RESUMO
Paramecium is a unicellular organism that possesses a specialized pathway for regulated secretion that is amenable to genetic studies. Numerous mutations affecting the process have been isolated over the years, among which is a subclass blocking the terminal step of fusion of the secretory granule with the plasma membrane. We report herein the cloning by functional complementation of one such gene, ND7. The 506-amino acid polypeptide encoded by ND7 is predicted to be a type I integral membrane protein with a highly charged cytosolic domain featuring amphipathic and coiled-coil regions. This structure is compatible with the physiological data on the mutant nd7-1 suggesting that the protein is anchored in the membrane of the secretory granule and that it may interact with other proteins. This work presents the first identification by a genetic approach of a novel gene involved in regulated secretion and establishes Paramecium as a powerful model system for the genetic dissection of this process.
Assuntos
Exocitose , Genes de Protozoários , Fusão de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Paramecium tetraurellia/genética , Proteínas de Protozoários/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Citoplasma/ultraestrutura , Teste de Complementação Genética , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
Paramecium is a unicell in which cellular processes are amenable to genetic dissection. Regulated secretion, which designates a secretory pathway where secretory products are first stored in intracellular granules and then released by exocytotic membrane fusion upon external trigger, is an important function in Paramecium, involved in defensive response through the release of organelles called trichocysts. In this review, we focus on recent advances in the molecular genetics of two major aspects of the regulated pathway in Paramecium, the biogenesis of the secretory organelles and their exocytosis.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Exocitose/fisiologia , Genes de Protozoários/fisiologia , Paramecium/fisiologia , Animais , Cálcio/metabolismo , Cálcio/fisiologia , Biologia Molecular , Paramecium/ultraestrutura , Proteínas de Protozoários/fisiologia , Transdução de Sinais/fisiologiaRESUMO
We report on an analysis of the constraints of PCR typing of field Plasmodium falciparum isolates by using a few highly polymorphic markers, MSA-1, MSA-2, TRAP, and CS. We show that the reactions are specific for the P. falciparum species. The detection threshold (minimum number of parasites required to detect a visible band by ethidium bromide) differed from one marker to the other and, within one locus, from one primer combination to the other. Importantly, the various MSA-1 and MSA-2 reference alleles were amplified with the same efficiency. Amplification from reconstituted allele mixtures indicated that at certain allele ratios, the most abundant allele interfered with the amplification of the less abundant one. An analysis of nine isolates collected from patients with acute malaria in Dielmo, Senegal, during a transmission season when the inoculation rate was one infective bite every second night is presented and discussed. All samples contained more than one parasite type. A significant polymorphism was observed for the four markers. Novel TaqI restriction fragment length polymorphisms were found for the TRAP gene, and TRAP gene typing alone allowed a distinction between the various isolates. MSA-1 and MSA-2 gave multiple band patterns specific for each sample.
Assuntos
Antígenos de Protozoários , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase/métodos , Alelos , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , DNA de Protozoário/genética , Amplificação de Genes , Genes de Protozoários , Marcadores Genéticos , Humanos , Malária Falciparum/parasitologia , Proteína 1 de Superfície de Merozoito , Dados de Sequência Molecular , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase/estatística & dados numéricos , Polimorfismo de Fragmento de Restrição , Precursores de Proteínas/genética , Proteínas de Protozoários/genética , Sensibilidade e EspecificidadeRESUMO
In an attempt to identify proteins that might underlie membrane trafficking processes in ciliates, calcium-dependent, phospholipid-binding proteins were isolated from extracts of Paramecium tetraurelia. The major protein obtained, named copine, had a mass of 55 kDa, bound phosphatidylserine but not phosphatidylcholine at micromolar levels of calcium but not magnesium, and promoted lipid vesicle aggregation. The sequence of a 920-base pair partial cDNA revealed that copine is a novel protein that contains a C2 domain likely to be responsible for its membrane active properties. Paramecium was found to have two closely related copine genes, CPN1 and CPN2. Current sequence data bases indicate the presence of multiple copine homologs in green plants, nematodes, and humans. The full-length sequences reveal that copines consist of two C2 domains at the N terminus followed by a domain similar to the A domain that mediates interactions between integrins and extracellular ligands. A human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein exhibited calcium-dependent phospholipid binding properties similar to those of Paramecium copine. An antiserum raised against a fragment of human copine I was used to identify chromobindin 17, a secretory vesicle-binding protein, as a copine. This association with secretory vesicles, as well the general ability of copines to bind phospholipid bilayers in a calcium-dependent manner, suggests that these proteins may function in membrane trafficking.