Investigation of GLUT4 sorting into the insulin responsive compartment: a role for ubiquitination and deubiquitination.

Lamb, Christopher A. (2011) Investigation of GLUT4 sorting into the insulin responsive compartment: a role for ubiquitination and deubiquitination. PhD thesis, University of Glasgow.

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GLUT4 is the insulin-regulated glucose transporter found in muscle and adipose tissue. On insulin stimulation, GLUT4 translocates from a slowly recycling storage compartment (GLUT4 storage vesicles or GSVs) to the plasma membrane.
This allows glucose to enter cells by diffusion down its concentration gradient, clearing glucose from the plasma. This response is defective in the disease states of insulin resistance and type 2 diabetes. The aim of this study is to
understand how GLUT4 enters GSVs, which will hopefully extend our knowledge of insulin responsive tissues.
Previous studies from our lab, expressing GLUT4 in yeast, have shown that GLUT4 is subject to the same nitrogen- and ubiquitin-dependent trafficking as the yeast amino acid permease Gap1p. In my thesis I have extended these
studies into 3T3-L1 adipocytes, and shown that GLUT4 is ubiquitinated in this insulin responsive cell line. A ubiquitin resistant version of GLUT4 (HA-GLUT4
7K/R) has an impaired ability to enter GSVs and does not translocate in response to insulin. However GLUT4 mutants with single ubiquitination sites outwith the large intracellular loop are ubiquitinated and traffic in an identical manner to wild type GLUT4, addressing concerns that mutation of the large intracellular loop of GLUT4 in HA-GLUT4 7K/R affects its trafficking.
The GGA family of clathrin adaptor proteins have previously been implicated in sorting of newly synthesised GLUT4 into GSVs. Our lab has shown previously that the two yeast Ggas are required for ubiquitin dependent trafficking of GLUT4 in
yeast, as is the case for Gap1p. I have gone on to show that the ubiquitin binding function of the GGA3 GAT domain is, at least partially, required for an in vitro interaction between GLUT4 and the VHS-GAT domains of GGA3. When expressed in adipocytes, a ubiquitin binding deficient mutant of myc-GGA3 reduces the proportion of GLUT4 loaded into a subcellular fraction enriched in GSVs, suggesting that GLUT4 ubiquitination is one of the signals for GGA
dependent sorting into GSVs.
As ubiquitination is usually thought of as a signal to direct lysosomal degradation, and only 0.1 % of total GLUT4 is ubiquitinated at any one time, there may be a role for a deubiquitination step in ubiquitin dependent GLUT4
traffic. Work by our collaborator (Nai-Wen Chi, UCSD) has demonstrated that the GSV cargo IRAP and its binding partner tankyrase-1 are required for normal insulin responsive GLUT4 traffic. An interaction between tankyrase and the deubiquitinase (DUB) USP25 has been demonstrated by yeast two hybrid analysis, and this DUB contains a putative tankyrase binding motif. USP25 may therefore be recruited to GSVs by IRAP, with tankyrase acting as a scaffold. I
demonstrated that GST-USP25 binds tankyrase-1 from an adipocyte lysate, and that a version of the enzyme with a mutation in the putative tankyrase binding motif (GST-USP25 R1049A) does not. I also used siRNA to deplete USP25 from
3T3-L1 adipocytes, and found that this results in a reduction of GLUT4 levels in these cells. A concomitant reduction in the fold change of insulin-stimulated
glucose transport into these cells suggests that GLUT4 is not sequestered in GSVs, but is rather directed to the lysosome.
In summary, my data show that ubiquitination of GLUT4 is required for the transporter to be loaded into its insulin responsive compartment (GSVs). I also began to characterise the role of the ubiquitin binding GAT domain of GGA3 and
the deubiquitinase USP25 in GLUT4 traffic, opening up two further avenues for research into the insulin regulated trafficking of GLUT4.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: GLUT4, ubiquitin, trafficking, Type II diabetes.
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Bryant, Dr. Nia
Date of Award: 2011
Depositing User: Mr Christopher A Lamb
Unique ID: glathesis:2011-2380
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 18 Feb 2011
Last Modified: 10 Dec 2012 13:54

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