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.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

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."]
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: 23418601External 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
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Cdc42 Affinity purification-mass spectrometry Mouse Full-length Mouse RAW264.7 macrophages Lysates 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ERIGC3 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Mouse Heart cDNA library 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 HSP40/DNAJB1 Affinity purification-mass spectrometry Mouse Full-length Mouse RAW264.7 macrophages Lysates 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NDKA Affinity purification-mass spectrometry Mouse Full-length Mouse RAW264.7 macrophages Lysates 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NovelProtein/NP9 Affinity purification-mass spectrometry Mouse Full-length Mouse RAW264.7 macrophages Lysates 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PEA-15 Affinity purification-mass spectrometry Mouse Full-length Mouse RAW264.7 macrophages Lysates 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Peroxisomal membrane protein 2 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Mouse Heart cDNA library 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PEX16 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Mouse Heart cDNA library 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PIP5K1A Split-Ubiquitin Yeast two-hybrid Mouse Full-length Mouse Heart cDNA library 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Rac1 Speculation Prediction 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Rac2 Affinity purification-mass spectrometry Mouse Full-length Mouse RAW264.7 macrophages Lysates 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RhoG Speculation Prediction 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 STOML1 Affinity purification-mass spectrometry Mouse Full-length Mouse RAW264.7 macrophages Lysates 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Unnamed protein (BAE) Split-Ubiquitin Yeast two-hybrid Mouse Full-length Mouse Heart cDNA library 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 YIF1B Split-Ubiquitin Yeast two-hybrid Mouse Full-length Mouse Heart cDNA library 23418601
(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
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Cdc42 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ERIGC3 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NDKA Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NovelProtein/NP9 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PEA-15 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Peroxisomal membrane protein 2 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PEX16 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Rac1 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Rac2 Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Unnamed protein (BAE) Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 YIF1B Split-Ubiquitin Yeast two-hybrid Mouse Full-length Yeast Mouse Full-length Yeast 23418601
(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
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Cdc42 Co-immunoprecipitation HeLa Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Cdc42 Co-immunofluorescence staining RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ERIGC3 Co-immunoprecipitation HeLa Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NovelProtein/NP9 Co-immunoprecipitation HeLa Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NovelProtein/NP9 Co-immunofluorescence staining RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PEA-15 Co-immunofluorescence staining RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PEX16 Split-Ubiquitin Yeast two-hybrid RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PIP5K1A Co-immunoprecipitation HeLa Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Rac1 Co-immunoprecipitation HeLa Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Rac2 Co-immunofluorescence staining RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RhoG Co-immunofluorescence staining RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 STOML1 Co-immunoprecipitation HeLa Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 STOML1 Co-immunoprecipitation RAW264.7 macrophage Mouse Full-length Not used 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 STOML1 Co-immunofluorescence staining RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Unnamed protein (BAE) Co-immunofluorescence staining RAW264.7 macrophage Mouse Full-length Mouse Full-length 23418601
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 YIF1B Co-immunoprecipitation HeLa Mouse Full-length Mouse Full-length 23418601
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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