Synaptobrevin 2 constructs. Untagged WT full-length syb2 (Syb2) was expressed from a bicistronic vector with EGFP expressed from an IRES2. N-tagged Syb2 fused to EGFP at the N terminus. C-tagged syb2 with mVenus fused to the C terminus with RST as the amino acid sequence of the linker (Syb2-RST-mVenus). Lipid-anchored syb2, and ΔTMD syb2 constructs, with EGFP fused to the N terminus. In the ΔTMD-CSPα construct syb2 was truncated at residue 92 and extended with the CSPα lipid-anchoring sequence. In the ΔTMD-CAC, ΔTMD-LC, and ΔTMD-CIIL constructs, syb2 was truncated at residue 94 and extended with the indicated lipid-anchoring motifs. Constructs used in liposome fusion did not contain EGFP, and their cysteines were conjugated to POPE-maleimide (see under “Experimental Procedures”). Lipid moieties are indicated as segmented lines connected to cysteines. ΔTMD-94 and ΔTMD-84 contain sequences of WT syb2 residues 1–94 and 1–84, respectively. The numbers on the right indicate the last residue of syb2 in each construct.