Identification, purification, and molecular cloning of N-1-naphthylphthalmic acid-binding plasma membrane-associated aminopeptidases from Arabidopsis
Identification, purification, and molecular cloning of N-1-naphthylphthalmic acid-binding plasma membrane-associated aminopeptidases from Arabidopsis
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DOI：
10.1104/pp.010519
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发表时间：
2002-03-01
期刊：
影响因子：
7.4
通讯作者：
Taiz, L
中科院分区：
文献类型：
--
作者：
Murphy, AS;Hoogner, KR;Taiz, L
植物激素生长素的极地运输在细胞水平上是通过抑制质膜(PM)载体的外流来调节的。生长素转运抑制剂N-1-萘基邻苯二甲酸(NPA)与载体相关的调节位点的结合已被表征，但NPA结合蛋白(S)尚未被鉴定。高亲和力NPA结合水平和生长素运输抑制水平之间的实验差异可以用低亲和力结合位点的存在和NPA在体内的水解来解释。在拟南芥中，NPA酰胺酶和氨基肽酶(AP)活性的共存，人工β-萘胺底物对生长素运输的抑制，以及AP抑制剂Bestatin对NPA的饱和置换，表明PM AP可能参与了低亲和力的NPA结合和水解。我们报道了拟南芥NPA结合的质膜蛋白及其相关蛋白的纯化和分子克隆。这是植物中PM AP的首次报道。PM蛋白通过凝胶渗透、阴离子交换和NPA亲和层析进行酪氨酸-AP活性检测。低亲和力组分包含两个哺乳动物AP的同源基因，它们参与信号转导和细胞表面-细胞外基质相互作用。ATAPM1和ATAPP1具有与其哺乳动物同源基因相似的底物特异性和抑制剂敏感性，并且具有与先前植物组织化学数据一致的时空表达模式。复制蛋白表明AP与分泌的细胞表面和细胞壁富含脯氨酸的蛋白相互作用。AtAPM1和AtAPP1由单基因编码。ATAPM1和AtAPP1的体外翻译产物具有与天然蛋白相似的酶活性。
dPolar transport of the plant hormone auxin is regulated at the cellular level by inhibition of efflux from a plasma membrane (PM) carrier. Binding of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) to a regulatory site associated with the carrier has been characterized, but the NPA-binding protein(s) have not been identified. Experimental disparities between levels of high-affinity NPA binding and auxin transport inhibition can be explained by the presence of a low-affinity binding site and in vivo hydrolysis of NPA. In Arabidopsis, colocalization of NPA amidase and aminopeptidase (AP) activities, inhibition of auxin transport by artificial beta-naphthylamide substrates, and saturable displacement of NPA by the AP inhibitor bestatin suggest that PM APs may be involved in both low-affinity NPA binding and hydrolysis. We report the purification and molecular cloning of NPA-binding PM APs and associated proteins from Arabidopsis. This is the first report of PM APs in plants. PM proteins were purified by gel permeation, anion exchange, and NPA affinity chromatography monitored for tyrosine-AP activity. Lower affinity fractions contained two orthologs of mammalian APs involved in signal transduction and cell surf ace-extracellular matrix interactions. AtAPM1 and ATAPP1 have substrate specificities and inhibitor sensitivities similar to their mammalian orthologs, and have temporal and spatial expression patterns consistent with previous in planta histochemical data. Copurifying proteins suggest that the APs interact with secreted cell surface and cell wall proline-rich proteins. AtAPM1 and AtAPP1 are encoded by single genes. In vitro translation products of ATAPM1 and AtAPP1 have enzymatic activities similar to those of native proteins.