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

The Ca2+ channel TRPML3 specifically interacts with the mammalian ATG8 homologue GATE16 to regulate autophagy.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Choi, S., Kim, H. J.,
["Department of Physiology, Sungkyunkwan University School of Medicine, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea; Center for Molecular Medicine, Samsung Biomedical Research Institute, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea.", "Department of Physiology, Sungkyunkwan University School of Medicine, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea; Center for Molecular Medicine, Samsung Biomedical Research Institute, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea. Electronic address: kimhyunjin@skku.edu."]
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.
PMID: 24269818External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPML3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GATE16 Yeast two-hybrid Human 1-312 Mouse Brain cDNA library 24269818
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPML3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GATE16 Fusion protein-pull down assay Human Full-length HEK293T lysates Mouse Full-length E. coli 24269818
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPML3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GATE16 Co-immunoprecipitation HEK293T Human Full-length Mouse Full-length 24269818
TRPML3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GATE16 Co-immunofluorescence staining HeLa Human Full-length Mouse Full-length 24269818
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPML3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GATE16 Fusion protein-pull down assay Human 1-312 Mouse Not determined 24269818
TRPML3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GATE16 Co-immunoprecipitation Human 1-312 Mouse Not determined 24269818
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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