy ofthis estimation, we tentatively conclude that the 105-kDa and 90-kDa subunits eachbind two FAD molecules per polypeptide, at F1 and F2.

The other characteristic regions, C1 and C2, quite unexpectedly showed similaritywith class III adenylyl cyclase (AC) catalytic domains, especially with bacterial ones.Although the sequence identity over the full length of the AC catalytic domains is notso high, four consensus amino acid sequences in class III ACs (Danchin, 1993) arewell conserved in C1 and C2 (Figure 22.4c). To examine whether the 400-kDa proteinis actually an adenylyl cyclase, we determined the AC activity after each of its consec-utive purification steps. Whereas the AC activity in each sample was low but signifi-cant in darkness, it was elevated up to 80-fold under blue light (Figure 22.5). The blue-


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light-activated AC activity of the 400-kDa f lavoprotein was saturated at 50 µM ATP,with a Vmax of 3500 pmol min–1 mg–1, and an estimated Km value of 0.5 µM for thesubstrate ATP, values comparable to those of cyanobacterial AC (Kasahara et al.,1997). From these results, we concluded that the f lavoprotein isolated from PFBs isan FAD-binding adenylyl cyclase with its activity regulated by blue light. We thus des-


Figure 22.3 Purification of the f lavoproteinfrom isolated paraflagellar bodies (Iseki et al.,2002) (a) Fluorescence micrograph of isolatedPFBs (green f luorescent particles) observedunder excitation by blue-violet light. Scale bar,10 µm. (b) Fluorescence excitation (left,530 nm emission) and emission (right,370 nm excitation) spectra of the boiled andundenatured 400-kDa fractions (25 µg ml–1

protein). (c) Fluorescence excitation (left,530 nm emission) and emission (right,

370 nm excitation) spectra of the boiled 400-kDa fraction at different pHs (pH 2.9, adjust-ed with citrate buffer; pH 7.3, the same pH asthe gel-filtration buffer at room temperature.)(d) SDS-PAGE of PFB proteins at consecutivepurification steps: CE, crude extract of PFB;AEX, the 530-nm fluorescence peak fraction ofanion exchange chromatography; GF, the 400-kDa fraction from gel filtration. A 5–20% acry-lamide gradient gel, stained by silver is shownin (d).

452 22 Discovery and Characterization of Photoactivated Adenylyl Cyclase (PAC)

105_F1 53 TQLRRLMYLSASTEPEKCNAEYLADMAHVATLRNKQIGVSGFLLYSSPFFFQVIEGTDEDLDFLFAKISADPRHERCIVLANGPCTGRMYG-EWHMKDSH 151

90_F1 54 TNLRRLMYLSKSTNPEECNPQFLAEMARVATIRNREIGVSGFLMYSSPFFFQVIEGTDEDLDFLFAKISADPRHERCIVLANGPCTGRMYG-DWHMKDSH 152

105_F2 465 GQLITLTYISQAAHPMSR--LDLASIQRIAFARNESSNITGSLLYVSGLFVQTLEGPKGAVVSLYLKIRQDKRHKDVVAVFMAPIDERVYGSPLDMTSAT 562

90_F2 469 TTLTTLTYISQATRPMSR--LDLSAIMRTATRRNAQQSITGTLLHVNGLFVQTLEGPKDAVVNLYLRIRQDPRHTDVTTVHMAPLQERVYPSEWTLTSAT 566

AppA 14 SDLVSCCYRSLAAPDLTL--RDLLDIVETSQAHNARAQLTGALFYSQGVFFQWLEGRPAAVAEVMTHIQRDRRHSNVEILAEEPIAKRRFA-GWHMQLSC 110

F403 1 MLTTLIYRSHIRDDEPV--KKIEEMVSIANRRNMQSDVTGILLFNGSHFFQLLEGPEEQVKMIYRAICQDPRHYNIVELLCDYAPARRFG-KAGMELFD 96

Slr1694 1 MSLYRLIYSSQGIPNLQP--QDLKDILESSQRNNPANGITGLLCYSKPAFLQVLEGECEQVNETYHRIVQDERHHSPQIIECMPIRRRNFE-VWSMQAIT 97

N N

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105_C1 205 VTFIYLVEF... 249 AKFITGICM... 297 SRCGVHYGRALLC... 321 LGDCINTASRI...

