2002 Global Researcher Conference Proceeding
April 26 - 28, 2002
| Conference: | 2002 Global Researcher Conference |
|---|---|
| Title: | N-linked glycosylation is essential for transport of the Aquaporin-2 water channel to the plasma membrane in MDCK cells |
| Authors: | Hendriks, Giel; Koudijs, Marco; van Balkom, Bas; Deen, Peter M.T.; van der Sluijs, Peter |
| Institutions: | Utrecht University School of Medicine, University of Nijmegen, Utrecht University |
Aquaporins are channel-forming proteins which allow highly efficient osmotic water transport across biological membranes. Aquaporin-2 (AQP2) is expressed in renal collecting duct epithelial cells where it is responsible for the reabsorption of water from the pro-urine. Mutations in AQP2 can result in the development of Nephrogenic Diabetes Insipidus. AQP2 is stored in intracellular vesicles which fuse with the apical cell surface in response to the anti-diuretic hormone vasopressin and subsequent phosphorylation of AQP2 by PKA. AQP2 is a 29 kD glycoprotein with 6 transmembrane domains and its N- and C-termini facing the cytosol. Little is known about the molecular mechanisms involved in the formation of functional AQP2 water channels. We show that up to 25% of the newly synthesized AQP2 is glycosylated. Both non-glycosylated and glycosylated forms of AQP2 have a half-life of › 12 hours and are both expressed at the cell surface. AQP2 forms a tetrameric complex in the endoplasmic reticulum containing glycosylated and non-glycosylated AQP2 subunits. A glycosylation knock-out mutant AQP2-N123Q is unable to reach the cell surface. Xenopus oocytes injected with AQP2-N123Q cDNA nevertheless show a water permeability comparable with wild type AQP2. AQP2-N123Q expressed in MDCK cells is able to form tetrameric complexes. Subcellular localization of AQP2 and AQP2-N123Q in MDCK cells show that glycosylation of AQP2 is essential for proper transport to the plasma membrane. The absence of glycosylation does not lead to a completely misfolded protein since AQP2-N123Q is able to exit the ER. AQP2-N123Q and AQP2 treated with the glycosylation inhibitor tunicamycin accumulate in the Golgi complex, suggesting that glycosylation is important for sorting of AQP2 in the Golgi and subsequent transport to the plasma membrane.
Glycosylation is the process wherein a molecular structure forms a link with a glycosyl group. Hendriks, et al., wanted to see if this process was essential to the movement of aquaporin-2 (AQP2) from the cell interior to the cell plasma. They experimented with a mutant AQP2 (AQP2 N123Q) that did not have the necessary parts for glycosylation to occur. This mutant is unable to reach the cell membrane, yet is still capable of letting water pass through it.
Analysis of the experimental cell cultures revealed that glycosylation is essential for AQP2's transport to the cell membrane. The researchers suggest that glycosylation needs to happen so a section of the cell interior called the Golgi complex can sort the AQP2 properly in order to send it on its way to the cell membrane.