Vacuolar SNARE transmembrane domains serve as non-specific membrane anchors with unequal roles in lipid mixing [Cell Biology]
March 27th, 2015 by Pieren, M., Desfougeres, Y., Michaillat, L., Schmidt, A., Mayer, A.
Membrane fusion is induced by SNARE complexes which are anchored in both fusion partners. SNAREs zipper up from the N- to C-terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3 and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial LV sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a pre-requisite for fusion. Lipid-anchored Vti1 was fully active, lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as non-specific membrane anchors in vacuole fusion.