Materials and Antibodies
DMEM, Myoclone-Plus fetal calf serum, and antibiotics were from Invitrogen (Paisley, United Kingdom). Normal sera were from DAKO (Carpinteria, CA). All other materials were obtained from SigmaAldrich (St. Louis, MO) unless stated otherwise.
Polyclonal rabbit antibodies were raised against glutathione S
-transferase-fusion proteins consisting of the cytosolic domain of Syntaxin 6 and insulin-responsive aminopeptidase (IRAP). Monoclonal antibodies raised against Syntaxin 6 were obtained from Transduction Laboratories (Lexington, KY) or were the generous gift of Dr. Jason Bock (Howard Hughes Medical Institute, Stanford University School of Medicine, Palo Alto, CA) (3D10). Rabbit antibodies against Syntaxin 13 were obtained from Dr. Rohan Teasdale (University of Queensland, Australia), anti-TGN38 antibodies were from Dr. Paul Luzio (University of Cambridge, Cambridge, United Kingdom), and anti-human endosomal antigen 1 protein (EEA1) antibodies were from Dr. Marvin Fritzler (University of Calgary, Calgary, Alberta, Canada). The monoclonal antibody 16B12, which recognizes the influenza hemagglutinin (HA) epitope, was purchased from Babco (Richmond, CA). A monoclonal antibody against the TfR was from Zymed Laboratories (South San Francisco, CA). Antibodies against GLUT4 (James et al., 1989
), Syntaxin 4 (Tellam et al., 1997
), Syntaxin 7 (Wade et al., 2001
), Syntaxin 16 (Mallard et al., 2002
), and GS15 (Xu et al., 1997
) have been described previously.
Cell Culture and Retroviral Transfection
3T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA) were cultured as described previously (Shewan et al., 2000
). Briefly, cells were grown in DMEM supplemented with 10% new born calf serum, 2 mM l
-glutamine, 100 U/l penicillin and 100 μg/l streptomycin at 37°C in 10% CO2
, and passaged at ~70% confluence. Confluent cells were then differentiated into adipocytes. Cells were used between days 6–10 postdifferentiation and between passages 4 and 12. To establish basal conditions before use, cells were incubated in serum-free DMEM for 2 h unless otherwise indicated.
The construction and generation of retroviral stocks of HA-GLUT4 and HA-TAIL have been described previously (Shewan et al., 2000
). Both constructs encode transporters harboring an HA epitope engineered in the large exofacial loop between transmembrane domains 1 and 2 of GLUT4. HA-GLUT4 encodes the full-length GLUT4 protein (Quon et al., 1994
). HA-TAIL encodes full-length GLUT4 in which the 12 carboxyl terminal residues have been replaced by the corresponding sequence from GLUT3 (Shewan et al., 2000
). HA-EXEY was generated by site-directed mutagenesis of pMEX shuttle HA-GLUT4 (pMS-HA-GLUT4) cDNA by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). To generate this mutant, we took advantage of the unique Nco
I site present in both human and rat GLUT4 cDNAs. Complimentary oligonucleotides were used to mutagenize E499
to ALAA, using primers 5′-gtgaaacccagtacagcacttgcagccttagggccagatgag-3′ and 5′-ctcatctggccctaaggctgcaagtgctgtactgggtttcac-3′. The Nco
RI fragment of pMS-HA-EXEY was fully sequenced before subcloning into pBabepuro for production of retrovirus (Pear et al., 1993
). For expression of the hTfR in adipocytes, the Bam
II fragment coding for hTfR was subcloned from pUC8-hTfR (T.E. McGraw, Cornell University, Ithaca, NY) into pBabepuro and retrovirus expressing pBabe-TfR was produced as described above (Pear et al., 1993
To generate 3T3-L1 adipocytes expressing each construct, 3T3-L1 fibroblasts (plated at a density of 5 × 105/100-mm plate 16 h prior) were infected with the relevant virus for 5 h in the presence of 4 μg/ml polybrene. After a 48-h recovery period, infected cells were selected in DMEM containing 10% fetal calf serum and supplemented with 2 μg/ml puromycin. Polyclonal pools of 3T3-L1 fibroblasts were then grown to confluence and subsequently differentiated into adipocytes as described above. Puromycin was not included in the differentiation media but was reapplied once the differentiation regime was completed.
