Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 May;17(5):2303-11.
doi: 10.1091/mbc.e06-01-0030. Epub 2006 Mar 8.

Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering

Mayumi Inoue  1 , Shian-Huey Chiang, Louise Chang, Xiao-Wei Chen, Alan R Saltiel
Affiliations

Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering

Mayumi Inoue et al. Mol Biol Cell. 2006 May.

Abstract

Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Knockdown of the exocyst complex blocks glucose uptake in 3T3L1 adipocytes. (A) Differentiated 3T3L1 adipocytes were transfected with Exo70 RNAi or scrambled oligo. Four days later, the cells were treated with or without 100 nM insulin for 30 min, and the rate of 2DG uptake was determined. Results are the mean ± SD of triplicate determinations and were reproduced five times. *Significant difference, p < 0.05. Protein expression levels were checked by Western blot for each assay. (B) The dose dependence of insulin on glucose uptake was assayed in the presence or absence of Exo70 RNAi or scrambled oligo as indicated. (C) Micrographs of 3T3L1 adipocytes 4 d after transfection of RNAi oligo as indicated. (D and E) Effect of Exo70 knockdown on insulin-stimulated Akt phosphorylation, insulin receptor and Glut4 expression. (F) Sec8, Sec6, or both were knocked down in 3T3L1 adipocytes using RNAi oligos, and 2DG uptake was assayed 4 d after transfection. Results are the mean ± SD of triplicate determinations and were reproduced three times. *Significant difference, p < 0.05. (G) The effect of Sec6 and Sec8 knockdown on each molecule and Exo70 were examined by immunoblot.
Figure 2.
Figure 2.
Insulin stimulates the translocation of exocyst proteins to lipid rafts. (A) HA-TC10 wild type or TC10 (Q67L) was transfected into 3T3L1 adipocytes with myc-Exo70 and caveolin-eGFP. To make plasma membrane sheets, cells were treated for 5 min with the Triton extraction buffer. The plasma membrane sheets were fixed and stained with an anti-HA mAb and anti-myc polyclonal antibody followed by Alexa633 goat anti-mouse IgG and Alexa568 goat anti-rabbit IgG. The cells transfected with HA-TC10WT were starved overnight and treated with or without insulin for 5 min (left 8 panels). The cells transfected with HA-TC10 Q67L were not starved or stimulated by insulin (right 4 panels). (B) After a 5-min insulin stimulation, 3T3L1 adipocytes were harvested in the nondetergent buffer followed by sucrose density gradient fractionation. The expression of caveolin, transferrin receptor and Sec8 were evaluated by immunoblot. (C) Cells treated with insulin for the indicated times were lysed and fractionated on sucrose gradients. Fractions 4 and 5 were pooled as the lipid raft fraction, and fraction 10-12 were pooled as the nonlipid raft fraction. These samples were resolved in 4-20% gradient SDS-PAGE and analyzed by immunoblotting with anti-Exo70, Sec8, and caveolin antibodies.
Figure 3.
Figure 3.
Time course of Glut4 translocation to lipid rafts. (A) Glut4 expression in lipid raft, nonlipid raft, or nonplasma membrane fractions were examined at different time points after insulin stimulation. The intensity of Glut4 levels in the raft, nonraft, and nonplasma membrane were quantified. (B) The lipid raft and nonlipid raft fractions purified by sucrose gradient from the different conditions (with or without insulin stimulation, Exo70 RNAi or scrambled oligo-transfected as indicated) were loaded on the same gel and blotted with anti-Exo70, Sec8, Glut4, transferrin receptor, and caveolin antibodies. (C) Four days after transfection of Exo70 or scrambled RNAi oligo, cells were treated with insulin for 10 min and incubated with Triton extraction buffer to make plasma membrane sheets. The plasma membrane sheets were fixed and stained with anti-Glut4 polyclonal antibody and anti-caveolin2 mAb followed by Alexa594 goat anti-rabbit IgG and Alexa488 goat anti-mouse IgG. Arrows show the rosette structures.
