Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct
Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct
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DOI：
10.1074/mcp.m500049-mcp200
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发表时间：
2005-08-01
影响因子：
7
通讯作者：
Knepper, MA
中科院分区：
文献类型：
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作者：
Barile, M;Pisitkun, T;Knepper, MA
加压素作用于肾集合管细胞以刺激含有水通道蛋白-2（AQP 2）的膜囊从整个细胞质移位到顶端区域。囊泡与质膜融合以增加水渗透性。为了确定含有AQP 2的细胞内膜室，我们进行了基于LC-MS/MS的蛋白质组学分析的免疫分离的含有AQP 2的细胞内囊泡从大鼠内髓集合管。免疫金电子显微镜和免疫印迹证实了免疫隔离囊泡的AQP 2标记。囊泡蛋白通过SDS-PAGE分离，然后在连续的凝胶切片中进行凝胶内胰蛋白酶消化，并通过LC-MS/MS进行鉴定。Rab GTP酶4、5、18和21（与早期内体相关）; Rab 7（晚期内体）; Rab 11和Rab 25（再循环内体）的鉴定表明，胞内AQP 2的大部分存在于内体隔室中。此外，还鉴定了几种内体相关SNARE蛋白，包括syntaxin-7、syntaxin-12、syntaxin-13、Vti 1a、囊泡相关膜蛋白2和囊泡相关膜蛋白3。Rab 3没有被发现，但是，无论是通过质谱或免疫印迹，这表明相对缺乏AQP 2的分泌囊泡。此外，我们确定了标记的trans-Golgi网络，组件的外囊复合物，和几个马达蛋白，包括肌球蛋白1C，非肌肉肌球蛋白IIA和IIB，肌球蛋白VI，肌球蛋白IXB。除此之外，多个内质网驻留蛋白和核糖体蛋白的鉴定表明，细胞内AQP 2的相当大一部分存在于粗面内质网中。这些结果表明，含有AQP 2的囊泡是异质性的，细胞内AQP 2主要存在于内体，trans-Golgi网络和粗面内质网。
Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2 (AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct. Immunogold electron microscopy and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 ( associated with early endosomes); Rab7 ( late endosomes); and Rab11 and Rab25 ( recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, vesicle-associated membrane protein 2, and vesicle-associated membrane protein 3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network, components of the exocyst complex, and several motor proteins including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB. Beyond this, identification of multiple endoplasmic reticulum-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum. These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, trans-Golgi network, and rough endoplasmic reticulum.