J Biol Chem. 2009 Dec 25;284(52):36357-66. doi: 10.1074/jbc.M109.047241. Epub 2009 Oct 28.

Identification of the penta-EF-hand protein ALG-2 as a Ca2+-dependent interactor of mucolipin-1.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Vergarajauregui, S., Martina, J. A., Puertollano, R.,
["Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA."]
Loss of function mutations in mucolipin-1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal storage disease characterized by severe neurological and ophthalmological abnormalities. MCOLN1 is an ion channel that regulates membrane transport along the endolysosomal pathway. It has been suggested that MCOLN1 participates in several Ca(2+)-dependent processes, including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes with lysosomes, and lysosomal biogenesis. Here, we searched for proteins that interact with MCOLN1 in a Ca(2+)-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction is direct, strictly dependent on Ca(2+), and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37 and 49. We further show that MCOLN1 and ALG-2 co-localize to enlarged endosomes induced by overexpression of an ATPase-defective dominant-negative form of Vps4B (Vps4B(E235Q)). In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that overexpression of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosome markers. Interestingly, aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that ALG-2 regulates MCOLN1 function. Overall, our data provide new insight into the molecular mechanisms that regulate MCOLN1 activity. We propose that ALG-2 acts as a Ca(2+) sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway.
PMID: 19864416External 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 ALG-2/PDCD6 Affinity purification-mass spectrometry Human 1-66 HeLa lysates 19864416
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 SEC13 Affinity purification-mass spectrometry Human 1-66 HeLa lysates 19864416
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 SEC31A Affinity purification-mass spectrometry Human 1-66 HeLa lysates 19864416
(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 ALG-2/PDCD6 Fusion protein-pull down assay Human 1-66 E. coli Human Full-length HeLa lysates 19864416
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 SEC31A Fusion protein-pull down assay Human 1-66 E. coli Human Full-length HeLa lysates 19864416
(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 ALG-2/PDCD6 Co-immunofluorescence staining ARPE-19 19864416
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ALG-2/PDCD6 Fusion protein-pull down assay Human 37-49 Human Not determined 19864416
TRPML1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 SEC31A Fusion protein-pull down assay Human 37-49 Human Not determined 19864416
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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