Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study.
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 integrated into the genome of HKC. Western blot indicates that the fusion proteins have been expressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics approach; 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 expression and the trans-membrane currents are measured by using two-microelectrode voltage-clamp technique. Na+ inward currents are present on cellular membrane of the injected oocytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lumen 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 localization signal of the SDCT1 protein maybe locate in the C-terminal sequence.
Bai
Xueyuan
X
Chinese PLA Kidney Center & Key Laboratory for Nephrology, Chinese PLA General Hospital & Military Medical Postgraduate College, Beijing 100853, China. baixy@301hospital.com.cn
Chen
Xiangmei
X
Fen
Zhe
Z
Wu
Di
D
Hou
Kai
K
Cheng
Genyang
G
Peng
Lixia
L
D000818:Animals; D002478:Cells, Cultured; D029382:Dicarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution; D014982:Xenopus laevis
D029382:Dicarboxylic Acid Transporters; D029363:Organic Anion Transporters, Sodium-Dependent; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C092524:SLC13A2 protein, human; D027981:Symporters