| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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
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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
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