(Taken directly from the application) Urothelium not only serves as an effective urine-blood barrier, but also has the remarkable ability to adjust reversibly its apical surface area during the micturition cycle. These urothelial functions are usually attributed to the highly specialized urothelial apical surface, which is covered with numerous, rigid-looking plaques. Such plaques, also known as the asymmetric unit membranes (AUM) due to the thickening of their outer leaflets, consist of 16-nm protein particles forming two-dimensional (2D) hexagonal crystals. This biomembrane offers unique opportunities to study how integral membrane proteins interact to form a highly organized structure, because (1) it has a relatively simple protein composition containing only 4 well-characterized major proteins, the uroplakins (UP) Ia, Ib, II and III; (2) it can be isolated in highly purified and morphologically intact form in tremendous quantities (greater than 10 mg per day); (3) it naturally forms 2D crystals already suitable for EM analysis; and (4) we have available a panel of antibodies to well-defined epitopes of all the uroplakins. Recent studies at 2.2 nm resolution revealed that the lumenal portion of the 16-nm AUM particle assumes a closed """"""""twisted ribbon"""""""" configuration. The goals of this project are to localize individual uroplakins in different domains of this ribbonlike structure, to better understand how the individual uroplakins traverse through the lipid bilayer, and to learn how they interact with one another to form the 16-nm AUM particle. Towards these goals, we will perform three series of studies. First, we will determine by EM coupled with image enhancement the association of various uroplakins and their sugar moieties with the six inner and six outer subdomains of the 16-nm particle using Fab fragments and specific lectins as the probes. Second, we will perform electron cryo-microscopy in order to assess the structure of the transmembrane domains of the uroplakins in naturally occurring 2D AUM crystals. Third, we will fractionate the detergent-solubilized uroplakin complexes and purify the two populations that contain UPIa/UPII and UPIb/UPIII to high purity and homogeneity suitable for crystallization, aiming at determining the high resolution structure of the AUM particles in the future. The detailed structural information that we will generate here will contribute to a better understanding of AUM structure, and may shed new light into the biological function of urothelial plaques.

Project Start
2001-12-01
Project End
2002-11-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
4
Fiscal Year
2002
Total Cost
$165,355
Indirect Cost
Name
New York University
Department
Type
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016
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Vieira, Neide; Deng, Fang-Ming; Liang, Feng-Xia et al. (2014) SNX31: a novel sorting nexin associated with the uroplakin-degrading multivesicular bodies in terminally differentiated urothelial cells. PLoS One 9:e99644
Desalle, Rob; Chicote, Javier U; Sun, Tung-Tien et al. (2014) Generation of divergent uroplakin tetraspanins and their partners during vertebrate evolution: identification of novel uroplakins. BMC Evol Biol 14:13
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