Novel Proteins Associated with GLUT4 Vesicle Subcellular Traffic

Abstract

GLUT4 mobilisation involves endocytic and exocytic processes that are profoundly enhanced by insulin. Insulin indirectly mediates GLUT4 sorting into storage or recycling pools via endosomal trafficking proteins and phosphoinositides. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery is involved in the endosomal sorting of ubiquitinated and phosphoinositide regulated cargo into subcellular pools. The findings in this thesis implicate the involvement of the ESCRT machinery in sorting ubiquitinated GLUT4 to direct it to its specialised storage compartment. Overexpression of ESCRT-III dominant negative constructs (CHMP3 1-179 and Vps4 E235Q) in rat adipocytes led to accumulation of GLUT4 in enlarged compartments. Consequently, GLUT4 seems to be trapped in the enlarged compartment and prevented from sorting to storage and recycling pools. The enlarged GLUT4 containing compartment was positive for early endosomal and TGN-associated markers, suggesting that it is a hybrid compartment formed during ESCRT-III- dependent sorting of GLUT4 into other subcellular pools.

Once sorted into its specialised storage compartment, GLUT4 movement to the plasma membrane requires exocytic proteins involved in vesicle translocation, tethering, docking and fusion. The study described in this thesis aimed to characterise the involvement of Rab3B as a potential regulator of GLUT4 exocytosis. Rab3B was identified as an insulin-responsive Rab that regulated GLUT4 trafficking. Overexpression of the constitutively active Rab3B construct led to an increase in cell surface GLUT4 in basal rat adipocytes compared to the wild type or dominant negative Rab3B constructs. Noc2 was explored as a potential Rab3B effector due to its known involvement in Rab3-mediated trafficking in other vesicular trafficking processes. Noc2 showed reduced interaction with Rab3B in insulin-treated adipocytes and the overexpression of wild type Noc2 in adipocytes led to a reduction in cell surface GLUT4 upon insulin stimulation. It was concluded that Noc2 has an inhibitory effect on Rab3B action in GLUT4 exocytosis.

The findings in this thesis uncover novel signalling and trafficking pathways that are important in regulating cell surface levels of GLUT4 in response to insulin. The proteins described in this thesis were shown to influence GLUT4 mobilisation making them important regulators of GLUT4 traffic.

Date of Award	16 Sept 2014
Original language	English
Awarding Institution	University of Bath
Supervisor	Geoff Holman (Supervisor) & Francoise Koumanov (Supervisor)