GTP hydrolysis is not required for the prefusion to postfusion conformational change. (A) Location of the C395 residue in ATL2 used to report on the postfusion conformation. The C395 side chain in ATL2 is highlighted as spheres in both pre- and postfusion ATL1 dimer structures rendered as detailed in Fig. 2. (B) The purified cytoplasmic domain of wild-type or C395N ATL2 (20 µM) was incubated in the presence or absence of the indicated nucleotides for 30 min at RT. Thereafter, samples were diluted, further incubated with or without BMOE for 1 h at RT, resolved by SDS-PAGE, and stained with Coomassie blue. The positions of non–cross-linked monomer and covalently cross-linked dimer ATL2 are indicated by single and double asterisks, respectively. The open circle indicates the position of ATL2 likely to have cysteine modifications not leading to dimer formation. (C) The purified cytoplasmic domains of the indicated ATL2 variants were subjected to the assay as described in B. Molecular masses are given in kilodaltons.