RESUMO
We determined the H+ and Ca(2+) uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca(2+) sequestering is due to Ca(2+)/H+ antiporter(s) localized to secretory pathway organelles while Ca(2+)-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca(2+) and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca(2+) signaling in a differential and local manner.
Assuntos
Antiporters/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Transporte de Cátions , Hidrogênio/metabolismo , Macrolídeos , ATPases Translocadoras de Prótons/fisiologia , Schizosaccharomyces/metabolismo , ATPases Vacuolares Próton-Translocadoras , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antiporters/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Immunoblotting , Ionóforos/farmacologia , Dobramento de Proteína , Transdução de Sinais , Sacarose/metabolismo , Vacúolos/metabolismoRESUMO
The Na+/H+ exchanger is an ubiquitous mammalian plasma membrane protein that is important for the regulation of intracellular pH and cell volume. In order to provide some insight into the molecular basis of NHE1 expression we have isolated and characterized genomic DNA clones containing the coding region and 5'-flanking region of the porcine NHE1 gene. The gene spans more than 30 kb in length and consists of twelve exons that are flanked by typical splice donor and acceptor sequences at the exon-intron boundaries. The positions of the splicing sites are conserved in relation to the human NHE1 gene. The 5' distal transcription initiation site, identified by primer extension analysis, is positioned 766 bp upstream of the translation initiation codon and 36 bp downstream of a TATA box. A 5'-flanking region of 1.62 kb in length contains a number of potential regulatory elements, and exhibits several features that distinguish the pig gene from those of rabbit, mouse and human. The NHE1 gene is located in a CpG island. The promoter sequence of 500 bp is compared with that for NHE1 genes from different species. The homology between the porcine and the human, rabbit and mouse genes is 78, 76 and 75%, respectively. Several consensus elements for transcription factors, including AP-1, C/EBP, and Sp1 are phylogenetically conserved between pig and human, while AP3 and PEA3 are found only in pig. Some conserved elements are found in the pig in multiple copies. These results suggest broadly similar regulatory mechanisms for NHE1 transcription among the different mammalian species but show some species or tissue-specific differences.
Assuntos
Regiões 5' não Traduzidas/genética , Sequências Reguladoras de Ácido Nucleico , Trocadores de Sódio-Hidrogênio/genética , Suínos/genética , Animais , Sequência de Bases , Clonagem Molecular , Sequência Consenso/genética , Ilhas de CpG , Éxons/genética , Humanos , Íntrons/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Isoformas de Proteínas , Sítios de Splice de RNA/genética , Alinhamento de SequênciaRESUMO
The basolateral Na+/H+ antiporter (NHE) from LLC-PK1 cells was expressed in Saccharomyces cerevisiae. Two different strategies were tested for expression. In the first, we used a yeast strain that contains a temperature-sensitive mutation in the SEC-6 gene, whose product is required for the fusion of secretory vesicles with the plasma membrane. This strain was transformed with a vector containing the coding region of the NHE1 isoform under control of a heat shock (HS) promoter (pYNHE1-HS). In the second strategy, we replaced the heat shock promoter from pYNHE1-HS with a galactose (GAL) promoter (pYNHEI-GAL) and transformed wild-type yeast. In both cases, Northern blots demonstrated a transcript that hybridized against a probe containing the membrane region of the exchanger. When an antibody against the last 40 amino acids of the carboxy-terminus of NHE1 was used for immunoblots, a protein with a Mr of 73000 was seen in total membranes from both yeast transformants. Subcellular fractionation revealed that NHE1 was expressed in the endoplasmic reticulum. In the case of the pYNHEI-GAL transformant, the 100000 x g membrane pellet was reconstituted in phosphatidylcholine liposomes, and ethylisopropylamiloride-sensitive Na+/H+ exchange was observed. These results have paved the way for expression of the Na+/H+ exchanger in a genetically well-known microorganism.