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. 2015 Aug 3;210(3):451–470. doi: 10.1083/jcb.201501043

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© 2015 Lee et al.

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Figure 2. — In vitro homotypic ER fusion requires the atlastin GTPase Sey1p. (A) GTP hydrolysis is required for ER fusion in vitro. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of the indicated nucleotide for 90 min. Fusion values were normalized to those obtained using an ATP/GTP-driven reaction incubated at 27°C. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P < 0.001, Tukey’s test). (B) Protein profiles of wild-type and sey1Δ microsomes. The expression of Gluc PCA fragments and ER-associated proteins was analyzed by immunoblotting using the indicated antibodies. The ER-associated proteins Sec22p and Yet3p were used as loading controls. (C) Sey1p in both of the fusing microsomes is required to support fusion. The indicated microsomes were incubated on ice or at 27°C in the presence of ATP and/or GTP for 90 min. Data represent the means ± SEM (error bars; n = 3). ***, P < 0.001, Tukey’s test between experiments performed at 27°C. (D) Anti-Sey1p antibodies specifically inhibit the in vitro ER fusion reaction. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of anti-Sey1p antibodies (88 nM) and/or GST-Sey1p (1 µM). Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P < 0.001, Tukey’s test). (E) Addition of the cytoplasmic domain of Sey1p inhibits ER fusion in vitro. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C in the presence of the Sey1p CD (Sey1p-CD). Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P < 0.001, Tukey’s test). (F) Sey1p-K50A, a GTP-binding-defective mutant, did not support in vitro ER fusion. The indicated microsomes were incubated on ice or at 27°C in the presence of ATP and/or GTP for 90 min. Data represent the means ± SEM (error bars; n = 3). ***, P < 0.001, between ATP/GTP-driven reactions; ###, P < 0.001, between GTP-only-driven reactions, Tukey’s test.