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The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination.


ABSTRACT: ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca2+ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca2+-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.

SUBMITTER: Zhang W 

PROVIDER: S-EPMC7504102 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination.

Zhang Wei W   Muramatsu Ayaka A   Matsuo Rina R   Teranishi Naoki N   Kahara Yui Y   Takahara Terunao T   Shibata Hideki H   Maki Masatoshi M  

International journal of molecular sciences 20200831 17


ALG-2 is a penta-EF-hand Ca<sup>2+</sup>-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of s  ...[more]

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