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. 2003 Mar;14(3):973-86.
doi: 10.1091/mbc.e02-06-0315.

GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif

Annette M Shewan  1 , Ellen M van Dam, Sally Martin, Tang Bor Luen, Wanjin Hong, Nia J Bryant, David E James
Affiliations

GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif

Annette M Shewan et al. Mol Biol Cell. 2003 Mar.

Abstract

Insulin stimulates glucose transport in fat and muscle cells by triggering exocytosis of the glucose transporter GLUT4. To define the intracellular trafficking of GLUT4, we have studied the internalization of an epitope-tagged version of GLUT4 from the cell surface. GLUT4 rapidly traversed the endosomal system en route to a perinuclear location. This perinuclear GLUT4 compartment did not colocalize with endosomal markers (endosomal antigen 1 protein, transferrin) or TGN38, but showed significant overlap with the TGN target (t)-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Syntaxins 6 and 16. These results were confirmed by vesicle immunoisolation. Consistent with a role for Syntaxins 6 and 16 in GLUT4 trafficking we found that their expression was up-regulated significantly during adipocyte differentiation and insulin stimulated their movement to the cell surface. GLUT4 trafficking between endosomes and trans-Golgi network was regulated via an acidic targeting motif in the carboxy terminus of GLUT4, because a mutant lacking this motif was retained in endosomes. We conclude that GLUT4 is rapidly transported from the cell surface to a subdomain of the trans-Golgi network that is enriched in the t-SNAREs Syntaxins 6 and 16 and that an acidic targeting motif in the C-terminal tail of GLUT4 plays an important role in this process.

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Figures

Figure 1
Figure 1
HA-GLUT4 traverses the same organelles as endogenous GLUT4 in 3T3-L1 adipocytes. 3T3-L1 adipocytes expressing HA-GLUT4 were stimulated with insulin, labeled on ice with an anti-HA antibody to label HA-GLUT4 at the cell surface and washed extensively to reverse the effects of insulin as described in MATERIALS AND METHODS. The degree of colocalization of internalized HA-GLUT4 (HA) with endogenous GLUT4 (G4) at various time points after the initiation of endocytosis was determined using confocal microscopy.
Figure 2
Figure 2
HA-GLUT4 trafficks through early endosomes before reaching a perinuclear compartment. Confluent 3T3-L1 adipocytes expressing either HA-GLUT4 or the hTfR were stimulated with insulin for 20 min. Cells were then either incubated at 4°C with the anti-HA antibody, washed, and chased for either 5 or 60 min at 37°C (HA, 5 min and HA, 60 min, respectively) or incubated with Alexa-488–conjugated transferrin for 60 min at 37°C (Tf, 60 min). Cells were then fixed and double labeled with antibodies specific for either EEA1, endogenous GLUT4, or Syntaxin 6 (Syn6). Note that not all cells in the fields shown were expressing either HA-GLUT4 or the TfR. This confirms the specificity of either HA antibody or transferrin in these experiment.
Figure 3
Figure 3
HA-GLUT4 cycles through a perinuclear Syntaxin 6/16-positive compartment. 3T3-L1 adipocytes expressing HA-GLUT4 were stimulated with insulin for 20 min. Cells were then chilled down and incubated with anti-HA antibody, washed, and chased for 60 min at 37°C (HA). Cells were then fixed and double labeled with antibodies specific for either Syntaxin 6 (Syn6), Syntaxin 16 (Syn16), or TGN38.
Figure 4
Figure 4
Syntaxins 6 and 16 are enriched in GLUT4 vesicles. GLUT4 antibody and nonspecific IgG-coupled beads were incubated with LDMs from 3T3-L1 adipocytes, washed and eluted with SDS sample buffer. LDMs and bound proteins were immunoblotted with antibodies specific for GLUT4, IRAP, Syntaxin 6, Syntaxin 16, and GS15.
Figure 5
Figure 5
IRAP is transported to the TGN after internalization. Adipocytes were incubated with insulin for 20 min and biotinylated on ice. Cells were then incubated with neuraminidase on ice and reincubated at 37°C for either 60 or 180 min. Solubilized lysates were incubated with streptavidin beads to recover biotinylated proteins which were subjected to SDS-PAGE followed by immunoblotting with antibodies specific for IRAP.
Figure 6
Figure 6
Insulin stimulates the translocation of Syntaxin 6 and Syntaxin 16 to the cell surface in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were incubated in the absence (−) or presence of insulin (+) for 20 min. Cells were then fractionated into PMs, LDMs, and HDMs as described in MATERIALS AND METHODS. Aliquots of each fraction (10 μg of protein) were then immunoblotted with antibodies specific for GLUT4; TGN38; and Syntaxins 6, 7, 13, and 16. (B) Adipocytes were grown on glass coverslips and incubated with or without insulin for 20 min. Cells were then sonicated as described in MATERIALS AND METHODS and fixed yielding a lawn of plasma membrane fragments attached to the glass coverslip. These fragments were then labeled with antibodies specific for GLUT4 or Syntaxins 4, 6, 13, or 16. Labeling was visualized by confocal immunofluorescence microscopy. (C) Adipocytes were incubated with insulin for different periods of time at 37°C (0–20 min) and fractionated into PMs and intracellular membranes (LDMs). Fractions (10 μg of protein) were immunoblotted with antibodies specific for either GLUT4 or Syntaxin 6. Immunoblots were quantified using densitometry. Data are mean ± SEM, n = 3.
Figure 7
Figure 7
Syntaxin 6 and 16 form a complex in adipocytes and are up-regulated during adipocyte differentiation. (A) 3T3-L1 adipocyte lysates were solubilized in 1% TX-100. Antibodies specific for Syntaxin 6 were used to immunoprecipitate protein complexes from the soluble fraction. After incubation at 95°C for 5 min, immunoblot analysis was used to determine the amount of Syntaxins 4, 6, and 16 present in the lysate (Lys), unbound fraction (Sup), and in the bound fraction (Syn6 IP). (B) Total membrane fractions were prepared from 3T3-L1 fibroblasts (80% confluent) (Fib) and 3T3-L1 adipocytes (day 8 postdifferentiated) (Adip), and immunoblot analysis was used to determine the levels of GLUT4 and Syntaxins 4, 6, 7, 13, and 16 in equivalent amounts of these fractions (5 μg of protein).
Figure 8
Figure 8
An acidic targeting motif is required for endosomal trafficking of GLUT4. 3T3-L1 adipocytes expressing either HA-GLUT4 or C-terminal mutants, HA-TAIL and HA-EXEY, were treated with insulin before labeling surface accessible recombinant transporter on ice with anti-HA. After either 5 or 60 min of endocytosis cells were fixed and double-labeled with an antibody specific for EEA1 to label early endosomes. HA was visualized by anti-mouse-Alexa-594 (red), EEA1 was visualized by anti-human-ALEXA488 (green). Merged images are shown.
Figure 9
Figure 9
Targeting of GLUT4 to the Syntaxin 16 compartment is mediated by an acidic targeting domain in its C-terminal tail. 3T3-L1 adipocytes expressing either HA-GLUT4 or C terminal mutants, HA-TAIL and HA-EXEY, were treated with insulin before labeling surface accessible recombinant transporter on ice with anti-HA. After 60 min of endocytosis cells were fixed and double labeled with an antibody specific for Syntaxin 16. High magnification images of the perinuclear area are shown.
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