Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes
Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes
复制标题
DOI：
10.1046/j.1365-313x.1999.00481.x
复制
发表时间：
1999-06-01
期刊：
影响因子：
7.2
通讯作者：
Maurel, C
中科院分区：
文献类型：
--
作者：
Gerbeau, P;Güclü, J;Maurel, C
主要内在蛋白（MIP）家族的成员，在植物中被描述为水选择通道（水通道蛋白），也可以在其他生物中运输小的中性溶质。在本研究中，我们描述了植物液泡膜（tonoplasast； TP）和质膜（PM）对非电解质的渗透性，并评估了MIP同源物对这种运输的贡献。采用自由流动电泳技术从烟草悬浮细胞中纯化出PM和TP囊泡，并采用停止流动荧光法研究了膜对尿素、不同分子大小的多元醇和氨基酸等多种中性溶质的透性。对于所有测试的溶质，发现TP囊泡比PM囊泡更具渗透性，例如在内流实验中，尿素的渗透性分别为74.9+/-9.6x10（-6）和1.0+/-0.3x10(-6) cm秒（-1）。甘油和尿素在TP囊泡中的转运表现出易于扩散的特点。这一点以及相同的TP囊泡对水的高通道介导的渗透性表明MIP蛋白在水和溶质运输中的共同作用。从烟草细胞中分离到一种新的烟草内源性蛋白（TIP）同源基因，命名为Nt-TIPa。纯化膜组分的Nt-TIPa免疫检测证实该蛋白定位于TP。Nt-TIPa在非洲爪蟾卵母细胞中的功能表达表明，该蛋白对水和尿素、甘油等溶质具有渗透性。这些特征可以解释在纯化TP囊泡中确定的转运选择性谱。这些结果支持了植物水通道蛋白在水和溶质运输中具有双重功能的观点。由于Mt-TIPa的序列与其他生物中溶质渗透性水通道蛋白的序列不同，它的鉴定也为研究水通道蛋白转运选择性的分子决定因素提供了一种新的工具。
Members of the major intrinsic protein (MIP) family, described in plants as water-selective channels (aquaporins), can also transport small neutral solutes in other organisms. In the present work, we characterize the permeability of plant vacuolar membrane (tonoplast; TP) and plasma membrane (PM) to non-electrolytes and evaluate the contribution of MIP homologues to such transport. PM and TP vesicles were purified from tobacco suspension cells by free-flow electrophoresis, and membrane permeabilities for a wide range of neutral solutes including urea, polyols of different molecular size, and amino acids were investigated by stopped-flow spectrofluorimetry. For all solutes tested, TP vesicles were found to be more permeable than their PM counterparts, with for instance urea permeabilities from influx experiments of 74.9+/-9.6x10(-6) and 1.0+/-0.3x10(-6) cm sec(-1), respectively. Glycerol and urea transport in TP vesicles exhibited features of a facilitated diffusion process. This and the high channel-mediated permeability of the same TP vesicles to water suggested a common role for MIP proteins in water and solute transport. A cDNA encoding a novel tonoplast intrinsic protein (TIP) homologue named Nicotiana tabacum TIPa (Nt-TIPa) was isolated from tobacco cells. Immunodetection of Nt-TIPa in purified membrane fractions confirmed that the protein is localized in the TP. Functional expression of Nt-TIPa in Xenopus oocytes showed this protein to be permeable to water and solutes such as urea and glycerol. These features could account for the transport selectivity profile determined in purified TP vesicles. These results support the idea that plant aquaporins have a dual function in water and solute transport. Because Mt-TIPa diverges in sequence from solute permeable aquaporins characterized in other organisms, its identification also provides a novel tool for investigating the molecular determinants of aquaporin transport selectivity.