RESUMO
Cyclic nucleotide-gated (CNG) ion channels mediate the response to light in retinal rods. They are tetramers of two homologous subunits (alpha and beta), each of which is essential for the function of the channels in vivo. We have investigated the stoichiometry and arrangement of these two subunits to determine how they come together within an individual channel complex. We exploited the very specific geometric and spatial requirements for forming a high-affinity Ni2+-binding site to examine the number and relative positions of the subunits. We found that only an order of alpha/alpha/beta/beta could account qualitatively and quantitatively for the observed intersubunit coordination of Ni2+ in wild-type and mutant alpha/beta channels. Furthermore, our results suggest a structural dimerization among like subunits, at least at the level of the Ni2+-binding site.
Assuntos
AMP Cíclico/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Ativação do Canal Iônico/genética , Canais Iônicos/biossíntese , Canais Iônicos/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação/fisiologia , Níquel/metabolismo , Níquel/farmacologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Segmento Externo da Célula Bastonete/fisiologia , Xenopus laevisRESUMO
The two-hybrid system was used to show that the Rep proteins from three members of the Dictyostelium discoideum Ddp2 plasmid family, Ddp2, Ddp5, and Ddp6, form homomultimers but not heteromultimers when expressed in yeast cells. The results with deletion mutations suggest that multiple regions of the Rep proteins are involved in the multimerization. Electrophoretic mobility shift assays with heterologously expressed and purified Ddp2 Rep protein showed that it is a DNA binding protein. The nucleosomal organization of Ddp2 and Ddp6 in their inverted repeat and promoter regions was investigated. Analysis of mutants derived from the Ddp6 plasmid revealed that its Rep protein is required for nucleosome positioning (i.e., phasing) to occur in the promoter region. On the other hand, nucleosome positioning in the inverted repeat regions of both plasmids is not dependent on Rep protein but on either a feature of the DNA sequence or the binding of cellular factors, perhaps the Dictyostelium origin recognition complex. Rep protein is likely involved in transcription regulation and control of DNA replication, specifically amplification of plasmid at low copy numbers. The formation of homomultimers may be required for their regulatory activity.
Assuntos
Dictyostelium/genética , Dictyostelium/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Genes de Protozoários , Nucleossomos/metabolismo , Plasmídeos/genética , Conformação Proteica , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/genéticaRESUMO
Ddp6 is a high-copy number, circular plasmid found in the nucleus of the simple eukaryote Dictyostelium discoideum wild isolate NC47.2. The complete nucleotide sequence, 5257 bp, shows that Ddp6 has a structure similar to that of other members of the Ddp2 plasmid family: a single long 2.8-kb open reading frame (rep gene) and an inverted repeat containing a pair of 654-bp elements. A single constitutively expressed 3.3-kb transcript of the rep gene was detected in RNA prepared from vegetative and developmental cells. Maintenance assays revealed that sequences within the inverted repeat and the intact Ddp6 ORF are essential for maintenance of the plasmid. Mutation of the inverted repeat, or of the rep gene, lowered the plasmid copy number but did not affect autonomous replication of shuttle-vector constructs. Comparisons of the predicted protein products of the rep genes of five members of the Ddp2 plasmid family identified a set of ten conserved features distributed throughout the peptides.
