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Title page for ETD etd-03212018-153959

Type of Document Dissertation
Author Frazier, Meredith N.
URN etd-03212018-153959
Title Structural and Functional Studies of AP4 and its Accessory Protein Tepsin
Degree PhD
Department Biological Sciences
Advisory Committee
Advisor Name Title
Todd R. Graham Committee Chair
Brandt F. Eichman Committee Member
Charles R. Sanders Committee Member
Kendal S. Broadie Committee Member
Lauren P. Jackson Committee Member
  • membrane trafficking
  • structural biology
Date of Defense 2018-03-12
Availability restricted
The adaptor protein 4 (AP4) heterotetrameric complex helps form vesicles and traffic cargo from the trans-Golgi Network. Tepsin, a member of the epsin family of trafficking proteins, is the only known accessory protein of AP4. My research focuses on understanding the interaction between AP4 and tepsin, and the function of tepsin.

Through analysis of the largely unstructured C-terminus of tepsin, I identified a conserved motif that interacts specifically with AP4 β4. Mutagenesis of the motif affected binding in vitro and lead to loss of tepsin at the membrane in cells. I also showed tepsin binds a surface on β4 conserved across AP β subunits. Slight differences in the surfaces give rise to specific epsin family binding interactions. These data provided the first molecular description of the AP4-tepsin interaction.

Unlike other epsins, tepsin contains a unique internal domain named VHS/ENTH-like (tVHS). I determined a high-resolution (1.85Å) structure of the tVHS domain and found it is smaller than other VHS domains. The structure explains negative binding data between tVHS and typical VHS interactors such as ubiquitin and acidic dileucine motifs. The first structure of an internal VHS domain, it raised interesting questions about the evolution of the ENTH/ANTH/VHS domain family. We showed through phylogenetic analyses that tVHS clusters with VHS domains, but tepsin diverges early from other epsins, in line with my functional data.

Commercial Y2H screens have revealed potential binding partners for tVHS and the N-terminal ENTH domain (tENTH). I have shown direct interactions between the tVHS domain and ISCA1, an iron-sulfur cluster assembly protein, as well as tENTH and c16orf62, a newly discovered retromer-like subunit, and we are validating the interactions in cells.

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