90_C1 206 VTFIYLVEF... 250 AKFITGICM... 298 SQAGVHYGRAMLC... 322 LGDCINTTSRI...

105_C2 616 MLATDICSF... 659 IKLIGDCVT... 708 CGVGLDFGQVIMA... 733 AGEVSARVMEV...

90_C2 620 MLATDICSF... 663 IKLIGDCVT... 712 CGVGLDYGQVVMA... 737 AGAVSARVMEV...

A.cyli 321 ILFCDIRGY... 364 DKYIGDAIM... 415 VGIGIHRGTVVMG... 440 IGDAVNVASRI...

7120_B1 602 VLFSDIRGY... 645 DKFIGDALM... 698 IGIGISSGEVVSG... 723 IGDGVNLSPRL...

T.pall 394 IFFSDVRSF... 437 DKFIGDAIM... 492 IGCGVNTGSCVAG... 517 IGDAVNTASRI...

Stig_A 231 LLFADIRDF... 274 DKFIGDALM... 326 IGVGVHTGPAVLG... 352 IGDTVNLASRI...

R.meli 1 -MFTDIYDF... 43 IQFHGDSVF... 95 TRFGIHTGTAVVG... 120 MGDTVNVASRL...

Bov_C1a 306 ILFADIVGF... 349 IKILGDCYY... 397 MRVGLHTGRVLCG... 421 WSNDVTLANVM...

Bov_C2a 872 VMFASIPNF... 922 IKTIGSTYM... 978 LRVGINVGPVVAG... 1002 WGNTVNVASRM...

453

ignated the f lavoprotein as photoactivated adenylyl cyclase (PAC) and its subunits,105-kDa and 90-kDa polypeptide, as PACα and PACβ, respectively (Iseki et al., 2002).

To examine whether PAC actually mediates the photophobic responses of Euglena,we suppressed expression of PACα and PACβ by RNA interference (RNAi) (Iseki etal., 2002): double stranded RNAs (dsRNAs) of PAC subunits were synthesized andelectroporated into Euglena cells. As a consequence, endogenous PAC mRNAs wereundetectable and PFBs were absent from the transformed cells (Figure 22.6a). Ob-servation by a computerized video motion analyzer (Matsunaga et al. 1998) showedthat the step-up photophobic response disappeared in the dsRNA-transformed cellseven at a high enough intensity of light sufficient to cause saturation of the responsein the control cells. However, the step-down response of the dsRNA-transformed cellswas essentially the same as that of the control cells (Figure 22.6b). Thus the intro-duction of the dsRNAs impaired only the photosensing system for the step-up pho-tophobic response without affecting the overall machinery that is needed for the cells


Figure 22.4 Sequences of the Mr 105K and90K polypeptides (Iseki et al., 2002). (a) Struc-tural features of the two polypeptides. Fourcharacteristic regions are shown as blue (F1,F2) and red (C1, C2) boxes. The DDBJ/EMBL/GenBank accession numbers for the Mr 105Kand 90K polypeptides are AB031225 andAB031226, respectively. (b) Sequence align-ment of F1 and F2 with the AppA f lavin (FAD)binding domain and its homologues. Regionsof similarity (blue) and identity (green) arehighlighted. Alignment and similarity identifi-cation were performed by the GCG software.AppA, a photoreceptor in Rhodobactersphaeroides that serves as a redox regulator(L42555); F403, E. coli hypothetical protein

(AE000215); Slr1694, Synechocystis sp.PCC6803 hypothetical protein (D90913).(c) Partial sequence alignment of C1 and C2with class III adenylate cyclase catalytic do-mains. Amino acids consistent with the fourconsensus sequences of class III adenylylcyclases (yellow) and those identical with im-portant amino acids for catalysis in mam-malian adenylyl cyclase (red) are highlighted.A. cyli, Anabaena cylindrica (D55650);7120_B1, Anabaena sp. PCC7120 CyaB1(D89623); T. pall, Treponema pallidum(AE001224); Stig_A, Stigmatella aurantiacaCyaA (AJ223796); R. meli, Rhizobium meliloti(M35096); Bov_C1a and Bov_C2a, bovineType I (M25579).

