Proximity ligation assay to study the GLUT4 membrane trafficking machinery

Dimitrios Kioumourtzoglou, Gwyn W. Gould, Nia J. Bryant

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages217-227
Number of pages11
DOIs
Publication statusPublished - 1 Jan 2018

Publication series

NameMethods in Molecular Biology
Volume1713
ISSN (Print)1064-3745

Keywords

  • GLUT4 trafficking
  • GSVs
  • Munc18c
  • PLA
  • protein-protein interactions
  • proximity ligation assay
  • SNAP23
  • SNARE proteins

Cite this

Kioumourtzoglou, D., Gould, G. W., & Bryant, N. J. (2018). Proximity ligation assay to study the GLUT4 membrane trafficking machinery. In Methods in Molecular Biology (pp. 217-227). (Methods in Molecular Biology; Vol. 1713). https://doi.org/10.1007/978-1-4939-7507-5_16