title: Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.
abstract: Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.
authors.affiliation: Department of Physiology, University of Hong Kong, Hong Kong, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Lu
authors.forename: Bai-Xia
authors.initials: BX
authors.lastname: Hao
authors.forename: Richard
authors.initials: R
authors.lastname: Graeff
authors.forename: Connie W M
authors.initials: CW
authors.lastname: Wong
authors.forename: Wu-Tian
authors.initials: WT
authors.lastname: Wu
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yue
mesh_terms: D000468:Alkalies / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000187:drug effects; D000090802:rab7 GTP-Binding Proteins
chemical_list: D000468:Alkalies; D015220:Calcium Channels; C546785:TPCN2 protein, mouse; D000090802:rab7 GTP-Binding Proteins; D009249:NADP; C024376:NAADP; D058570:TOR Serine-Threonine Kinases; D020691:rab GTP-Binding Proteins; D002118:Calcium
keywords: Autophagy; Calcium; Calcium Channels; Calcium Intracellular Release; Calcium Signaling; Lysosomes; NAADP; TPC2; pH