CE AEX GF Control0

1,000

2,000

3,000

4,000

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tivity

(pm

o l/m

in/m

g)

Figure 22.5 Adenylyl cyclase activity of theflavoprotein purified from PFBs (Iseki et al.,2002). Adenylyl cyclase activity of proteins atconsecutive purification steps: CE, crude ex-tract of PFB; AEX, the 530-nm fluorescencepeak fraction of anion exchange chromatogra-

phy; GF, the Mr 400 Kd fraction of gel filtra-tion; Control, gel-filtration buffer as a control.Means and standard error of the mean fromthree measurements in darkness (black) orunder blue light (gray) at 10 µmol m–2 s–1 areshown.

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454

to exhibit phobic responses. From these results, we conclude that PAC is the majorconstituent of the PFB and that it acts as the photoreceptor for the step-up photo-phobic response in Euglena, whereas it is not involved in the step-down photophobicresponse.


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Figure 22.6 Suppression of gene expressionof PAC by RNAi. (a) Photomicrographs of an-terior portion of Euglena cells electroporatedwith buffer only (I, III) or dsRNAs of PACαand PACβ (II, IV). I, II: Nomarski optics; III,IV: Fluorescence images, under excitation byblue-violet light, superimposed with brightfield images of the same cell. S, stigma; F, f la-

gellum; P, PFB. Scale bar, 1 µm. (b) Fluencerate-response curves for the step-up photo-phobic response of the dsRNAs-introducedcells (circles) and control cells (squares) at450 nm. Fluence rate-response curves for thestep-down photophobic response are alsoshown (inset). Means and standard deviationfrom three measurements are shown.

455

Considering the fact that Ca2+ affects f lagellar movements of Euglena (Doughty andDiehn, 1979), the process of step-up photophobic response might be explained sim-ply as follows (Figure 22.7): blue light activates PAC to induce a local increase in the


Figure 22.7 Working hypothesis for the mechanism of the Euglenastep-up photophobic response mediated by PAC in PFB.

456

cAMP concentration around the PFB. The elevated cAMP level induces Ca2+ entry in-to the f lagellar apparatus and the Ca2+ increase somehow modifies the direction off lagellar beating. Another possibility is that the elevated cAMP level causes phos-phorylation of f lagellar proteins by activating a cAMP-dependent protein kinase(PKA), which affects the f lagellar motility as reported in other unicellular organisms(Hasegawa et al., 1987, Noguchi et al., 2000). Further work is needed to elucidate theexact mechanism.

22.4PAC Involvement in Phototaxis

The PAC discovery stimulated a close collaboration between our laboratory and thatof Donat Häder. This collaboration yielded discovery of the first PAC homologues(AlPAC alpha and beta) from Astasia, a close colorless relative of Euglena. It alsoshowed by RNAi that PAC mediates the Euglena phototaxis response, an oriented,steering, behavioral response toward or away from the light source (Figure 22.8; Nte-fidou et al., 2003).


Figure 22.8 Tracks of Euglena cells at high ir-radiances summarized in circular histograms(Ntefidou et al. 2003). Control cells swim with

high precision away from the light source(800 W m–2) whereas RNAi-treated cells showrandom swimming.

