Sorting and transport of secretory and membrane proteins occur at the trans-Golgi network (TGN). Carriers of the TGN to the cell surface (CARTS) are one of the carriers that mediate the transport of certain proteins from the TGN to the plasma membrane. Recent studies have shown that CARTS formation is dependent on membrane contact sites between the Golgi apparatus and the endoplasmic reticulum (ER). Here, we describe a method to visualize by fluorescence microscopy the formation of CARTS at the TGN. This method combines a reverse dimerization system for synchronized export from the ER of a CARTS-specific cargo, pancreatic adenocarcinoma upregulated factor, together with the halt of export from the TGN by a 20 °C block. Incubation of cells at 37 °C releases the 20 °C block and allows to monitor the formation of CARTS at the TGN. Finally, we also present a workflow to quantify CARTS formation using ImageJ software.
Keywords: Membrane contact sites; Membrane trafficking; Protein kinase D; Reverse dimerization system; Trans-Golgi network.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.