J Cell Physiol. 2009 May;219(2):344-53.

Chaperone-mediated autophagy is defective in mucolipidosis type IV.External

Venugopal, B., Mesires, N. T., Kennedy, J. C., Curcio-Morelli, C., Laplante, J. M., Dice, J. F., Slaugenhaupt, S. A.,
["Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA."]
Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin-1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two-hybrid and co-immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone-mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex- and age-matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal-associated membrane protein type 2A (LAMP-2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly-Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV.
PMID: 19117012External
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Screening Toggle
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPML1 Link HOP/STI1 Inference Prediction 19117012
TRPML1 Link HSC70 Yeast two-hybrid Human 94-298 Human Brain cDNA library 19117012
TRPML1 Link HSP40/DNAJC14 Yeast two-hybrid Human 94-298 Human Brain cDNA library 19117012
TRPML1 Link HSP90 Inference Prediction 19117012
TRPML2 Link HSC70 Inference Prediction 19117012
TRPML3 Link HSC70 Inference Prediction 19117012
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Validation: In vitro validation Toggle
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPML1 Link HSC70 Fusion protein-pull down assay Human 94-298 In vitro translation Human Full-length In vitro translation 19117012
TRPML1 Link HSP40/DNAJC14 Fusion protein-pull down assay Human 94-298 In vitro translation Human Full-length In vitro translation 19117012
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Validation: In vivo validation Toggle
  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 HOP/STI1 Co-immunoprecipitation CHO Human Full-length Not used 19117012
TRPML1 Link HSC70 Co-immunoprecipitation CHO Human Full-length Human Full-length 19117012
TRPML1 Link HSC70 Co-immunoprecipitation CHO Human Full-length Not used 19117012
TRPML1 Link HSP40/DNAJC14 Co-immunoprecipitation CHO Human Full-length Human Full-length 19117012
TRPML1 Link HSP40/DNAJC14 Co-immunoprecipitation CHO Human Full-length Not used 19117012
TRPML1 Link HSP40/DNAJC14 Co-immunofluorescence staining CHO Human Full-length Human Full-length 19117012
TRPML1 Link HSP90 Co-immunoprecipitation CHO Human Full-length Not used 19117012
TRPML2 Link HSC70 Co-immunoprecipitation CHO Human Full-length Not used 19117012
TRPML3 Link HSC70 Co-immunoprecipitation CHO Human Full-length Not used 19117012
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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