457

22.5PAC Origin

To gain an insight into the evolution of this unique photoreceptor protein, wesearched for similar sequences in several euglenoids by RT-PCR using degenerateprimers (Koumura et al., 2004). Two similar transcripts were thus detected in each ofthe four phototrophic euglenoids, Euglena stellata, Colacium sideropus, Eutreptiaviridis, Eutreptiella gymnastica, and in an osmotrophic species (i. e., obtaining nutri-ents by absorption), Khawkinea quartana, but not in a phagotrophic species (i. e. ob-taining nutrients by phagocytosis), Petalomonas cantuscygni. Each of the PAC-like se-quences detected appeared orthologous to PACα and PACβ, respectively, and had thesame domain structure as PAC subunits each of which is composed of two f lavinbinding domains, F1 and F2, each followed by an adenylyl cyclase catalytic domain,C1 and C2, respectively. These observations imply that the the encoded proteins con-stitute a functional photoactivated adenylyl cyclase similar to the Euglena PAC. Phy-logenetic analysis of the adenylyl cyclase catalytic domains revealed that they belongto a bacterial cluster, not to a trypanosomal one (Figure 22.9). In addition, two try-panosome-type adenylyl cyclases were discovered in E. gracilis. In contrast to PAC, de-duced amino acid sequences of the trypanosome-type adenylyl cyclases indicated thatthey are integral membrane proteins with a membrane-spanning region at theirmidpoint, followed by an adenylyl cyclase catalytic domain which seems cytoplasmic.Thus, we propose that PAC might have been transferred to euglenoids on the occa-sion of secondary endosymbiosis (Figure 22.10).

22.6Future Prospects

The molecular identity of the putative photoreceptor for the step-down photophobicresponse still presents a challenging open question. This photoreceptor may be a sen-sor for a light-off signal whereas PAC is a sensor for a light-on signal. The elucidationof the mechanisms allowing both sensors to bring about the abrupt changes in f la-gellar motion resulting in the turn in the direction of cell movement will also requiremuch interdisciplinary research, and will also provide a sound basis for biotechno-logical applications of these complimentary light sensors (see below).

PAC is a unique protein that can act both as a photoreceptor and an effector to cat-alyze cAMP synthesis in contrast with G-protein-coupled receptor systems in whichthree different proteins sequentially act to modulate the cyclic nucleotide level (Torreet al., 1995). This simple mechanism indicates that PAC may be a promising tool tophoto-manipulate the intracellular cAMP level in various heterologous cell systemsfor cell biological and biotechnological purposes. In nerve and other cells, this wouldenable us to pinpoint control of cellular processes regulated by cAMP such as axonguidance, synaptic long term potentiation, synaptic long term depression, and celldifferentiation. It would be even more fascinating if we succeeded in mutating a PACinto a PGC (photoactivated guanylyl cyclase, which produces cGMP), and replacing

22.6 Future Prospects

458

the f lavin chromophore with analogs that have different absorption peaks. Thiswould enable us to use these combinations as wavelength-sensitive switches of bio-logical activities (Lewis, 2002).

The above idea is actually encouraged by a recent success in functional heterolo-gous expression of PAC subunits in the Xenopus oocyte, demonstrating that either ofthem alone is sufficient for photoactivated adenylyl cyclase activity (Schröder-Lang etal., 2004).

It is extremely interesting and surprising that recently an archaerhodopsin-typeprotein, Cop5, containing either an adenylyl cyclase or a guanylyl cyclase domain(AC/GC) was found in the Chlamydomonas genome (Kateriya et al., 2004). This dis-covery suggests further possibilities for finding examples of adenylyl cyclase- orguanylyl cyclase-containing photoreceptor proteins, whether membrane-bound (likeCop5), cytosolic, or paracrystaline (like PAC in PFB).


0.1 substituions/site

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PACα C1AlPACα C1

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Sa cyaB

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Bacteria

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100

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Figure 22.9 Phylogenetic tree for catalytic do-mains of class III adenylyl cyclases (Koumuraet al., 2004). The tree was generated by theNeighbor-Joining method using Clustal X.

Bootstrap values for the trypanosome-cladeand the bacterial/PAC-clade are shown as per-centage of 1000 replications.

459

Acknowledgements

We acknowledge grant supports from Ministry of Education, Science, Cultue, andSports of Japan and Japan Space Forum. M. I. Acknowledges postdoctoral fellow-ships from National Institute for Basic Biology (NIBB) and Bio-oriented TechnologyResearch Advancement Institution during part of the studies summarized here. Partof the studies was done under the NIBB Cooperative Research Program to use theOkazaki Large Spectrograph.