Subcellular membrane fractions from basal and insulin-treated 3T3-L1 adipocytes were prepared using a previously described differential centrifugation procedure (Piper et al., 1991
; Marsh et al., 1995
). Briefly, the plasma membrane fraction was obtained after a 20-min centrifugation at 17,200 × g
followed by centrifugation through sucrose. The high-density microsomes (HDMs) were obtained by centrifuging the 17,200 × g
supernatant at 38,700 × g
for 20 min and the low-density microsomes (LDMs) were obtained by spinning the 38,700 × g
supernatant at 150,000 × g
for 75 min. These fractions have previously been characterized in detail (Piper et al., 1991
). The HDM fraction contains large endosomal components and endoplasmic reticulum (ER), whereas the LDM fraction contains small vesicles, including those enriched in GLUT4. All fractions were resuspended in HES buffer (20 mM HEPES, 1 mM EDTA, 250 mM sucrose, pH 7.4), protein quantified using the bicinchoninic acid assay (Pierce Chemical
, Rockford, IL) and stored at −80°C before use. Total membrane fractions were prepared from 3T3-L1 fibroblasts and adipocytes after homogenization in HES buffer containing protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin, 250 μM phenylmethylsulfonyl fluoride). Homogenates were subjected to centrifugation at 50,000 rpm in a Beckman Coulter TLA100–3 rotor for 60 min. The membrane pellet was resuspended in HES buffer and stored at −80°C before use.
Resialylation studies were performed essentially as described by Teuchert et al. (1999)
. Cells were incubated in serum-free medium overnight and insulin (20 nM) was added for 30 min at 37°
C. Cells were then washed five times with ice-cold phosphate-buffered saline (PBS) containing 0.1 mM CaCl2
, 1 mM MgCl2
) and biotinylated twice for 20 min in 2 ml of PBS++
containing 0.5 mg/ml sulfo-NHS-biotin (Pierce Chemical
). Cells were washed three times with ice-cold PBS++
containing 0.1 M glycine to quench free biotin, incubated with neuraminidase Vibrio cholerae
(80 mU/ml; Roche Diagnostics, Indianapolis, IN) on ice for 1 h and then washed three times with PBS++
. Cells were then incubated with prewarmed DMEM containing fetal calf serum (10%) for different times as indicated at 37°
C. Cells were washed twice with PBS++
at 4°C and incubated with Triton X-100 (1%) containing protease inhibitors (see above) for 20 min at 4°
C. Cells were scraped from the dish and centrifuged at 14,000 rpm for 10 min at 4°
C. The cell lysate was incubated with streptavidin agarose beads at 4°
C for 16 h and washed three times in PBS++
containing 1% Triton X-100/0.1% SDS. Samples were then heated to 60°C for 10 min and subjected to SDS-PAGE and immunoblotted with anti-IRAP.
Immunoprecipitation of SNARE Complexes
Basal 3T3-L1 adipocytes were homogenized by passaging twice through a 25-gauge needle followed by passaging twice through a 27-gauge needle in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA containing protease inhibitors. Cell lysates were solubilized using 1% Triton X-100 on ice for 30 min. The solubilized lysate was cleared by centrifugation for 30 min at 4°C in a microcentrifuge. Aliquots of the soluble proteins were incubated overnight with relevant antibodies bound to protein A-Sepharose. Immunoprecipitated proteins were resolved by SDS-PAGE together with aliquots of the supernatant and starting material.
