Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg²⁺ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca²⁺ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg²⁺ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca²⁺. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mM Mg²⁺, other free divalent ions are not needed. Reactions containing low Mg ²⁺ concentrations are strongly inhibited by the rapid Ca²⁺ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca²⁺ or Mg²⁺, suggesting that Ca²⁺ does not perform a unique signaling function. When the need for Mg²⁺, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.