Figure 1.

Human ATL1 has fusion activity. (A) His-tagged ATL1 fractions eluted from a Ni⁺² column and resolved on SDS-PAGE. AH, amphipathic helix. (B) ATL1 was reconstituted into donor and acceptor vesicles (1:2 donor/acceptor), and fusion was monitored during the dequenching of NBD-labeled lipid present in the donor vesicles over time at 37°C after addition of 2 mM GTP or buffer (no nuc). Loss of NBD fluorescence without GTP was attributed to photobleaching, because it mimicked NBD loss from protein-free liposomes (pf no nuc). (C) Lipid mixing by DATL from E. coli with or without GTP performed as in B. (D) ATL1 lipid mixing after reconstitution at varying protein/lipid ratios. (E) Lipid mixing by WT, R77E, Δtail ATL1(1–496), and I507D showing a requirement for GTP binding, the amphipathic helix, and the nonpolar face of the amphipathic helix. (F) Comparison of ATL1 HSP mutant variants H258R, R239C, and R217Q to WT. All lipid mixing was performed at a 1:1,000 protein/lipid ratio unless otherwise stated. The data are the average of at least two independent traces.