PLoS One. 2013;8(2):e56780. doi: 10.1371/journal.pone.0056780. Epub 2013 Feb 13.
Systematic screens for proteins that interact with the mucolipidosis type IV protein TRPML1.
Spooner, E., McLaughlin, B. M., Lepow, T., Durns, T. A., Randall, J., Upchurch, C., Miller, K., Campbell, E. M., Fares, H.,
["Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, United States of America."]
["Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, United States of America."]
Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRPML1 will facilitate the elucidation of its cellular and biochemical functions. We report here two saturation screens for proteins that interact with TRPML1: one that is based on immunoprecipitation/mass spectrometry and the other using a genetic yeast two-hybrid approach. From these screens, we identified largely non-overlapping proteins, which represent potential TRPML1-interactors., Using additional interaction assays on some of the potential interactors from each screen, we validated some proteins as candidate TRPML1 interactors In addition, our analysis indicates that each of the two screens not only identified some false-positive interactors, as expected from any screen, but also failed to uncover potential TRPML1 interactors. Future studies on the true interactors, first identified in these screens, will help elucidate the structure and function of protein complexes containing TRPML1.
PMID: 23418601

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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 | |||
TRPML1 |
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Cdc42 | Affinity purification-mass spectrometry | Mouse | Full-length | Mouse | RAW264.7 macrophages | Lysates | 23418601 | |
TRPML1 |
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ERIGC3 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Mouse | Heart | cDNA library | 23418601 | |
TRPML1 |
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HSP40/DNAJB1 | Affinity purification-mass spectrometry | Mouse | Full-length | Mouse | RAW264.7 macrophages | Lysates | 23418601 | |
TRPML1 |
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NDKA | Affinity purification-mass spectrometry | Mouse | Full-length | Mouse | RAW264.7 macrophages | Lysates | 23418601 | |
TRPML1 |
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NovelProtein/NP9 | Affinity purification-mass spectrometry | Mouse | Full-length | Mouse | RAW264.7 macrophages | Lysates | 23418601 | |
TRPML1 |
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PEA-15 | Affinity purification-mass spectrometry | Mouse | Full-length | Mouse | RAW264.7 macrophages | Lysates | 23418601 | |
TRPML1 |
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Peroxisomal membrane protein 2 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Mouse | Heart | cDNA library | 23418601 | |
TRPML1 |
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PEX16 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Mouse | Heart | cDNA library | 23418601 | |
TRPML1 |
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PIP5K1A | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Mouse | Heart | cDNA library | 23418601 | |
TRPML1 |
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Rac1 | Speculation | Prediction | 23418601 | |||||
TRPML1 |
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Rac2 | Affinity purification-mass spectrometry | Mouse | Full-length | Mouse | RAW264.7 macrophages | Lysates | 23418601 | |
TRPML1 |
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RhoG | Speculation | Prediction | 23418601 | |||||
TRPML1 |
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STOML1 | Affinity purification-mass spectrometry | Mouse | Full-length | Mouse | RAW264.7 macrophages | Lysates | 23418601 | |
TRPML1 |
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Unnamed protein (BAE) | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Mouse | Heart | cDNA library | 23418601 | |
TRPML1 |
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YIF1B | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Mouse | Heart | cDNA library | 23418601 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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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 | ||
TRPML1 |
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Cdc42 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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ERIGC3 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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NDKA | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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NovelProtein/NP9 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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PEA-15 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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Peroxisomal membrane protein 2 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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PEX16 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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Rac1 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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Rac2 | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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Unnamed protein (BAE) | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
TRPML1 |
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YIF1B | Split-Ubiquitin Yeast two-hybrid | Mouse | Full-length | Yeast | Mouse | Full-length | Yeast | 23418601 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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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 | ||||
TRPML1 |
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Cdc42 | Co-immunoprecipitation | HeLa | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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Cdc42 | Co-immunofluorescence staining | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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ERIGC3 | Co-immunoprecipitation | HeLa | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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NovelProtein/NP9 | Co-immunoprecipitation | HeLa | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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NovelProtein/NP9 | Co-immunofluorescence staining | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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PEA-15 | Co-immunofluorescence staining | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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PEX16 | Split-Ubiquitin Yeast two-hybrid | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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PIP5K1A | Co-immunoprecipitation | HeLa | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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Rac1 | Co-immunoprecipitation | HeLa | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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Rac2 | Co-immunofluorescence staining | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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RhoG | Co-immunofluorescence staining | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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STOML1 | Co-immunoprecipitation | HeLa | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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STOML1 | Co-immunoprecipitation | RAW264.7 macrophage | Mouse | Full-length | Not used | 23418601 | ||
TRPML1 |
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STOML1 | Co-immunofluorescence staining | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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Unnamed protein (BAE) | Co-immunofluorescence staining | RAW264.7 macrophage | Mouse | Full-length | Mouse | Full-length | 23418601 | |
TRPML1 |
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YIF1B | Co-immunoprecipitation | HeLa | Mouse | Full-length | Mouse | Full-length | 23418601 |
(
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
