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Expression of EGFP/SDCT1 fusion protein,subcellular localization signal analysis,tissue distribution and electrophysiological function study

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Science China(Life Sciences) 2004年第6期47卷 530-539页

作者： BAI Xueyuan CHEN Xiangmei FEN Zhe WU Di HOU Kai CHENG Genyang PENG Lixia Chinese PLA Kidney Center & Key Laboratory for Nephrology Chinese PLA General Hospital & Military Medical Postgraduate CollegeBeijing 100853China

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 se

关键词： dicarboxylate co-transporter tricarboxylic acid cycle renal tubule subcellular localization.

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