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 & mu;M supports maximal membrane attachment, and 15 & mu;M 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 & alpha;-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.