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.

Autophagy; Ca2+ channel; PI4P; SYT5; TRPML1/3.