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.
Okumura
Mayumi
M
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Ichioka
Fumitaka
F
Kobayashi
Ryota
R
Suzuki
Hironori
H
Yoshida
Haruna
H
Shibata
Hideki
H
Maki
Masatoshi
M
D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000099177:Tsg101 Protein
D051017:Apoptosis Regulatory Proteins; D002118:Calcium; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D020033:Protein Isoforms; D014157:Transcription Factors; D000099177:Tsg101 Protein; C097753:PDCD6 protein, human; C117020:PDCD6IP protein, human