Wild-type strains of the protozoan ciliate Euplotes collected from different locations on the coasts of Antarctica, Tierra del Fuego and the Arctic were taxonomically identified as the morpho-species Euplotes nobilii, based on morphometric and phylogenetic analyses. Subsequent studies of their sexual interactions revealed that mating combinations of Antarctic and Arctic strains form stable pairs of conjugant cells. These conjugant pairs were isolated and shown to complete mutual gene exchange and cross-fertilization. The biological significance of this finding was further substantiated by demonstrating that close homology exists among the threedimensional structures determined by NMR of the water-borne signaling pheromones that are constitutively secreted into the extracellular space by these interbreeding strains, in which these molecules trigger the switch between the growth stage and the sexual stage of the life cycle. The fact that Antarctic and Arctic E. nobilii populations share the same gene pool and belong to the same biological species provides new support to the biogeographic model of global distribution of eukaryotic microorganisms, which had so far been based exclusively on studies of morphological and phylogenetic taxonomy.ciliate mating types and sexual interactions | microbial ecology | NMR structures | polar biology | signaling by water-borne pheromones

Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er-1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix-bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding.

The ciliate Euplotes raikovi produces a family of diffusible signal proteins (pheromones) that function as prototypic growth factors. They may either promote cell growth, by binding to pheromone receptors synthesized by the same cells from which they are secreted (autocrine activity), or induce a temporary cell shift from the growth stage to a mating (sexual) one by binding to pheromone receptors of other, conspecific cells (paracrine activity). In cells constitutively secreting the pheromone Er-1, it was first observed that the expression of the Er-1 receptor "p15," a type II membrane protein of 130 amino acids, is quantitatively correlated with the extracellular concentration of secreted pheromone. p15 expression on the cell surface rapidly and markedly increased after the removal of secreted Er-1 and gradually decreased in parallel with new Er-1 secretion. It was then shown that p15 is internalized through endocytic vesicles following Er-1 binding and that the internalization of p15/Er-1 complexes is specifically blocked by the paracrine p15 binding of Er-2, a pheromone structurally homologous to, and thus capable of fully antagonizing, Er-1. Based on previous findings that the p15 pheromone-binding site is structurally equivalent to Er-1 and that Er-1 molecules polymerize in crystals following a pattern of cooperative interaction, it was proposed that p15/Er-1 complexes are internalized as a consequence of their unique property (not shared by p15/Er-2 complexes) of undergoing clustering.In association with the evolution of systems of multiple cell (mating) types controlling self/nonself recognition phenomena, species of Euplotes synthesize families of structurally homologous soluble proteins that confer on cells a chemical specificity, with each protein representing a diffusible chemical signal (pheromone) that distinguishes one cell type from all the others (17). Euplotes raikovi is the species with which these pheromones, designated Er-1, Er-2, and so forth, have been better characterized with regard to their structure and function. Pheromones are small molecules of 38 to 50 amino acids with a common architecture based on a bundle of three ␣-helices fastened together by three conserved disulfide bonds (16,26,35). They have at least two activities each, acting as both prototypic autocrine growth factors and paracrine mating (sexual) signals (31). Upon binding their own pheromones, which cells constitutively secrete into the extracellular environment throughout the cell cycle, cells grow vegetatively and divide mitotically. When cells bind a nonself pheromone from another cell type, they temporarily arrest their growth and develop competence for uniting in mating pairs (23). We refer to the response to self pheromones as an autocrine response and the response to nonself pheromones as a paracrine response.The field addressing the issue of how cells can discriminate between autocrine and paracrine pheromone binding and accordingly mount a growth or mating response was advanced by the identification of the pher...

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The high-multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating-type substance (pheromone). In strain L-2D, now known to be homozygous at the mating-type locus, we previously identified two pheromones (Ec-α and Ec-1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR-73 characterized by a heterozygous genotype and strong mating compatibility with L-2D strain. It was found that POR-73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec-α pheromone of L-2D cells. The other two pheromones were shown to be new and were designated Ec-2 and Ec-3 to denote their structural homology with the Ec-1 pheromone of L-2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating-type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.

Ciliates comprise species synthesizing water-diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane-bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone-secreting ciliate, like other Euplotes species. From cell-free filtrate preparations of the E. crassus strain L-2D, we isolated two distinct pheromones, designated as Ec-α and Ec-1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec-α pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec-1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full-length Ec-α and Ec-1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec-α and Ec-1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome.

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SummaryCell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear 1 H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-a-helix bundle with an up-down-up topology and a 3 10 -helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters. IUBMB Life, 59: 578-585, 2007