Sevinc_Akin.pdf (2.44 MB)
Download file

Non-Erythroid Beta Spectrin: Effects of Mutations and of Interacting Proteins on Tetramerization

Download (2.44 MB)
posted on 15.04.2014, 00:00 authored by Akin Sevinc
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.



Fung, Leslie



Degree Grantor

University of Illinois at Chicago

Degree Level


Committee Member

Kassner, Richard J. Burns, Richard P. Miller, Lawrence Mehboob, Shahila

Submitted date




Issue date