Acknowledgements

Common ancestry

Phagotrophic euglenoids

Phototrophic euglenoids

Osmotrophic euglenoids(secondarily derived)

Trypanosomes

Euglena gracilisEuglena stellataColacium sideropusEutoreptia viridisEutreptiella gymnastica

Petalomonas cantuscygni

Astasia longaKhawkinea quartana

Acquisition of

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Figure 22.10 Working hypothesis on the ori-gin of PAC (Koumura et al., 2004). Presence oforthologues of PAC subunits (‘PAC’ in anoval) and trypanosome-type adenylyl cyclases

(‘TAC’ in a rectangle) are indicated on thewell-accepted evolutionary history of eugle-noids.

460

References


Brodhun, B., and Häder, D.-P. (1990) Pho-tochem. Photobiol. 52, 865–871.

Checcucci, A., Colombetti, G., Ferrara, R., andLenci, F. (1976) Photochem. Photobiol. 23, 51–54.

Danchin, A. (1993) Adv. Second Message Phos-phoprotein Res. 27, 109–162.

Diehn, B. (1969) Exp. Cell Res. 56, 375–381.Doughty, M.J., and Diehn, B. (1979) Biochem.

Biophys. Acta 588, 148–168.Gomelsky, M., and Kaplan, S. (1998) J. Biol.

Chem. 52, 35319–35325.Gomelsky, M., and Klug, G. (2002) Trends

Biochem. Sci. 27, 497–500.Gressel, J. (1979) Photochem. Photobiol. 30:

749–754.Hasegawa, E., Hayashi, H., Asakura, S., and

Kamiya, R. (1987) Cell Motil. Cytoskeleton 8,302–311.

Iseki, M., Matsunaga, S., Murakami, A., Ohno,K., Shiga, K., Yoshida, K., Sugai, M., Taka-hashi, T., Hori, T., and Watanabe, M. (2002)Nature 415, 1047–1051.

Kasahara, M., Yashiro, K., Sakamoto, T., andOhmori, M. (1997) Plant Cell Physiol. 38,828–836.

Kateriya, S., Nagel, G., Bamberg, E., and Hege-mann, E. (2004) News Physiol. Sci. 19, 133–137.

Kondo, T., Kubota, M., Aono, Y., and Watan-abe, M. (1988) Protoplasma Suppl. 1, 185–192.

Koumura, Y., Suzuki, T., Yoshikawa, S., Watan-abe, M., and Iseki, M. (2004) Photochem.Photobiol. Sci. 3, 580–586.

Lebert, M. (2001) in Comprehensive Series inPhotosciences 1: Photomovement (edsHäder, D.-P. and Lebert, M.) 297–341 (Else-vier, Amsterdam)

Lewis, R. A. (2002) Biophotonics InternationalMay 2002, 40–41.

Masuda, S., and Bauer, C.E. (2002) Cell 110,613–623.

Matsunaga, S., Hori, T., Takahashi, T., Kubota,M., Watanabe, M., Okamoto, K., Masuda, K.,and Sugai, M. (1998) Protoplasma 201, 45–52.

Matsunaga, S., Takahashi, T., Watanabe, M.,Sugai, M., Hori, T. (1999) Plant Cell Physiol.40, 213–221.

Noguchi, M., Ogawa, T., and Taneyama, T.(2000) Cell Motil. Cytoskeleton 45, 263–271.

Ntefidou, M., Iseki, M., Watanabe, M., Lebert,M., and Häder, D.-P. (2003) Plant Physiol.133, 1517–1521.

Schmidt, W., Galland, P., Senger, H., and Fu-ruya, M. (1990) Planta 182, 375–381.

Schröder-Lang, S., Ollig, D., Schiereis, T.,Hegemann, P., and Nagel, G. (2004) Abst.11th Internatl. Conf. Retinal Proteins,Frauenchiemsee, Germany, June 20–24, P60.

Torre, V., Ashmore, J.F., Lamb, T.D., and Meni-ni, A. (1995) J. Neurosci. 15, 7757–7768.

Watanabe, M. (2004) in CRC Handbook of Or-ganic Photochemistry and Photobiology, 2nded. (eds. Horspool, W., and Lenci, F.) 115–1~115–16 (CRC Press, Boca Raton)

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