Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion

Phagosomes mature into phagolysosomes by sequential fusion with early endosomes, late endosomes, and lysosomes. Phagosome-with-lysosome fusion (PLF) results in the delivery of lysosomal hydrolases into phagosomes and in digestion of the cargo. The machinery that drives PLF has been little investigated. Using a cell-free system, we recently identified the phosphoinositide lipids (PIPs) phosphatidylinositol 3-phosphate (PI(3) P) and phosphatidylinositol 4-phosphate (PI(4) P) as regulators of PLF. We now report the identification and the PIP requirements of four distinct subreactions of PLF. Our data show that (i) PI(3) P and PI(4) P are dispensable for the disassembly and activation of (phago) lysosomal soluble N-ethylmaleimide- sensitive factor attachment protein receptors, that (ii) PI(3) P is required only after the tethering step, and that (iii) PI(4) P is required during and after tethering. Moreover, our data indicate that PI(4) P is needed to anchor Arl8 (Arf-like GTPase 8) and its effector homotypic fusion/vacuole protein sorting complex (HOPS) to (phago) lysosome membranes, whereas PI(3) P is required for membrane association of HOPS only. Our study provides a first link between PIPs and established regulators of membrane fusion in late endocytic trafficking.