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dc.contributor.authorRay, Gregoryen_US
dc.identifier.otherbibid: 8641142
dc.description.abstractThe Arabidopsis thaliana synaptotagmin SYTA (AT2G20990) regulates endocytosis at the plasma membrane and virus movement protein-mediated cellto-cell movement. As with all synaptotagmin proteins, SYTA is predicted to consist of a transmembrane domain, a cytosolic variable domain, and two calcium/lipid binding domains (C2A and C2B) at its COOH-terminus. Deletion of the C2B domain abolishes SYTA function. The C2B deleted mutant of SYTA also acts as a dominant-negative mutant as evidenced by its interference with endogenous, wild-type SYTA. This finding is consistent with the unproven hypothesis that synaptotagmin proteins in animals potentially function as dimers or tetramers. However, the existence of a SYTA C2B domain in plants that is functionally similar to those in animal synaptotagmins has been questioned by some research groups. In this project, I utilized molecular modeling to predict how a homodimer of SYTA may function, and cell-based functional assays and in vitro biochemical approaches to demonstrate the relevance of the model I created. I modeled SYTA-C2B to explain how the C2B domains from the individual proteins within a dimer could function to bind calcium. I demonstrated that key residues from this model (E430, D431, and E433) were functionally relevant by expressing alanine point mutants of each in protoplasts and observing that they did not localize to endosomes effectively. My research was consistent with the prediction that E430 and D431 are essential for SYTA function, possibly forming the core of a calcium-binding site. Although it is not essential in this activity, I also concluded that E433 may improve the calciumsensing ability of C2B. By utilizing dynamic and static light scattering, I observed that purified SYTA is a dimer, which indicated calcium binding via the C2B domain is not required for the formation of this dimer. This research is the first direct observation of a synaptotagmin protein, plant or animal, forming a dimer.en_US
dc.titleArabidopsis Thaliana Syta As A Model To Address Whether Synaptotagmin Proteins Function As Dimers Or Tetramersen_US
dc.typedissertation or thesisen_US and Cell Biology Universityen_US of Philosophy D., Molecular and Cell Biology
dc.contributor.chairLazarowitz, Sondra Galeen_US
dc.contributor.committeeMemberVogt, Volker Men_US
dc.contributor.committeeMemberParrish, Colin Rossen_US

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