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.
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."]
["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: 24269818
 Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
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Screening
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| Experimental screening | Non-experimental screening | Reference | ||||||||
| TRP channel construct | Interactor source | |||||||||
| TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
| TRPML3 |
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GATE16 | Yeast two-hybrid | Human | 1-312 | Mouse | Brain | cDNA library | 24269818 | |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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| Validation: In vitro validation
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| Assay with recombinant proteins | Reference | |||||||||
| TRP channel construct | Interactor construct | |||||||||
| TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
| TRPML3 |
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GATE16 | Fusion protein-pull down assay | Human | Full-length | HEK293T lysates | Mouse | Full-length | E. coli | 24269818 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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top
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| Validation: In vivo validation
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| 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 |
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GATE16 | Co-immunoprecipitation | HEK293T | Human | Full-length | Mouse | Full-length | 24269818 | |
| TRPML3 |
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GATE16 | Co-immunofluorescence staining | HeLa | Human | Full-length | Mouse | Full-length | 24269818 | |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
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Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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top
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| Characterization
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| Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
| TRP channel | Interactor | |||||||||
| TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
| TRPML3 |
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GATE16 | Fusion protein-pull down assay | Human | 1-312 | Mouse | Not determined | 24269818 | ||
| TRPML3 |
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GATE16 | Co-immunoprecipitation | Human | 1-312 | Mouse | Not determined | 24269818 | ||
(:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)