Expression of a mammalian Na+/H+ antiporter in Saccharomyces cerevisiae.
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.
Montero-Lomelí
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Departmento de Bioquímica Medica, Instituto Ciencias Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Río de Janeiro, Brasil. montero@bioqmed.ufrj.br
Okorokova Façanha
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AL
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012313:RNA / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D012313:RNA