Proximity Ligation Assay to Study the GLUT4 Membrane Trafficking Machinery.

In this chapter a detailed protocol of proximity ligation assay (PLA) is described thoroughly. PLA is a technique that allows detection of protein associations in situ, providing a sensitive and selective approach for protein-protein interaction studies. We demonstrate the technique by applying it for trafficking studies of the facilitative glucose transporter GLUT4. Trafficking of GLUT4 from perinuclear depots to the plasma membrane is regulated by insulin in adipocytes and muscle cells, and mediated by formation of functional SNARE complexes containing Syntaxin4, SNAP23, and VAMP2. The Sec1/Munc18 (SM) protein Munc18c also plays a key role in insulin-stimulated GLUT4 translocation via a series of different interactions with the SNARE complex and/or with the SNARE proteins individually. Studying the interactions that occur between SNARE proteins themselves and also with Munc18c in insulin-responsive cells is critical to further understand SNARE protein function and GLUT4 trafficking mechanism in general.