Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along wi...Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been in- tegrated into the genome of HKC. Western blot indicates that the fusion proteins have been ex- pressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics ap- proach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for ex- pression and the trans-membrane currents are measured by using two-microelectrode volt- age-clamp technique. Na+ inward currents are present on cellular membrane of the injected oo- cytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lu- men membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane lo- calization signal of the SDCT1 protein maybe locate in the C-terminal sequence.展开更多
基金This work was supported by the National Key Basic Research Program of China(973 Program)(Grant No.G2000057003)National Natural Science Foundation of China(NSFC)(Grant Nos.30070288 and 30270505)creative research group fund from NSFC(Grant No.30121005).
文摘Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been in- tegrated into the genome of HKC. Western blot indicates that the fusion proteins have been ex- pressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics ap- proach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for ex- pression and the trans-membrane currents are measured by using two-microelectrode volt- age-clamp technique. Na+ inward currents are present on cellular membrane of the injected oo- cytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lu- men membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane lo- calization signal of the SDCT1 protein maybe locate in the C-terminal sequence.
