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

Biochem Biophys Res Commun. 2014 Jan 3;443(1):56-61. doi: 10.1016/j.bbrc.2013.11.044. Epub 2013 Nov 20.

Abstract

TRPML3 is a Ca(2+) 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 Ca(2+) in the fusion process.

Keywords: ATG8; Autophagy; GATE16; LC3; Split-ubiquitin membrane yeast two-hybrid (MY2H) system; TRPML3.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Autophagy*
  • Autophagy-Related Protein 8 Family
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism*
  • Microtubule-Associated Proteins / metabolism
  • Protein Interaction Mapping
  • Transient Receptor Potential Channels / genetics
  • Transient Receptor Potential Channels / metabolism*
  • Ubiquitin / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Protein 8 Family
  • Calcium Channels
  • GABARAPL2 protein, human
  • MAP1LC3B protein, human
  • MCOLN3 protein, human
  • Microfilament Proteins
  • Microtubule-Associated Proteins
  • Transient Receptor Potential Channels
  • Ubiquitin