J Cell Physiol. 2009 May;219(2):344-53. doi: 10.1002/jcp.21676.
Chaperone-mediated autophagy is defective in mucolipidosis type IV.
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."]
["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: 19117012
 Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
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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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HOP/STI1 | Inference | Prediction | 19117012 | |||||
| TRPML1 |
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HSC70 | Yeast two-hybrid | Human | 94-298 | Human | Brain | cDNA library | 19117012 | |
| TRPML1 |
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HSP40/DNAJC14 | Yeast two-hybrid | Human | 94-298 | Human | Brain | cDNA library | 19117012 | |
| TRPML1 |
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HSP90 | Inference | Prediction | 19117012 | |||||
| TRPML2 |
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HSC70 | Inference | Prediction | 19117012 | |||||
| TRPML3 |
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HSC70 | Inference | Prediction | 19117012 | |||||
(:
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)
|
Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
|
top
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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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HSC70 | Fusion protein-pull down assay | Human | 94-298 | In vitro translation | Human | Full-length | In vitro translation | 19117012 |
| TRPML1 |
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HSP40/DNAJC14 | Fusion protein-pull down assay | Human | 94-298 | In vitro translation | Human | Full-length | In vitro translation | 19117012 |
(:
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)
|
Screening
Validation: In vitro validation
Validation: In vivo validation
Characterization
Functional consequence
|
top
|
| 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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HOP/STI1 | Co-immunoprecipitation | CHO | Human | Full-length | Not used | 19117012 | ||
| TRPML1 |
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HSC70 | Co-immunoprecipitation | CHO | Human | Full-length | Human | Full-length | 19117012 | |
| TRPML1 |
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HSC70 | Co-immunoprecipitation | CHO | Human | Full-length | Not used | 19117012 | ||
| TRPML1 |
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HSP40/DNAJC14 | Co-immunoprecipitation | CHO | Human | Full-length | Human | Full-length | 19117012 | |
| TRPML1 |
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HSP40/DNAJC14 | Co-immunoprecipitation | CHO | Human | Full-length | Not used | 19117012 | ||
| TRPML1 |
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HSP40/DNAJC14 | Co-immunofluorescence staining | CHO | Human | Full-length | Human | Full-length | 19117012 | |
| TRPML1 |
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HSP90 | Co-immunoprecipitation | CHO | Human | Full-length | Not used | 19117012 | ||
| TRPML2 |
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HSC70 | Co-immunoprecipitation | CHO | Human | Full-length | Not used | 19117012 | ||
| TRPML3 |
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HSC70 | Co-immunoprecipitation | CHO | Human | Full-length | Not used | 19117012 | ||
(:
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)