The GLUT4 storage vesicle pool is maintained by intracellular nanovesicle traffic

Glucose transporter 4 (GLUT4) is sequestered intracellularly until the action of insulin causes its redistribution to the cell surface. How cells sequester GLUT4, generating and maintaining GLUT4 storage vesicles (GSVs), is still unclear. Intracellular nanovesicles (INVs) operate on various membrane trafficking pathways and show molecular similarity to GSVs. Here we show that GLUT4 and associated GSV cargo proteins (IRAP/LNPEP, cellugyrin/SYNGR2, sortilin/SORT1, and VAMP2) are trafficked by INVs, which we term GLUT4-flavor INVs. The majority of GLUT4 is carried in GLUT4-flavor INVs which represent a small fraction of the total INV pool. We use a method to capture specific vesicles and test their involvement in the redistribution of GLUT4 in response to insulin. Direct capture of defined GSVs ablated the response, however capture of INVs had a smaller effect. We show that the function of GLUT4-flavor INVs is in the sorting of GSV proteins to supply the insulin-responsive GSV pool and that interfering with INV function by TPD54/TPD52L2 depletion results in mistrafficking of GLUT4 to the plasma membrane. These findings establish GLUT4-flavor INVs as the precursor GSVs that are responsible for intracellular sequestration of GLUT4.