Two Pore Channel 2 (TPC2) Inhibits Autophagosomal-Lysosomal Fusion by Alkalinizing Lysosomal pH

Yingying Lu,Bai-Xia Hao,Richard Graeff,Connie W.M Wong,Wu-Tian Wu,Jianbo Yue

doi:10.1074/jbc.m113.484253

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.

Highlights

The role and mechanism of NAADP, an endogenous Ca2ϩ mobilizing nucleotide, in autophagic regulation remain to be determined
Antibodies and Reagents—LC3 antibody (antibody from MBL was used in study in the embryonic stem (ES) cells, and the one from Novus was used in the study in HeLa cells; 1:500 for the immunofluorescence analysis and 1:1000 for the Western blotting analysis (WB)), p62 antibody (Novus; 1:1000 WB), cathepsin-L antibody (BD Bioscience; 1:250 WB), Lamp-1 and EGF binds to its receptors (EGFR) antibodies (Santa Cruz; 1:250 WB), Lamp-1 antibody (Cell Signaling; 1:500 immunofluorescence analysis), phosphomTOR and mTOR antibodies (Cell Signaling; 1:500 WB), GAPDH antibody (Sigma; 1:5000 WB), and TPC2 antibody
The Effects of Ca2ϩ on TPC2-induced Autophagy Inhibition— NAADP can induce Ca2ϩ release from lysosomes [25], and we found that NAADP treatment inhibited autophagosomal-lysosomal fusion (Fig. 4C and supplemental Fig. S3A)

Summary

Background

The role and mechanism of NAADP, an endogenous Ca2ϩ mobilizing nucleotide, in autophagic regulation remain to be determined. Results: Activation of NAADP/TPC2 signaling induced the accumulation of autophagosomes. Conclusion: The NAADP/TPC2 signaling inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH. NAADP mobilizes Ca2ϩ from acidic lysosome-related stores in a wide variety of cells, from plant to animal, including human [25]. Several recent papers found that NAADP does not directly bind to TPC2, suggesting that NAADP first binds to accessory proteins, which subsequently activate TPC2 or other ion channels for Ca2ϩ mobilization depending upon the cell type (40 – 42). Here we found that the increased LC3-II levels by TPC2 signaling are not due to the enhanced autophagy activity but result from the inhibition of basal autophagy progression, which leads to the accumulation of autophagosomes. Inhibition of TPC2 signaling either by TPC2 knockdown or by treatment with a NAADP antagonist, on the other hand, facilitates autophagosomal-lysosomal fusion, thereby decreasing LC3-II levels

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION