McHaffie Gavin S, Graham Chris, Kohl Beate, Strunck-Warnecke Ursula, Werner Andreas
Epithelial Research Group, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
Biochim Biophys Acta. 2007 Sep;1768(9):2099-106. doi: 10.1016/j.bbamem.2007.05.017. Epub 2007 May 23.
The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [(3)H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.
II型钠/磷酸盐共转运体（NaPi-II）对于控制脊椎动物的血浆磷酸盐水平至关重要。NaPi-IIb介导从小肠摄取磷酸盐，随后通过肾小球滤过以及由NaPi-IIa和NaPi-IIc从肾近端小管进行选择性重吸收。NaPi-IIb亚型的C末端一段半胱氨酸残基是其特征。该基序在NaPi-IIb型转运体中高度保守，但在其他膜蛋白中未发现。为了研究该基序的作用，我们在瞬时和稳定转染的MDCK细胞中分析了NaPi-II构建体。该细胞系将来自比目鱼和小鼠的NaPi-IIb亚型靶向至顶端膜，而小鼠IIa亚型则没有质膜偏好。将小鼠NaPi-IIa和NaPi-IIb C末端的不同部分与绿色荧光蛋白标记的比目鱼NaPi-II融合。构建体显示质膜有强烈染色，与NaPi-IIb相关的构建体主要分选至顶端，IIa构建体定位于顶端和基底外侧，有轻微的细胞内滞留。当将半胱氨酸片段插入NaPi-IIa C末端时，构建体保留在细胞质区室中。2-溴棕榈酸酯，一种特异性棕榈酰化抑制剂，可将转运体释放至顶端和基底外侧膜。该药物还导致NaPi-IIb构建体重分布至两个质膜区室。从[³H]-棕榈酸酯标记的MDCK细胞中对标记的NaPi-II构建体进行免疫沉淀表明半胱氨酸片段被棕榈酰化。我们的结果表明，修饰的半胱氨酸基序可防止构建体进行基底外侧分选。位于半胱氨酸片段下游的其他分选决定因素可能将货物释放至顶端区室。
