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.
Highlights
Eukaryotes have a group of proteins that possess a domain of five serially repeated helix-loop-helix Ca2+-binding motifs [1]
ALG-2 was first shown to bind to an ALG-2-interacting protein named AIP1/ALIX [8,9], which was later shown to play roles as one of key associating factors of endosomal sorting complex required for transport (ESCRT) by binding to CHMP4s and TSG101 [10,11,12]
Our subsequent studies including sequence comparison and mutational analysis of ALG-2-binding sites based on X-ray crystal structures revealed the presence of three different ALG-2-binding motifs (ABMs) in the Pro-rich regions (PRRs) (See Ref. [17]) for review: type 1 (ABM-1, represented by ALIX); type 2 (ABM-2, represented by Sec31A); type 3 (ABM-3), MetPro repeats in IST1
Summary
Eukaryotes have a group of proteins that possess a domain of five serially repeated helix-loop-helix Ca2+-binding motifs (penta-EF-hand proteins, PEF proteins) [1]. The PEF protein ALG-2 (gene name: PDCD6) was originally identified as one of the factors associated with cell death of T cells [2], the roles of ALG-2 in apoptosis are not clearly understood. Our subsequent studies including sequence comparison and mutational analysis of ALG-2-binding sites based on X-ray crystal structures revealed the presence of three different ALG-2-binding motifs (ABMs) in the PRRs We demonstrated a Ca2+-dependent interaction between SARAF and ALG-2 by co-immunoprecipitation (co-IP) assays of the endogenous proteins in mammalian cells. We investigated roles of the two PPXY motifs present in the SARAF cytosolic domain (CytD) for ubiquitination and clarified the relationship between ALG-2 binding to SARAF and ubiquitination interference. We have found that the ALG-2 dimer bridges two SARAF molecules in a Ca2+-dependent manner by binding to the respective CytDs and we discussed the correlation with ubiquitination interference
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