Large Scale Protein Identification in Intracellular Aquaporin-2 Vesicles from Renal Inner Medullary Collecting Duct

Abstract

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. Molecular & Cellular Proteomics 4:1095-1106, 2005. Aquaporin-2 (AQP2)1 is the vasopressin-regulated molecular water channel of the renal collecting duct where it constitutes the major route of water movement across the apical plasma membrane. Vasopressin rapidly increases the water permeability of the collecting duct epithelium by binding to V 2 receptors in the basolateral plasma membrane and inducing the cAMP-dependent trafficking of AQP2-containing vesicles from throughout the cytoplasm to the apical region of collecting duct principal cells followed by fusion of these vesicles with the apical membrane of collecting duct cells (1). Although this fundamental mechanism is well established, there is little information about the specific intracellular protein trafficking pathways involved and the nature of the intracellular compartments in which AQP2 resides.Until now, studies to identify the intracellular localization of AQP2 in collecting duct cells have depended largely on two fundamental approaches, namely immunoelectron microscopy and immunofluorescence immunocytochemistry with confocal microscopy. Immunoelectron microscopy (1, 2) has demonstrated ...

Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion

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Proteomic Analysis of GLUT4 Storage Vesicles Reveals LRP1 to Be an Important Vesicle Component and Target of Insulin Signaling

“…Moreover, it is striking that vasopressin-sensitive aquaporin-2 containing vesicles that regulate water retention in the collecting duct of the kidney have many of the same protein constituents as GSVs, including IRAP, sortilin, and both mannose 6-phosphate receptors, as well as many or all of the same membrane trafficking machinery components that might be expected (62). Thus, the mass action and self-assembly model may be a common pathway for the formation of several types of tissue-specific, regulated vesicular traffic.…”

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

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