Mays R W, Siemers K A, Fritz B A, Lowe A W, van Meer G, Nelson W J
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305, USA.
J Cell Biol. 1995 Sep;130(5):1105-15. doi: 10.1083/jcb.130.5.1105.
We have studied mechanisms involved in generating a polarized distribution of Na/K-ATPase in the basal-lateral membrane of two clones of MDCK II cells. Both clones exhibit polarized distributions of marker proteins of the apical and basal-lateral membranes, including Na/K-ATPase, at steady state. Newly synthesized Na/K-ATPase, however, is delivered from the Golgi complex to both apical and basal-lateral membranes of one clone (II/J), and to the basal-lateral membrane of the other clone (II/G); Na/K-ATPase is selectively retained in the basal-lateral membrane resulting in the generation of complete cell surface polarity in both clones. Another basal-lateral membrane protein, E-cadherin, is sorted to the basal-lateral membrane in both MDCK clones, demonstrating that there is not a general sorting defect for basal-lateral membrane proteins in clone II/J cells. A glycosyl-phosphatidylinositol (GPI)-anchored protein (GP-2) and a glycosphingolipid (glucosylceramide, GlcCer) are preferentially transported to the apical membrane in clone II/G cells, but, in clone II/J cells, GP-2 and GlcCer are delivered equally to both apical and basal-lateral membranes, similar to Na/K-ATPase. To examine this apparent inter-relationship between sorting of GlcCer, GP-2 and Na/K-ATPase, sphingolipid synthesis was inhibited in clone II/G cells with the fungal metabolite, Fumonisin B1 (FB1). In the presence of FB1, GP-2 and Na/K-ATPase are delivered to both apical and basal-lateral membranes, similar to clone II/J cells; FB1 had no effect on sorting of E-cadherin to the basal-lateral membrane of II/G cells. Addition of exogenous ceramide, to circumvent the FB1 block, restored GP-2 and Na/K-ATPase sorting to the apical and basal-lateral membranes, respectively. These results show that the generation of complete cell surface polarity of Na/K-ATPase involves a hierarchy of sorting mechanisms in the Golgi complex and plasma membrane, and that Na/K-ATPase sorting in the Golgi complex of MDCK cells may be regulated by exclusion from an apical pathway(s). These results also provide new insights into sorting pathways for other apical and basal-lateral membrane proteins.
我们研究了在MDCK II细胞的两个克隆的基底外侧膜中产生Na/K-ATP酶极化分布所涉及的机制。在稳态下，两个克隆均呈现顶端和基底外侧膜标记蛋白（包括Na/K-ATP酶）的极化分布。然而，新合成的Na/K-ATP酶从高尔基体复合体被转运至一个克隆（II/J）的顶端和基底外侧膜，以及另一个克隆（II/G）的基底外侧膜；Na/K-ATP酶被选择性地保留在基底外侧膜，从而在两个克隆中产生完整的细胞表面极性。另一种基底外侧膜蛋白E-钙黏蛋白在两个MDCK克隆中均被分选至基底外侧膜，这表明II/J细胞中不存在基底外侧膜蛋白的普遍分选缺陷。一种糖基磷脂酰肌醇（GPI）锚定蛋白（GP-2）和一种糖鞘脂（葡萄糖神经酰胺，GlcCer）在II/G细胞中优先被转运至顶端膜，但在II/J细胞中，GP-2和GlcCer被同等地转运至顶端和基底外侧膜，类似于Na/K-ATP酶。为了研究GlcCer、GP-2和Na/K-ATP酶分选之间这种明显的相互关系，用真菌代谢产物伏马菌素B1（FB1）抑制II/G细胞中的鞘脂合成。在FB1存在的情况下，GP-2和Na/K-ATP酶被转运至顶端和基底外侧膜，类似于II/J细胞；FB1对E-钙黏蛋白分选至II/G细胞的基底外侧膜没有影响。添加外源性神经酰胺以绕过FB1阻断，分别恢复了GP-2和Na/K-ATP酶向顶端和基底外侧膜的分选。这些结果表明，Na/K-ATP酶完整的细胞表面极性的产生涉及高尔基体复合体和质膜中分选机制的层次结构，并且MDCK细胞高尔基体复合体中Na/K-ATP酶的分选可能通过排除顶端途径来调节。这些结果还为其他顶端和基底外侧膜蛋白的分选途径提供了新的见解。
