ATL2 is the major atlastin in HEK293T cells and is required for ER fusion. (A) Schematic representation of the in vitro membrane fusion assay using ER microsomes isolated from cultured HEK293 cells. ER microsomes containing ZIP-Gluc1-KDEL (Gluc1 microsome) or ZIP-Gluc2-KDEL (Gluc2 microsome) were isolated from HEK293 cells transfected with genes encoding ssZIP-Gluc1-KDEL or ssZIP-Gluc2-KDEL, mixed, and incubated in the presence of a GTP-regenerating system at 37°C for 90 min. Fusion was measured as reconstituted luciferase activity, which produces luminescence in the presence of coelenterazine. (B) ER microsome fusion in vitro requires GTP hydrolysis. Gluc1 and Gluc2 microsomes were mixed and incubated in the absence or presence of GTP or GTPγS on ice or at 37°C for 90 min. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (C) ER microsome fusion is inhibited by an anti-ATL2 antibody. Gluc1 and Gluc2 microsomes were mixed and incubated in the presence of GTP with increasing concentrations of an anti-ATL2 antibody at 37°C for 90 min. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (D) Gluc1 and Gluc2 microsomes were isolated from HEK293 cells treated with indicated siRNAs and incubated in the presence of GTP at 37°C for 90 min. Fusion values were normalized to fusion between microsomes isolated from HEK293 cells treated with control siRNA. The data represent the means ± SEM (error bars; n = 4). n refers to the number of independent experiments. ****P < 0.0001, ***P < 0.001, and **P < 0.01, one-way ANOVA with Tukey’s multiple comparisons test. (E) Protein profiles of microsomes used in D. The expression levels of ATL2, ATL3, ZIP-Gluc1-KDEL, and ZIP-Gluc2-KDEL were analyzed by immunoblotting using the indicated antibodies. Calnexin, an ER-resident protein, was used as a loading control. Source data are available for this figure: SourceData F8.
Figure 8.

ATL2 is the major atlastin in HEK293T cells and is required for ER fusion. (A) Schematic representation of the in vitro membrane fusion assay using ER microsomes isolated from cultured HEK293 cells. ER microsomes containing ZIP-Gluc1-KDEL (Gluc1 microsome) or ZIP-Gluc2-KDEL (Gluc2 microsome) were isolated from HEK293 cells transfected with genes encoding ssZIP-Gluc1-KDEL or ssZIP-Gluc2-KDEL, mixed, and incubated in the presence of a GTP-regenerating system at 37°C for 90 min. Fusion was measured as reconstituted luciferase activity, which produces luminescence in the presence of coelenterazine. (B) ER microsome fusion in vitro requires GTP hydrolysis. Gluc1 and Gluc2 microsomes were mixed and incubated in the absence or presence of GTP or GTPγS on ice or at 37°C for 90 min. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (C) ER microsome fusion is inhibited by an anti-ATL2 antibody. Gluc1 and Gluc2 microsomes were mixed and incubated in the presence of GTP with increasing concentrations of an anti-ATL2 antibody at 37°C for 90 min. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (D) Gluc1 and Gluc2 microsomes were isolated from HEK293 cells treated with indicated siRNAs and incubated in the presence of GTP at 37°C for 90 min. Fusion values were normalized to fusion between microsomes isolated from HEK293 cells treated with control siRNA. The data represent the means ± SEM (error bars; n = 4). n refers to the number of independent experiments. ****P < 0.0001, ***P < 0.001, and **P < 0.01, one-way ANOVA with Tukey’s multiple comparisons test. (E) Protein profiles of microsomes used in D. The expression levels of ATL2, ATL3, ZIP-Gluc1-KDEL, and ZIP-Gluc2-KDEL were analyzed by immunoblotting using the indicated antibodies. Calnexin, an ER-resident protein, was used as a loading control. Source data are available for this figure: SourceData F8.

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