Volume 3, Issue 6, Pages 751-760 (June 1999) Synip  Jing Min, Shuichi Okada, Makoto Kanzaki, Jeffrey S Elmendorf, Kenneth J Coker, Brian P Ceresa, Li-Jyun Syu, Yoichi Noda, Alan R Saltiel, Jeffrey E Pessin  Molecular Cell  Volume 3, Issue 6, Pages 751-760 (June 1999) DOI: 10.1016/S1097-2765(01)80007-1

Figure 1 Cloning, Characterization of Synip Expression, and Specificity of Binding (A) Deduced amino acid sequences of the single open reading frame in the isolated Synip cDNA. (B) Predicted structural organization of Synip functional domains. The numbers on the top indicate the amino acid residues that define the boundaries of these domains. (C) Northern blot analysis of Synip mRNA expression in mouse tissues. The mouse multiple-tissue mRNA blot was probed with the coding sequence of the Synip cDNA. H, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; Sk, skeletal muscle; K, kidney; Te, testis. (D) Specificity of Synip/WT and Synip/CT binding to syntaxin 4. Cell lysates from 293T cells overexpressing Flag epitope-tagged wild-type Synip (Synip/WT) or the carboxy-terminal Synip (Synip/CT) were incubated with equal amounts of GST (lane 1), GST–Syn1A (lane 2), GST–Syn1B (lane 3), GST–Syn2 (lane 4), GST–Syn3 (lane 5), and GST–Syn4 (lane 6) proteins immobilized on glutathione-agarose beads. The retained proteins were immunoblotted with the Flag M2 monoclonal antibody. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)

Figure 2 Insulin Stimulation Induces a Dissociation of Synip from Syntaxin 4 In Vivo CHO/IR cells were transfected with the full-length Synip (Synip/WT), the amino-terminal Synip domain (Synip/NT), or the carboxy-terminal Synip domain (Synip/CT) and incubated in the absence or presence of 100 nM insulin for 20 min at 37°C. (A) Cell lysates were prepared and immunoblotted with the Flag antibody. (B) Cell lysates were immunoprecipitated with a syntaxin 4 antibody and immunoblotted with the Flag antibody. (C) The immunoprecipitates in (B) were immunoblotted with the syntaxin 4 antibody. These are representative experiments independently performed five times. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)

Figure 3 Insulin Stimulation Results in a Decreased Affinity of Synip for Syntaxin 4 CHO/IR cells were transfected with the full-length Synip (Synip/WT), the amino-terminal Synip domain (Synip/NT), or the carboxy-terminal Synip domain (Synip/CT) and stimulated with and without insulin for 15 min at 37°C. (A) Cell lysates were prepared and immunoblotted with the Flag antibody. (B) The cell lysates were incubated with the GST–Syn4 fusion protein (20 μg) for 2 hr at 4°C, and the resultant precipitates were immunoblotted with the Flag antibody. (C) The cell lysates were incubated with the GST–Synip fusion protein (20 μg) for 2 hr at 4°C, and the resultant precipitates were immunoblotted with the syntaxin 4 antibody. These are representative experiments independently performed 4–6 times. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)

Figure 4 Synip and VAMP2 Binding to Syntaxin 4 Are Mutually Exclusive (A) GST–Synip/WT or GST–Synip/CT (5 nM) was premixed with various amounts of GST–SNAP23 for 15 min prior to the addition of amylose resin–bound MBP–Syn4 (3 nM) for 2 hr at 4°C. The samples were then washed, resuspended in Laemmli sample buffer, and subjected to immunoblotting with a carboxy-terminal Synip antibody. (B) GST–Synip/WT or GST–Synip/CT (5 nM) was premixed with various amounts of GST–VAMP2 for 15 min prior to the addition of amylose resin–bound MBP–Syn4 (3 nM) for 2 hr at 4°C. The samples were then washed, resuspended in Laemmli sample buffer, and subjected to immunoblotting with a carboxy-terminal Synip antibody. These are representative experiments independently performed five times. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)

