Voltage-gated proton channel in a dinoflagellate.

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH(V)1: it is proton-specific and activated by depolarization, its g(H)-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp(51)) results in loss of proton-specific conduction. Indirect evidence suggests that kH(V)1 is monomeric, unlike other proton channels. Furthermore, kH(V)1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E(H). This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.

A type-1 phosphoprotein phosphatase from a dinoflagellate as a possible component of the circadian mechanism.

Indicative of the importance of protein phosphorylation in the core circadian clock mechanism, chronically applied inhibitors of both protein kinases and phosphoprotein phosphatases have significant effects on the period, phase, and light-dependent regulation of circadian rhythms in the dinoflagellate Lingulodinium polyedrum. This study was aimed at identifying the presence of the affected phosphatase(s). Dephosphorylation of a PP1/PP2A-specific substrate by L. polyedrum extracts was inhibited by okadaic acid only at concentrations greater than 100 nM, as in vivo, by mammalian inhibitor-2 (I-2), and by an endogenous inhibitor with properties similar to I-2, indicating that a type-1 protein phosphatase (PP1) was predominant. A cDNA encoding a highly conserved PP1 was isolated, the 1st such signaling molecule identified in dinoflagellates. Antisera specific for this type of phosphatase recognized a 34 kDa protein in L. polyedrum extract, this being the same size as the PP1 encoded by the isolated cDNA. These findings are consistent with the suggestion that the L. polyedrum PP1 may be a part of the clock mechanism in this species.

Genome-wide analysis of redox-regulated genes in a dinoflagellate.

In this study, the effects of 1 mM sodium nitrite, a reactive nitrogen species (RNS) generator, and 0.5 mM paraquat, which produces reactive oxygen species (ROS), on gene expression in the marine dinoflagellate species Pyrocystis lunula were investigated using microarrays containing 3500 complementary DNAs (cDNAs). A total of 246 differentially expressed genes were identified under these treatments: 204 genes were specifically regulated in response to nitrite and 37 genes specifically to paraquat. Only six genes showed a dependence on both nitrite and paraquat, indicating that the two agents act predominantly via distinct pathways. Although many of these redox-regulated genes encode proteins from a diverse range of functional categories, the majority of them (68%) represent novel sequences. Temporary abnormal spherical cells occurred in nitrite-treated cultures, but not in those exposed to paraquat, suggesting that this response involves a specific pathway triggered by RNS. The genes involved include one that encodes a transcription factor unique to dinoflagellates (HPl), and genes encoding proteins similar to those regulating developmental processes in plants and animals such as NYD-SP5, shaggy and calcium-dependent kinases, the COP9 signalosome complex, ubiquitin-related proteases and a metacaspase.

Two bioluminescent diptera: the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems.

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (lambda(max) = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin-luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of approximately 140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin-luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.

Lifetimes of mRNAs for clock-regulated proteins in a dinoflagellate.

Both pulsed and continuous applications of the RNA polymerase II inhibitor thiolutin cause a dramatic but reversible loss of bioluminescence and its overt rhythmicity in cells of the dinoflagellate Lingulodinium polyedrum (formerly Gonyaulax polyedra). Such cells remain alive, and the rhythm resumes after an interval, the length of which depends on the concentration of thiolutin used. The period and phase of the resumed rhythm were not systematically altered following such treatments, and the effects were not different at different circadian phases. For three different genes, luciferin binding protein (lbp), luciferase (lcf), and glyceraldehyde-3-phosphate dehydrogenase (gapdh), which are circadian-regulated at the level of translation, the amounts of their mRNAs were determined by Northern blots for times up to 12.5 h following the addition of 1.5 microM thiolutin. Consistent with previous reports that their abundances do not change with circadian time, their levels remained high for several hours after thiolutin addition, but then did diminish.

Circadian Rhythms in Dinoflagellates: What Is the Purpose of Synthesis and Destruction of Proteins?

There is a prominent circadian rhythm of bioluminescence in many species of light-emitting dinoflagellates. In Lingulodinium polyedrum a daily synthesis and destruction of proteins is used to regulate activity. Experiments indicate that the amino acids from the degradation are conserved and incorporated into the resynthesized protein in the subsequent cycle. A different species, Pyrocystis lunula, also exhibits a rhythm of bioluminescence, but the luciferase is not destroyed and resynthesized each cycle. This paper posits that synthesis and destruction constitutes a cellular mechanism to conserve nitrogen in an environment where the resource is limiting.

Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species.

Noctiluca scintillans, a heterotrophic unarmored unicellular bioluminescent dinoflagellate, occurs widely in the oceans, often as a bloom. Molecular phylogenetic analysis based on 18S ribosomal DNA sequences consistently has placed this species on the basal branch of dinoflagellates. Here, we report that the structural organization of its luciferase gene is strikingly different from that of the seven luminous species previously characterized, all of which are photosynthetic. The Noctiluca gene codes for a polypeptide that consists of two distinct but contiguous domains. One, which is located in the N-terminal portion, is shorter than but similar in sequence to the individual domains of the three-domain luciferases found in all other luminous dinoflagellates studied. The other, situated in the C-terminal part, has sequence similarity to the luciferin-binding protein of the luminous dinoflagellate Lingulodinium polyedrum, encoded there by a separate gene. Western analysis shows that the native protein has the same size (approximately 100 kDa) as the heterologously expressed polypeptide, indicating that it is not a polyprotein. Thus, sequences found in two proteins in the L. polyedrum bioluminescence system are present in a single polypeptide in Noctiluca.

Crystal structure of a pH-regulated luciferase catalyzing the bioluminescent oxidation of an open tetrapyrrole.

The luciferase of Lingulodinium polyedrum, a marine bioluminescent dinoflagellate, consists of three similar but not identical domains in a single polypeptide. Each encodes an active luciferase that catalyzes the oxidation of a chlorophyll-derived open tetrapyrrole (dinoflagellate luciferin) to produce blue light. These domains share no sequence similarity with any other in the GenBank database and no structural or motif similarity with any other luciferase. We report here the 1.8-A crystal structure of the third domain, D3, at pH 8, and a mechanism for its activity regulation by pH. D3 consists of two major structural elements: a beta-barrel pocket putatively for substrate binding and catalysis and a regulatory three-helix bundle. N-terminal histidine residues previously shown to regulate activity by pH are at the interface of the helices in the bundle. Molecular dynamics calculations indicate that, in response to changes in pH, these histidines could trigger a large molecular motion of the bundle, thereby exposing the active site to the substrate.

Molecular evolution of dinoflagellate luciferases, enzymes with three catalytic domains in a single polypeptide.

Enzymes with multiple catalytic sites are rare, and their evolutionary significance remains to be established. This study of luciferases from seven dinoflagellate species examines the previously undescribed evolution of such proteins. All these enzymes have the same unique structure: three homologous domains, each with catalytic activity, preceded by an N-terminal region of unknown function. Both pairwise comparison and phylogenetic inference indicate that the similarity of the corresponding individual domains between species is greater than that between the three different domains of each polypeptide. Trees constructed from each of the three individual domains are congruent with the tree of the full-length coding sequence. Luciferase and ribosomal DNA trees both indicate that the Lingulodinium polyedrum luciferase diverged early from the other six. In all species, the amino acid sequence in the central regions of the three domains is strongly conserved, suggesting it as the catalytic site. Synonymous substitution rates also are greatly reduced in the central regions of two species but not in the other five. This lineage-specific difference in synonymous substitution rates in the central region of the domains correlates inversely with the content of GC3, which can be accounted for by the biased usage toward C-ending codons at the degenerate sites. RNA modeling of the central region of the L. polyedrum luciferase domain suggests a function of the constrained synonymous substitutions in the circadian-controlled protein synthesis.

Characterization and crystallization of active domains of a novel luciferase from a marine dinoflagellate.

Lingulodinium polyedrum luciferase is a bioluminescent protein found in the marine dinoflagellate formerly known as Gonyaulax. It is located in organelles called scintillons that emit brief and bright flashes of light that are regulated by an endogenous circadian clock. The complete luciferase molecule has a molecular mass of 136 994 Da and contains three homologous domains, each of which is a separately active luciferase. Two of these domains, D2-LCF and D3-LCF, have been cloned, expressed and crystallized. Crystals of D2-LCF were obtained from PEG 10 000 in space group P2(1)2(1)2(1), with unit-cell parameters a = 49.1, b = 104.7, c = 180.3 A. They diffract to 2.9 A on a rotating anode. Crystals of D3-LCF were grown from PEG 2000 in space group P2(1)2(1)2(1), with unit-cell parameters a = 58.86, b = 63.98, c = 95.76 A. They diffract to 2.3 A on a rotating anode.