Jiang L, Rogers J C
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
J Cell Biol. 1998 Nov 30;143(5):1183-99. doi: 10.1083/jcb.143.5.1183.
Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of alpha-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of gamma-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of gamma-TIP did not alter this traffic. In contrast, the alpha-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and alpha-TIP protein colocalized in them with the alpha-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.
植物细胞可能含有两个功能不同的液泡区室。蛋白质储存液泡（PSV）的膜以α-液泡膜内在蛋白（TIP）的存在为标志，而溶酶体液泡（LV）则以γ-TIP的存在为标志。将整合膜蛋白分选到不同液泡的机制尚未阐明。在这里，我们研究了一种在烟草悬浮培养原生质体中表达的嵌合整合膜报告蛋白，通过跟踪复杂的天冬酰胺连接聚糖修饰的获得和蛋白水解加工对其运输进行生化评估，并通过共聚焦免疫荧光确定其细胞内定位。我们表明，植物液泡分选受体BP-80的跨膜结构域将报告蛋白通过高尔基体导向LV前液泡区室，并且连接γ-TIP的细胞质尾巴（CT）不会改变这种运输。相反，α-TIP CT阻止报告蛋白通过高尔基体运输，并使其定位在与内质网、高尔基体和LV前液泡区室标记物分开的细胞器中。这些细胞器的浮力密度与液泡一致，并且当α-TIP蛋白和α-TIP CT报告蛋白在原生质体中共同表达时，它们在其中共定位。这些结果与膜蛋白进入液泡的两条独立途径一致：一条是从内质网直接到PSV的途径，另一条是通过高尔基体到LV的独立途径。
