Identification of the Penta-EF-hand Protein ALG-2 as a Ca2+-dependent Interactor of Mucolipin-1

Silvia Vergarajauregui,Jose A Martina,Rosa Puertollano

doi:10.1074/jbc.m109.047241

Silvia Vergarajauregui, Jose A Martina + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m109.047241

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Abstract

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.

Highlights

Mucolipidosis type IV (MLIV)2 is an autosomal recessive disorder characterized by severe neurological and ophthalmological abnormalities
Consistent with the lysosomal defects observed in MLIV, MCOLN1 localizes to late endosomes-lysosomes via two acidic di-leucine motifs individually located near the ends of the amino- and carboxyl-terminal tails [15, 16]
The accumulation of enlarged vacuolar structures observed in MLIV patients led to the suggestion that MCOLN1 may be involved in the regulation of the biogenesis of lysosomes, in the reformation of lysosomes from endosome-lysosome hybrid organelles [23]

Summary

Introduction

Mucolipidosis type IV (MLIV)2 is an autosomal recessive disorder characterized by severe neurological and ophthalmological abnormalities. Given the primary location of MCOLN1 to late endosomes/ lysosomes [16] and the fact that ALG-2 is the only Ca2ϩ-binding protein identified in the eluted fractions, we reasoned that MCOLN1 interacts with ALG-2, the presence of Sec31A and Sec13 at the GST-MCOLN1-NTail pulldowns is indirect and due to the ability of Sec31A to bind ALG-2.

Results

Conclusion