Figure 5 Insulin Induces Dissociation of the Synip:Syntaxin 4 Complex in Differentiated 3T3L1 Adipocytes Differentiated 3T3L1 adipocytes were transfected with the full-length Synip (Synip/WT) or the carboxy-terminal Synip domain (Synip/CT) and stimulated with and without insulin for 15 min at 37°C. (A) Cell lysates were incubated with the GST–Syn4 fusion protein (20 μg) for 2 hr at 4°C, and the resultant precipitates were immunoblotted with the Flag antibody. (B) The cell lysates were immunoprecipitated with the syntaxin 4 antibody and immunoblotted with the Flag antibody (lanes 1–4) or the syntaxin 4 antibody (lanes 5–8). These are representative experiments independently performed 3–5 times. (C) Nontransfected 3T3L1 adipocytes were either unstimulated (lanes 1 and 3) or stimulated with 100 nM insulin for 15 min at 37°C (lanes 3 and 4). Cell lysates were prepared and immunoprecipitated with a carboxy-terminal polyclonal Synip antibody (8 μg) and immunoblotted with either the syntaxin 4 antibody (lanes 1 and 2) or the Synip antibody (lanes 3 and 4). This is a representative experiment independently performed four times. (D) The cell lysates isolated from unstimulated (lane 1) or insulin-stimulated 3T3L1 adipocytes (lane 2) indicated above (C) were immunoblotted for the presence of syntaxin 4 (Pre). The same unstimulated (lane 3) and insulin-stimulated (lane 4) cell lysates were then immunoblotted for syntaxin 4 following immunoprecipitation with the Synip antibody (Post). This is a representative experiment independently performed four times. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)

Figure 6 Expression of a Dominant-Interfering Mutant of Synip Inhibits Insulin-Stimulated Glucose Transport (A) Differentiated 3T3L1 adipocytes were electroporated with various amounts of pcDNA3.1-LacZ, and transfection/expression efficiency was assessed by X-Gal staining for β-galactosidase expression. Under each electroporation condition, the total amount of plasmid DNA was 600 μg maintained by the addition of the pcDNA3.1 empty vector. (B) Differentiated 3T3L1 adipocytes were electroporated with either empty vector or various Synip cDNA constructs, and the rate of basal (open bars) and insulin-stimulated (closed bars) 2-deoxyglucose transport was determined. This data represents the average with the standard error of the mean from 5–9 independent determinations. The asterisk indicates p < 0.01. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)

Figure 7 Microinjection of the GST–Synip/CT Fusion Protein Inhibits Insulin-Stimulated GLUT4 but Not GLUT1 Translocation Fully differentiated 3T3L1 adipocytes were microinjected with approximately 0.1 pl of MBP–Ras (6 mg/ml) mixed 1:1 with 8 mg/ml GST (panels 1–4), 8 mg/ml GST–Synip/WT (panels 5 and 6), 8 mg/ml GST–Synip/NT (panels 7 and 8), and 6 mg/ml GST–Synip/CT (panels 9 and 10). Following microinjection, the cells were either left untreated (panels 1 and 2) or insulin-stimulated (panels 3–10) for 15 min at 37°C. (A) Plasma membrane sheets were prepared and subjected to confocal immunofluorescence microscopy with an MBP-specific antibody (panels 1, 3, 5, 7, and 9) or a GLUT4-specific antibody (panels 2, 4, 6, 8, and 10). (B) Plasma membrane sheets were prepared and subjected to confocal immunofluorescence microscopy with an MBP-specific antibody (panels 1, 3, 5, 7, and 9) or a GLUT1-specific antibody (panels 2, 4, 6, 8, and 10). These are representative fields from 3–4 independent experiments in which a total of 255 plasma membrane sheets for GLUT4 and 212 plasma membrane sheets for GLUT1 were scored for translocation. The plasma membrane sheets derived from the microinjected cells are indicated by the arrows. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)

Figure 7 Microinjection of the GST–Synip/CT Fusion Protein Inhibits Insulin-Stimulated GLUT4 but Not GLUT1 Translocation Fully differentiated 3T3L1 adipocytes were microinjected with approximately 0.1 pl of MBP–Ras (6 mg/ml) mixed 1:1 with 8 mg/ml GST (panels 1–4), 8 mg/ml GST–Synip/WT (panels 5 and 6), 8 mg/ml GST–Synip/NT (panels 7 and 8), and 6 mg/ml GST–Synip/CT (panels 9 and 10). Following microinjection, the cells were either left untreated (panels 1 and 2) or insulin-stimulated (panels 3–10) for 15 min at 37°C. (A) Plasma membrane sheets were prepared and subjected to confocal immunofluorescence microscopy with an MBP-specific antibody (panels 1, 3, 5, 7, and 9) or a GLUT4-specific antibody (panels 2, 4, 6, 8, and 10). (B) Plasma membrane sheets were prepared and subjected to confocal immunofluorescence microscopy with an MBP-specific antibody (panels 1, 3, 5, 7, and 9) or a GLUT1-specific antibody (panels 2, 4, 6, 8, and 10). These are representative fields from 3–4 independent experiments in which a total of 255 plasma membrane sheets for GLUT4 and 212 plasma membrane sheets for GLUT1 were scored for translocation. The plasma membrane sheets derived from the microinjected cells are indicated by the arrows. Molecular Cell 1999 3, 751-760DOI: (10.1016/S1097-2765(01)80007-1)