MCOLN1/TRPML1-MCOLN3/TRPML3 heteromer and its coupling to Ca²⁺ sensor SYT5 regulates autophagosome-lysosome fusion in a PtdIns4P-dependent manner

Macroautophagy/autophagy progresses through Ca²⁺-dependent multiple fusion events. Recently, we reported that the intracellular Ca²⁺ channel MCOLN3/TRPML3 provides Ca²⁺ for membrane fusion during autophagosome formation as a downstream effector of phosphatidylinositol-3-phosphate (PtdIns3P). However, the molecular mechanism of Ca²⁺ signaling in the late stage of autophagy remains unknown. Here, we show that the MCOLN1/TRPML1-MCOLN3/TRPML3 heteromer is the Ca²⁺ provider for autophagosome-lysosome fusion. MCOLN1-MCOLN3 functions downstream of PtdIns4P to release Ca²⁺ from autophagosomes, and the Ca²⁺ signal via PtdIns4P-MCOLN1-MCOLN3 is decoded by the Ca²⁺ sensor SYT5 (synaptotagmin 5). The binding of Ca²⁺ and PtdIns4P to SYT5 is critical for autophagosome-lysosome fusion by forming a fusion complex. Collectively, these results reveal that PtdIns4P-MCOLN1-MCOLN3-SYT5 constitutes the Ca²⁺ signaling complex in autophagosome-lysosome fusion and that MCOLN3 also regulates the late stage of autophagy through heteromerization with MCOLN1 in a phosphoinositide (PI)-dependent manner. Abbreviations: ATG, autophagy related; CPA, cyclopiazonic acid; DN, dominant-negative; GPN, glycyl-L-phenylalanine-beta-naphthylamide; KO, knockout; NH₄Cl, ammonium chloride; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; PI, phosphoinositide; SYT5, synaptotagmin 5; tfLC3, mRFP-GFP tandem fluorescent-tagged LC3; WT, wild-type.