Figure 4.
Figure 4.
Dominant negative Exo70 inhibits Glut4 movement to lipid rafts. (A) Myc-Exo70-WT and -N were overexpressed in 3T3L1 adipocytes by electroporation and harvested 3 d later without starvation or stimulation by insulin. Sucrose gradients fractionations were performed as described above, and fractions were blotted with anti-myc mAb and anti-caveolin antibody. (B) Vector control, myc-Exo70-WT and -N overexpressed 3T3L1 adipocytes were treated with or without insulin for 5 min, and sucrose gradient fractionation were performed as in A. The fractions were loaded in SDS-PAGE and blotted with anti-Sec8 mAb, anti-Exo70 mAb, anti-Glut4 polyclonal antibody, and anti-caveolin polyclonal antibody.
Figure 5.
Figure 5.
SAP97 binds to Sec8 through its PDZ-binding motif. (A) HA-Sec8 WT, HA-Sec8Δ4 amino acids and empty vector were cotransfected with eYFP-myc SAP97 in Cos-1 cells. Cells were harvested in HNTG buffer and incubated with anti-HA mAb for 2 h at 4°C. The immune complexes were precipitated with protein G beads for 1 h at 4°C, washed extensively with lysis buffer, resolved in a 4-20% gradient SDS-PAGE, and analyzed by immunoblotting with anti-YFP/eGFP polyclonal antibody. (B) Plasma membrane sheets were stained with anti-SAP97 mAb and anti-caveolin polyclonal antibody followed by Alexa594 goat anti-mouse IgG and Alexa488 goat anti-rabbit IgG. (C) Sucrose gradient samples from Figure 2B were blotted with anti-SAP97 mAb and anti-caveolin antibody.
Figure 6.
Figure 6.
SAP97 anchors the exocyst complex to lipid rafts. (A) SAP97 RNAi, Sec8 RNAi, or scrambled oligo were transfected into 3T3L1 adipocytes. Four days later, cells were harvested without starvation or stimulation by insulin and blotted with anti-SAP97 mAb, anti-Sec8 mAb, and anti-caveolin polyclonal antibody. (B) Four days after transfection of SAP97 RNAi or scrambled RNAi, 3T3L1 adipocytes were harvested and sucrose gradient fractionation was performed. SAP97, Sec8, Exo70, Glut4, and caveolin were examined by immunoblot. (C) Differentiated 3T3L1 adipocytes were transfected with SAP97 RNAi or scrambled oligo, and 4 d later were assayed for 2DG uptake. Results are the mean ± SD of triplicate determinations and were reproduced four times. *Significant difference, p < 0.05. (D) Effect of SAP97 knockdown on insulin-stimulated Akt phosphorylation and Glut4 expression.
See this image and copyright information in PMC

References

    1. Anderson, R. G. (1998). The caveolae membrane system. Annu. Rev. Biochem. 67, 199-225. - PubMed
    1. Baumann, C. A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S., Bickel, P. E., Pessin, J. E., and Saltiel, A. R. (2000). CAP defines a second signalling pathway required for insulin-stimulated glucose transport [see comments]. Nature 407, 202-207. - PubMed
    1. Bickel, P. E. (2002). Lipid rafts and insulin signaling. Am. J. Physiol. Endocrinol. Metab. 282, E1-E10. - PubMed
    1. Bryant, N. J., Govers, R., and James, D. E. (2002). Regulated transport of the glucose transporter GLUT4. Nat. Rev. Mol. Cell. Biol. 3, 267-277. - PubMed
    1. Chamberlain, L. H., and Gould, G. W. (2002). The v-and t-SNARE proteins that mediate Glut4 vesicle fusion are localised in detergent-insoluble lipid rafts present on distinct intracellular membranes. J. Biol. Chem. 9, 9. - PubMed

Publication types

MeSH terms

LinkOut - more resources