STRUCTURAL ANALYSIS OF SPRINTER

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recently, we have identified a novel multi-transmembrane protein in Drosophila we named Sprinter (Srt), which is required for the secretion of active Wg/Wnt from ligand expressing cells. Although it is known that Srt is necessary for secretion of active Wg/Wnt, its actual function in the complex maturation process of Wnts is not yet understood. Analysis of the Srt amino acid suggests it could have several possible topologies. We hypothesize Srt has 4 transmembrane domains with a large N-terminal globular extracellular/luminal domain which likely constitutes the predominate functional region of this protein. This region includes a conserved structural domain of unknown function, DUFF1141. The overall objective of this proposal is to initiate structural and functional characterization of the Srt globular extracellular/luminal domain and determine the overall topology of Srt. To do this we will first perform topological studies to confirm our predicted structure. Next, we will develop constructs that will allow for high levels of expression of N-terminal Srt for its purification, as well as its in vivo expression. We will use these tools in order to ascertain the function of the N-terminal region in vivo and determine if Srt and its N-terminal region interact with Wg/Wnt and possibly other proteins involved in Wnt processing. Ultimately we believe these studies will lead to collaboration with a laboratory here at the University of Delaware to perform structural analysis of the purified Srt N-terminal globular domain by X-ray crystallography or nuclear magnetic resonance to the integrate structural and functional aspects of Srt.