Penta-EF-hand protein ALG-2 functions as a Ca2+-dependent adaptor that bridges Alix and TSG101.
Alix and TSG101, known to physically interact with each other, have Pro-rich regions that are bound by ALG-2 Ca2+-dependently. We investigated the role of ALG-2 in the Alix-TSG101 association by pulldown assays using Strep-tagged Alix and its various mutants. The ALG-2-binding site was required for the Ca2+-dependent pulldown of TSG101 ... using HEK293T cells, whereas the PSAP sequence, a binding motif for the UEV domain of TSG101, was dispensable. Alix-TSG101 association was not observed using ALG-2-knockdown cells but became detectable by addition of the purified recombinant ALG-2 protein in the assay mixtures. Exogenous expression of mGFP-fused ALG-2 also restored the pulldown capability of Strep-Alix, but an alternatively spliced shorter ALG-2 isoform and a dimerization-defective mutant were incompetent. Based on the X-ray crystal structure model showing the presence of one ligand-binding site in each molecule of an ALG-2 dimer, we propose that Ca2+-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein.
Mesh Terms:
Apoptosis Regulatory Proteins, Calcium, Calcium-Binding Proteins, Cell Cycle Proteins, Cell Line, Crystallography, X-Ray, DNA-Binding Proteins, Endosomal Sorting Complexes Required for Transport, Humans, Protein Isoforms, Transcription Factors
Apoptosis Regulatory Proteins, Calcium, Calcium-Binding Proteins, Cell Cycle Proteins, Cell Line, Crystallography, X-Ray, DNA-Binding Proteins, Endosomal Sorting Complexes Required for Transport, Humans, Protein Isoforms, Transcription Factors
Biochem. Biophys. Res. Commun.
Date: Aug. 14, 2009
PubMed ID: 19520058
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