Muth T R, Gottardi C J, Roush D L, Caplan M J
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Am J Physiol. 1998 Mar;274(3):C688-96. doi: 10.1152/ajpcell.1998.274.3.C688.
Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the alpha-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric alpha-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1 cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase alpha-subunit contain a basolateral sorting signal. H-K-ATPase beta-subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase beta-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase alpha-subunit masks or is dominant over any apical sorting information present in the beta-polypeptide. In gastric parietal cells, the H-K-ATPase beta-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase beta-subunit complex is directed to a similar compartment in LLC-PK1 cells, we treated transfected cells with drugs that raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase beta-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.
钠钾ATP酶和氢钾ATP酶是高度同源的离子泵，在上皮细胞中表现出不同的质膜分布。我们研究了这些异二聚体泵的α亚基，以确定负责其极化分选的蛋白质结构域。构建了一种嵌合α亚基构建体（N519H），其中前519个氨基酸残基对应于钠钾ATP酶序列，其余500个氨基酸来自氢钾ATP酶序列。在稳定转染的LLC-PK1细胞系中，我们发现N519H嵌合体在稳态条件下局限于基底外侧表面，这表明钠钾ATP酶α亚基氨基末端的519个氨基酸内的残基包含基底外侧分选信号。单独在LLC-PK1细胞中表达的氢钾ATP酶β亚基在顶端表面积累。当与N519H共表达时，氢钾ATP酶β亚基与该嵌合体组装并伴随其到达基底外侧表面。因此，钠钾ATP酶α亚基中的氨基末端基底外侧信号掩盖或优于β多肽中存在的任何顶端分选信息。在胃壁细胞中，氢钾ATP酶β亚基将氢钾ATP酶靶向细胞内囊泡区室，该区域在促分泌剂刺激下与质膜融合。为了测试嵌合体-氢钾ATP酶β亚基复合物是否被导向LLC-PK1细胞中的类似区室，我们用提高细胞内3',5'-环磷酸腺苷（cAMP）水平的药物处理转染细胞。胞质cAMP的升高增加了N519H嵌合体和氢钾ATP酶β亚基的表面表达。然而，这种表面表达的增加似乎是转录上调的结果，而不是嵌合体从cAMP诱导区室募集到表面的结果。
