Date of Award


Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Jonathan Bogan, Advisor

Second Advisor

Raymond Russell

Third Advisor

Thomas Melia


REGULATION OF GLUT4 GLUCOSE TRANSPORTER TRAFFICKING. Leah McNally and Jonathan Bogan, Departments of Internal Medicine and Cellular Biology, Yale University School of Medicine, New Haven CT. In fat and muscle cells, insulin stimulates glucose uptake by causing the translocation of GLUT4 glucose transporters out of intracellular membranes and to the plasma membrane. Impaired GLUT4 translocation results in insulin resistance, and contributes to the pathogenesis of type 2 diabetes. Yet, how insulin signaling and protein trafficking pathways intersect remains poorly understood. In 3T3-L1 adipocytes, data support a model in which TUG ("tether", containing a UBX domain, for GLUT4) binds GLUT4 and retains it intracellularly in the absence of insulin. Insulin then signals the release GLUT4 from TUG, which mobilizes GLUT4 to the cell surface. How and where TUG retains GLUT4 intracellularly remains unknown. Previous data show that the TUG carboxyl terminus is required for intracellular retention of GLUT4, but is dispensable for binding to GLUT4 itself. Therefore we hypothesized that this region interacts with an unidentified, intracellular "anchoring" protein. Here, we tested if GCC185 may be this sought-after protein, to which TUG links GLUT4 in unstimulated cells. GCC185 is a Golgi matrix protein that captures vesicles arriving at the trans-Golgi network (TGN) from endocytic or biosynthetic pathways. It tethers the vesicles to the TGN membrane and promotes their fusion at the TGN. It has previously been suggested that GLUT4 may by retained by an intracellular cycle of fusion and budding at the TGN in unstimulated cells. To test the hypothesis that TUG cooperates with GCC185 to facilitate such a cycle, we performed coimmunoprecipitation experiments. We found that TUG interacted with GCC185 in cotransfected 293 cells. Importantly, this interaction required the TUG carboxyl terminus, as predicted for the "anchoring" protein. The TUG-GCC185 interaction was confirmed in reciprocal coimmunoprecipitation experiments. Mutagenesis identified a particular residue in TUG that is likely involved in this interaction, which may be modified to control the binding of TUG and GCC185. A second project was prompted by the observation that Ubc9 is another protein that binds GLUT4 and promotes its accumulation in insulin-responsive storage vesicles. Because Ubc9 is a conjugating enzyme for the ubiquitin-like protein, SUMO, we tested the hypothesis that TUG is a target of SUMO modification. However, no data were obtained to support this hypothesis. In summary, our data show that GCC185 and TUG interact, and support a model in which GCC185 participates in targeting GLUT4 to vesicles that are mobilized acutely by insulin to control glucose uptake.