The Ca²⁺ channel TRPML3 specifically interacts with the mammalian ATG8 homologue GATE16 to regulate autophagy

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• 作者：Suzy Choi ; Hyun Jin Kim
• 关键词：TRPML3 ; GATE16 ; Autophagy ; ATG8 ; LC3 ; Split-ubiquitin membrane yeast two-hybrid (MY2H) system
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：3 January, 2014
• 年：2014
• 卷：443
• 期：1
• 页码：56-61
• 全文大小：1330 K

文摘

TRPML3 is a Ca²⁺ permeable cation channel expressed in multiple intracellular compartments. Although TRPML3 is implicated in autophagy, how TRPML3 can regulate autophagy is not understood. To search interacting proteins with TRPML3 in autophagy, we performed split-ubiquitin membrane yeast two-hybrid (MY2H) screening with TRPML3-loop as a bait and identified GATE16, a mammalian ATG8 homologue. GST pull-down assay revealed that TRPML3 and TRPML3-loop specifically bind to GATE16, not to LC3B. Co-immunoprecipitation (co-IP) experiments showed that TRPML3 and TRPML3-loop pull down only the lipidated form of GATE16, indicating that the interaction occurs exclusively at the organellar membrane. The interaction of TRPML3 with GATE16 and GATE16-positive vesicle formation were increased in starvation induced autophagy, suggesting that the interaction facilitates the function of GATE16 in autophagosome formation. However, GATE16 was not required for TRPML3 trafficking to autophagosomes. Experiments using dominant-negative (DN) TRPML3(D458K) showed that GATE16 is localized not only in autophagosomes but also in extra-autophagosomal compartments, by contrast with LC3B. Since GATE16 acts at a later stage of the autophagosome biogenesis, our results suggest that TRPML3 plays a role in autophagosome maturation through the interaction with GATE16, by providing Ca²⁺ in the fusion process.