Non-Erythroid Beta Spectrin: Effects of Mutations and of Interacting Proteins on Tetramerization
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Non-Erythroid Beta Spectrin: Effects of Mutations and of Interacting Proteins on Tetramerization Akin Sevinc Department of Chemistry University of Illinois at Chicago Chicago, IL (2011) Dissertation Chairperson: Dr. Leslie Wo-Mei Fung Spectrin performs its fundamental role by forming a filamentous network beneath the plasma membrane, where the association of α and β subunits to form tetramers is crucial. We employed yeast two-hybrid (Y2H) methods to study the mutational effects of non-erythroid alpha spectrin (II) at position 22 in tetramer formation with non-erythroid beta spectrin (II). Colony growth and β-galactosidase assays of Y2H system qualitatively showed that wild-type and three mutants (V22W, V22M, and V22F) interacted with βII-C, whereas V22D mutant did not. These results correlated with isothermal titration calorimetry (ITC) results. We also screened a human brain cDNA library using the C-terminal fragment (residues 1697-2145) of βII (βII-C), which includes the tetramerization region, as the bait, to identify proteins interacting with the bait protein. Library screening results showed that 17 proteins interacted with βII-C (IPβII-C s). These proteins are a fragment (residues 38-284) of "THAP domain containing, apoptosis associated protein 3, isoform CRA g", "glioma tumor suppressor candidate region gene 2" (residues 1-478), a fragment (residues 74-442) of septin 8 isoform c, a fragment (residues 704-953) of "coatomer protein complex, subunit beta 1", a fragment (residues 146-614) of zinc-finger protein 251, and a fragment (residues 284-435) of syntaxin binding protein 1, as well as 4 unknown proteins. Using yeast three-hybrid system to determine the effects of IPβII-C s on spectrin tetramer formation, we found that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N. We also found that one of these proteins, the syntaxin binding protein 1 fragment, abolished spectrin tetramerization. This suggests that this protein may be implicated in the regulation of the non-erythroid spectrin tetramer formation. Similar studies were also done on 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) (Oh and Fung, 2007), and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. Our results demonstrate the effects of mutations and of interacting proteins on the interaction of α and β spectrin isoforms to form the spectrin tetramers. Given the importance of spectrin tetramerization for its cellular functions, these effects may provide a better understanding of the physiology and pathophysiology of neuronal cells.
yeast two-hybrid system
yeast three-hybrid system