An investigation into the role of syntaxin 16 in GLUT4 trafficking

QH301 Biology;Q Science (General)

Hamilton, Nicola Samantha ; Gould, Gwyn

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: GLUT4, SNARE, syntaxin 16, membrane trafficking;   

Others  :  http://theses.gla.ac.uk/2724/1/2011hamiltonmscr.pdf

来源: University of Glasgow

【 摘 要 】

In muscle and fat cells, insulin stimulates the translocation of GLUT4 from unique intracellular storage site(s) to the cell surface. In type II diabetes this translocation is impaired. The translocation of GLUT4 in response to insulin is an example of regulated membrane trafficking. Membrane trafficking involves the fusion of vesicles with specific target membranes and is mediated by SNARE proteins. The SNARE proteins involved in the intracellular trafficking of GLUT4 are poorly understood. Stx 16 is a t-SNARE located in the TGN which has been proposed to have a role in GLUT4 trafficking. Previous work has shown that expression of the cytosolic domain of stx 16 significantly slows the reversal of insulin-stimulated glucose transport and depletion of stx 16 using morpholino antisense oligonucleotides reduces glucose transport over a range of insulin concentrations. Stx 16 knockdown also reduces total cellular levels of GLUT4. It has been suggested that stx 16 has a role in sequestration of GLUT4 in its storage site(s). In this thesis the role of stx 16 in GLUT4 trafficking was further investigated.First of all, stx 16 expression was stably depleted in 3T3-L1 adipocytes using a shRNA approach. Knockdown of stx 16 reduced the total GLUT4 cellular levels by 20% and the majority of GLUT4 appeared to be lost from fractions which included GSVs. Stx 16 depletion also resulted in a reduction in stx 6 levels and reduced recruitment of mVps45 to the membrane.Also, assays were developed to investigate GLUT4 trafficking kinetics using 3T3-L1 adipocytes expressing HA-GLUT4-GFP.Stx 16 is phosphorylated under basal conditions and insulin stimulation decreases this phosphorylation. It is thought that this dephosphorylation regulates an important membrane trafficking event in GLUT4 sorting. Potential phosphorylation sites have been previously identified. Screening of phosphorylation site mutants in yeast suggested that stx 16 may be phosphorylated at Ser 95. Unfortunately it was not possible to confirm this in 3T3-L1 adipocytes. In summary the data in this thesis suggests that stx 16 has a role in sorting GLUT4 to its storage site(s). The precise role of stx 16 will need to be further examined to determine the how stx 16 depletion affects GLUT4 trafficking kinetics. The work presented in this thesis also suggests that stx 16 may be phosphorylated at Ser 95. This will need to be confirmed in 3T3-L1 adipocytes and the effect of stx 16 phosphorylation on GLUT4 trafficking examined.