(A) s-Opa1 alone is capable of tethering bilayers, but insufficient for close membrane docking and hemifusion. (B) l-Opa1, in a heterotypic arrangement, can tether bilayers, and upon GTP stimulation promote low levels of lipid mixing, but no full fusion, pore opening or content release. (C) Homotypic l-Opa1-l-Opa1 tethered bilayers can mediate full content release (i). This activity is greatly stimulated by the presence of s-Opa1, with peak activity at 1:1 s-Opa1:l-Opa1 (ii). Excess levels of s-Opa1 suppress fusion, likely through competing with the l-Opa1-l-Opa1 homotypic tethering interface (iii).