Immunoabsorption of GLUT4 and Syntaxins 6 and 16 Vesicles
Protein G and protein A beads (Pierce Chemical) were incubated with 1% bovine serum albumin (BSA) for 30 min. Beads were then incubated with either monoclonal GLUT4 antibody 1F8, nonspecific mouse IgG, anti-Syntaxin 6, or anti-Syntaxin 16 antibodies. Antibodies were cross-linked to the beads by using 20 mM dimethyl suberimidate (Pierce Chemical) for 30 min at room temperature and cross-linked antibodies were saturated with 1% BSA for 30 min at room temperature. LDMs from noninfected, HA-GLUT4–infected or HA-TAIL–infected 3T3-L1 adipocytes were incubated separately with each of the specific and nonspecific antibody-coupled beads overnight at 4°C. The beads were washed and adsorbed material was eluted with SDS sample buffer and subjected to SDS-PAGE together with aliquots of the starting material.
Electrophoresis and Immunoblotting
Proteins were subjected to electrophoresis on 7.5 or 12% SDS-polyacrylamide gels and transblotted onto polyvinylidene difluoride. Immunolabeled proteins were visualized using horseradish peroxidase-conjugated secondary antibody and either the enhanced chemiluminescence system (Amersham Biosciences, Aylesbury, United Kingdom) or Supersignal (Pierce Chemical). Bands were quantitated by densitometry or by using a Lumi-Imager (Roche Diagnostics, Castle Hill, New South Wales, Australia).
Indirect Immunofluorescence Microscopy
The preparation of plasma membrane (PM) lawns was performed as described in Robinson et al. (1992)
. Briefly, after incubating cells on coverslips with the appropriate treatment, adipocytes were sonicated yielding a lawn of PM fragments attached to the coverslip. Coverslips were then incubated with the relevant antibodies directed against C-terminal domains, followed by fluorescein isothiocyanate-conjugated secondary antibody (Molecular Probes, Eugene, OR). Cells were viewed using either a 63×/1.4 oil immersion objective on an Axiovert fluorescence microscope (Carl Zeiss, Thornwood, NY), equipped with an MRC-600 laser confocal imaging system (Bio-Rad
, Hercules, CA), or a 100×/1.4 Plan Apo oil immersion objective on an Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan), equipped with a Radiance 2000 laser confocal imaging system (Bio-Rad
Endocytosis of HA-GLUT4 and Transferrin in 3T3-L1 Adipocytes
Adipocytes expressing HA-GLUT4, HA-TAIL, HA-EXEY, or hTfR were serum starved for 2 h in Krebs-Ringer phosphate buffer (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.4 mM NaH2PO4, 0.6 mM Na2HPO4, pH 7.4) containing 0.2% BSA (KRP/B) and stimulated with insulin (20 nM) for 20 min to bring a cohort of GLUT4 molecules to the cell surface. Cells were then washed with ice-cold KRP/B and incubated on ice with monoclonal anti-HA for 60 min. To reverse the insulin stimulation, cells were rinsed five times in ice-cold KRP/B, and endocytosis was initiated by transfer to 37°C in prewarmed KRP/B. 3T3-L1 adipocytes expressing hTfR were incubated with Tf-Alexa-488 (Molecular Probes) during the chase at a final concentration of 50 μg/ml. At the times indicated, cells were fixed using 3% paraformaldehyde in PBS for at least 30 min at room temperature. Free aldehyde groups were quenched in 50 mM NH4Cl in PBS. Cells were permeabilized and labeled in PBS containing 2% BSA and 0.1% saponin by using standard procedures. Cells were double labeled for endocytosed markers (HA or Tf) and either endogenous GLUT4, Syntaxin 6, Syntaxin 16, TGN38, or EEA1, followed by Alexa-488 or Alexa-594–conjugated secondary antibodies (Molecular Probes). Optical sections were analyzed by confocal scanning laser microscopy by using a TCS SP system (Leica Microsystems, Deerfield, IL).