Abstract
To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.
Highlights
GLUT4 glucose transporters are trapped and sequestered intracellularly in adipocytes by TUG
Based on an initial immunoblot suggesting that TUG is cleaved, we considered that TUG contains a third ubiquitin-like domain and that it may be cleaved at the 164 –165 bond (Fig. 1A)
Glucose uptake is tightly regulated by insulin, which controls the number of GLUT4 glucose transporters present in plasma membranes of fat and muscle cells
Summary
GLUT4 glucose transporters are trapped and sequestered intracellularly in adipocytes by TUG. The notion that insulin stimulates the release of sequestered GSVs to the cell surface and that GLUT4 participates in an endosomal recycling pathway is consistent with previous data suggesting a “quantal release” mechanism for GLUT4 mobilization [12, 13]. According to this model, discrete packets of GLUT4 molecules are liberated from a sequestered storage compartment into the recycling pool, yet the mechanism by which GLUT4 is released from TUG is not understood. Our data support the concept that TUG proteolysis is a novel enzymatic activity by which insulin mobilizes GSVs to stimulate glucose uptake
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