Rab GTPase regulation of phagosome-lysosome fusion is bypassed in the presence of micromolar Ca2.

Several ATP- and cytosol-dependent fusion processes between membranes of the endocytic and exocytic pathways have been biochemically reconstituted. Here, we present a phagosome-lysosome fusion reaction that is driven by micromolar concentrations of Ca2+ in the absence of ATP and cytosol. Investigating classical fusion and Ca2+-driven fusion (CaFu) side-by-side in vitro, using the same membrane preparations, we show that CaFu is faster than standard fusion (StaFu), leads to larger fusion products and is not blocked by established inhibitors of StaFu. A Ca2+ concentration of ∼120 µM supports maximal membrane attachment, and 15 µM Ca2+ supports maximal membrane fusion, indicating that Ca2+ has both a membrane-binding activity and a fusion-promoting activity. StaFu and CaFu are inhibited by a mutant form of α-SNAP (NAPA) that does not support soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) activation, and both are inhibited by a mixture of the cytosolic domains of three cognate Q-SNARE proteins, demonstrating a role of SNAREs in Ca2+-driven membrane merger. CaFu is independent of the Ca2+-regulated proteins synaptotagmin-7, calmodulin, and annexins A2 and A7. We propose that CaFu corresponds to the last step of phagosome-lysosome fusion, when a raised Ca2+ concentration from the compartment lumen activates SNAREs for fusion.