Sun An-Qiang, Salkar Rachita, Xu Shuhua, Zeng Lei, Zhou Ming-Ming, Suchy Frederick J
Department of Pediatrics and Structural Biology Program, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
J Biol Chem. 2003 Feb 7;278(6):4000-9. doi: 10.1074/jbc.M207163200. Epub 2002 Nov 14.
The rat ileal sodium-dependent bile acid transporter (Asbt) is a polytopic membrane glycoprotein, which is specifically expressed on the apical domain of the ileal brush-border membrane. In the present study, an essential 14-amino acid (aa 335-348) sorting signal was defined on the cytoplasmic tail of Asbt with two potential phosphorylation sites motifs for casein kinase II ((335)SFQE) and protein kinase C (PKC) ((339)TNK). Two-dimension NMR spectra analysis demonstrated that a tetramer, (340)NKGF, which overlaps with the potential PKC site within the 14-mer signal sequence, adopts a type I beta-turn conformation. Replacement of the potential phosphorylation residue Ser(335) and Thr(339) with alanine or deletion of either the 4 ((335)SFQE) or 10 aa (338-348, containing (339)TNKGF) from the C terminus of Asbt resulted in a significantly decreased initial bile acid transport activity and increased the basolateral distribution of the mutants by 2-3-fold compared with that of wild type Asbt. Deletion of the entire last 14 amino acids (335-348) from the C terminus of Asbt abolished the apical expression of the truncated Asbt. Moreover, replacement of the cytoplasmic tail of the liver basolateral membrane protein, Na(+)/taurocholate cotransporting polypeptide, with the 14-mer peptide tail of Asbt redirected the chimera to the apical domain. In contrast, a chimera consisting of the 14-mer peptide of Asbt fused with green fluorescent protein was expressed in an intracellular transport vesicle-like distribution in transfected Madin-Darby canine kidney and COS 7 cells. This suggests that the apical localization of the 14-mer peptide requires a membrane anchor to support proper targeting. The results from biological reagent treatment and low temperature shift (20 degrees C) suggests that Asbt follows a transport vesicle-mediated apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift.
大鼠回肠钠依赖性胆汁酸转运蛋白（Asbt）是一种多次跨膜糖蛋白，特异性表达于回肠刷状缘膜的顶端结构域。在本研究中，在Asbt的胞质尾上确定了一个由14个氨基酸组成的关键分选信号（第335 - 348位氨基酸），其中包含酪蛋白激酶II的两个潜在磷酸化位点基序（第335位氨基酸处的SFQE）和蛋白激酶C（PKC）的潜在磷酸化位点基序（第339位氨基酸处的TNK）。二维核磁共振光谱分析表明，一个四聚体（第340位氨基酸处的NKGF），它与14聚体信号序列中的潜在PKC位点重叠，呈I型β-转角构象。将潜在磷酸化残基丝氨酸（Ser335）和苏氨酸（Thr339）替换为丙氨酸，或者从Asbt的C末端缺失4个氨基酸（第335位氨基酸处的SFQE）或10个氨基酸（第338 - 348位氨基酸，包含第339位氨基酸处的TNKGF），均导致初始胆汁酸转运活性显著降低，且与野生型Asbt相比，突变体的基底外侧分布增加了2 - 3倍。从Asbt的C末端缺失最后14个氨基酸（第335 - 348位氨基酸）则消除了截短型Asbt的顶端表达。此外，用Asbt的14聚体肽尾替换肝基底外侧膜蛋白钠/牛磺胆酸盐共转运多肽的胞质尾，可使嵌合体重新定位于顶端结构域。相反，由Asbt的14聚体肽与绿色荧光蛋白融合而成的嵌合体，在转染的Madin - Darby犬肾细胞和COS 7细胞中以类似细胞内运输囊泡的分布形式表达。这表明14聚体肽的顶端定位需要膜锚定来支持正确的靶向。生物试剂处理和低温转移（20℃）的结果表明，Asbt遵循一种由运输囊泡介导的顶端分选途径，该途径对布雷菲德菌素A敏感，对蛋白质糖基化、莫能菌素处理和低温转移不敏感。
