Chemoorientation in male gametes of Ectocarpus siliculosus in response to sexual pheromones is effected by two distinct mechanisms, chemokinesis and chemoklinotaxis. These are characterized by a strongly asymmetric bending pattern of the anteriorly-directed flagellum and transient unilateral bending of the hind flagellum, respectively. Removal of extracellular Ca2+ showed that normal flagellar movement and chemokinesis require millimolar concentrations ol Ca2+ in the medium. The response to pheromones is strongly inhibited by La3+, whereas the Ca2+-channel drugs, verapamil and nifedipine, have only little effect. Nifedipine nethertheless effectively inhibited accumu. lation at pheromone sources. These results are interpreted as an indication for the involvement of t w c pharmacologically distinct Ca2+-channels in chemokinesis and chemoklinotaxis. The calmodulin-anta. gonist, trifluoperazine, induces, at low concentrations the same flagellar response in chemokinesis as tht pheromone, the mechanism of action remaining un. known.

Little is known about phototactic signal transduction in flagellate green algae; therefore, eyespot apparatuses, which are the light-sensitive "organelles" involved in photoorientation of these algae, were isolated and analyzed for the presence of heterotrimeric guanine nucleotide binding proteins (G proteins) and their coupling to the retinal-based photoreceptor. Specific high-affinity 35 S-GTP-␥ -S binding and GTPase activity, with sensitivity toward antibodies raised against vertebrate/invertebrate G ␣ subunits and fluoroaluminates, were detected. In one-and two-dimensional immunoblot analyses, an antiserum directed against G i ␣ -type subunits exhibited cross-reactivity at 42 kD, whereas a 43-kD protein cross-reacted with antisera directed against G q ␣ subunits. Green light below 1 E m ؊ 2 sec ؊ 1 suppressed cholera toxin-dependent ADP ribosylation at these apparent molecular masses and modulated a significant proportion of the GTPase activity in a reversible manner. Antisera against Chlamydomonas rhodopsin and the G ␣ subunits completely impaired light modulation. Both light sensitivity and dark recovery of the GTPase were affected by changes in free Ca 2 ؉ . Dissociation of the putative G ␣ subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of G ␣ subunits in rhodopsin-based signaling of flagellate green algae.

Knowledge about possible elements involved in signal transduction and adaptational phenomena during phototaxis of flagellate green algae is limited. In order to identify such putative elements we present here: (i) the isolation and characterisation of an enriched plasma membrane fraction of Spermatozopsis similis, and (ii) a comparison of the distribution of Ca2+-dependent putative kinases and G proteins, as recognised by antibodies directed against mammalian Gc~-subunits, in the plasma membrane preparation and isolated eyespot apparatuses of this green alga. In western blot analyses a 54 kDa protein reacted with an antiserum against a conserved sequence of G protein c~-subunits (anti-c~ common). A 43 kDa protein was recognised by a serum against the G~-subunit of transducin. These proteins were enriched in the eyespot fraction and to a lesser extent in the plasma membrane fraction. Two polyclonal antisera against C-terminal peptides of animal G(~-subunits also labelled a 43 kDa and 54 kDa protein in the eyespot preparations. Additionally, four putative Ca2+-dependent kinases or their catalytic subunits (47, 48, 77 and 82 kDa) were identified in the plasma membrane fraction by a gel kinase assay following electrophoretic separation. The 82 kDa protein was specifically enriched in the plasma membrane fraction, while the other putative kinases were also enriched in the eyespot fraction but not in other subcellutar fractions. Monoclonal antibodies directed against a soybean CaZ+-dependent protein kinase cross-reacted with a 6I kDa protein in the plasma membrane and a 60 kDa protein in the soluble fraction. Only a very faint response was observed in the fraction enriched in eyespot apparatuses. Our results indicate (i) a low cross-contamination of the eyespot apparatus preparation by normal plasma membrane constituents and (ii) the regulatory potential for the identified kinases and G proteins in blue/green-light-dependent photoresponses of Spermatozopsis similis.

Isolated intact eyespot apparatuses, the photoreceptive organelles involved in blue-light-mediated photoresponses of flagellate green algae, were analyzed regarding their carotenoid composition. Carotenoids from the eyespot apparatuses of Spermatozopsis similis were identified by high-performance liquid chromatography, visible-light absorption spectra, mass spectroscopy and by 1H-nuclear magnetic resonance spectroscopy (carotenes), and compared with those of whole-cell extracts. Both extracts contained fl,fl-carotene, fl, O-carotene (formerly 7-carotene), lycopene, lutein, zeaxanthin, violaxanthin and all-E-and 9'-Z-neoxanthin. The relative carotenoid compositions, however, differed significantly. A twofold relative increase in the total carotene level was evident in the fraction enriched in eyespot apparatuses. This was mainly due to an increase in the monocyclic fl,~9-carotene and the aliphatic lycopene, whereas the relative content of fl,fl-carotene remained unchanged. On the other hand a relative decrease in the total xanthophyll content, especially of lutein and the epoxidic carotenoid neoxanthin, was observed in the eyespot apparatuses compared with the whole-cell extracts. The decrease of the latter resulted almost solely from a reduction of the 9"-Z-rather than the all-E-isomer. The bulk of the carotenes is thought to be localized in the highly organized eyespot lipid globules, which act as a combined quarter-wave interference reflector and absorption screen for the photoreceptor in green algae. The enrichment offl,~-carotene and lycopene in the eyespot apparatuses, extending the range of visible light absorption to longer wavelengths, represents an adaptation of the screen to the retinal-based photoreceptor of flagellate green algae and is one of the prerequisites for maximal directional sensitivity of the eyespot apparatus.Abbreviations: 1H-NMR = nuclear magnetic resonance; IUPAC -International Union of Pure and Applied Chemistry; VIS =visible absorption spectra Correspondence to-S. Liaaen-Jensen; FAX: 47(0)73594256

Little is known about phototactic signal transduction in flagellate green algae; therefore, eyespot apparatuses, which are the light-sensitive "organelles" involved in photoorientation of these algae, were isolated and analyzed for the presence of heterotrimeric guanine nucleotide binding proteins (G proteins) and their coupling to the retinal-based photoreceptor. Specific high-affinity 35 S-GTP-␥ -S binding and GTPase activity, with sensitivity toward antibodies raised against vertebrate/invertebrate G ␣ subunits and fluoroaluminates, were detected. In one-and two-dimensional immunoblot analyses, an antiserum directed against G i ␣ -type subunits exhibited cross-reactivity at 42 kD, whereas a 43-kD protein cross-reacted with antisera directed against G q ␣ subunits. Green light below 1 E m ؊ 2 sec ؊ 1 suppressed cholera toxin-dependent ADP ribosylation at these apparent molecular masses and modulated a significant proportion of the GTPase activity in a reversible manner. Antisera against Chlamydomonas rhodopsin and the G ␣ subunits completely impaired light modulation. Both light sensitivity and dark recovery of the GTPase were affected by changes in free Ca 2 ؉ . Dissociation of the putative G ␣ subunits from the eyespot membranes was not observed when the membranes were illuminated. Our results emphasize the regulatory potential of G ␣ subunits in rhodopsin-based signaling of flagellate green algae.