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

哺乳动物2型镁离子非依赖性磷脂酸磷酸酶（PAP-2）的几种同工酶最近已被克隆出来，并预测它们的催化位点暴露在细胞表面膜上。我们研究了在HEK 293细胞中表达的人PAP-2b作为绿色荧光融合蛋白（GFP）对细胞外脂质底物的利用模式。有趣的是，PAP-2b对长链磷脂酸的活性可以忽略不计，无论是外源加入时还是用细菌磷脂酶D处理细胞膜内产生的。这些研究结果表明，PAP-2b的行为在细胞表面双层的外小叶，并可以解释的各种类型的细胞中先前描述的外-PAP活动。接下来，我们详细研究了PAP 2a和PAP 2b的洗涤剂溶解度。虽然如前所述Triton X-100溶解了PAP 2a而不是PAP 2b，但两种PAP 2同工酶在用非离子去污剂CH 关于我们 APS。CHAPS不溶性的PAP 2在密度梯度离心后被分离成低密度的膜组分，这表明PAP 2a和PAP 2b分别以Triton X-100敏感和抗性的方式与筏相关联。共聚焦显微镜分析揭示了两种类型的运输囊泡，较大的一个含有PAP 2a和2b，较小的一个只含有PAP 2b。这些观察结果表明，PAP 2同工酶通过不同的机制运输到质膜，促使我们研究PAP 2s在极化细胞中的定位。我们已经发现，与GFP融合的人PAP 2a和PAP 2b分别靶向极化的Mardin-Darby犬肾（MDCK）细胞中的相反膜结构域。PAP 2a的顶端分布由PAP 2a序列N端的6个氨基酸残基（Phe 2-Leu 7）组成的新的顶端分选信号决定。PAP 2b在极化的MDCK细胞中基底侧分布，分选信号位于序列Ser 72-Arg 114的部分。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