The cell surface localization and the physiological roles of type 2 phosphatidic acid phosphatase

最近克隆了哺乳动物2型磷酸酶(PAP-2)的几种同工酶，它们的催化部位暴露在细胞表面膜上。我们研究了在HEK293细胞中表达的人PAP-2b作为绿色荧光融合蛋白(GFP)对细胞外脂底物的利用方式。有趣的是，PAP-2b对长链磷脂酸的活性可以忽略不计，无论是外源添加到细胞膜中，还是通过细菌磷脂酶D处理细胞内。这些发现表明，PAP-2b作用于细胞表面双层的外叶，可以解释先前描述的各种类型细胞的ecto-PAP活性。接下来，我们详细研究了PAP2a和PAP2b的洗涤剂溶解度。尽管Triton X-100能溶解PAP2a，但不能如前所述溶解PAP2b，但这两种PAP2同工酶在非离子洗涤剂CH…的处理中都不能溶解不溶于CHAPS的PAP2s经密度梯度离心法分离成低密度膜组分，表明PAP2a和PAP2b分别以Triton X-100敏感和抗性的方式与RAFT结合。共聚焦显微镜分析发现了两种类型的运输囊泡，较大的一个同时含有PAP2a和2b，较小的一个只含有PAP2b。这些观察结果表明，PAP2同工酶通过不同的机制运输到质膜上，促使我们研究PAP2在极化细胞中的定位。我们发现，人PAP2a和PAP2b分别与GFP融合在极化的Mardin-Darby犬肾(MDCK)细胞中，靶向相反的膜结构域。PAP2a的顶端分布是由PAP2a序列N端6个氨基酸残基(Phe2-Leu7)组成的新的顶端排序信号决定的。PAP2b在极化的MDCK细胞中呈基线分布，分选信号位于Ser72-Arg114序列的一部分。较少

Several isozymes of mammalian type 2, Mg^<2+>-independent phosphatidic acid phosphatase (PAP-2) have recently been cloned, and they are predicted to have their catalytic sites exposed at the cell surface membranes. We investigated the mode of utilization of extracellular lipid substrates by the human PAP-2b expressed in HEK293 cells as a green fluorescent fusion protein (GFP).Interestingly, PAP-2b exhibited negligible activities toward long-chain phosphatidic acid either exogenously when added or generated within the membranes by treating the cells with bacterial phospholipase D. These findings indicate that PAP-2b acts at the outer leaflet of cell surface bilayers and can account for the ecto-PAP activities previously described for various types of cells. Next, we investigated the detergent solubilities of PAP2a and PAP2b in detail. Although Triton X-100 solubilized PAP2a but not PAP2b as described previously, both PAP2 isozymes were insoluble in treatment with a nonionic detergent CH … More APS. The CHAPS-insoluble PAP2s were fractionated into low-density membrane fractions after density gradient centrifugation, suggesting that PAP2a and PAP2b associates with raft in a Triton X-100-sensitive and resistant manner, respectively. Confocal microscopic analysis has revealed two types of transport vesicles, larger one containing both PAP2a and 2b and smaller one containing only PAP2b. These observations, suggesting that the PAP2 isozymes are transported to plasma membranes by distinct mechanisms, prompted us to investigate the localization of the PAP2s in polarized cells. We has found that the human PAP2a and PAP2b fused with GFP, respectively, were targeted to opposite membrane domains in the polarized Mardin-Darby canine kidney (MDCK) cells. The apical distribution of PAP2a is determined by a novel apical sorting signal composed with six amino acid residues (Phe2-Leu7) in the N-terminus of PAP2a sequence. PAP2b is basolateral distributed in polarized MDCK cells and the sorting signal lies in the portion of the sequence Ser72-Arg114. Less