VPS37 isoforms differentially modulate the ternary complex formation of ALIX, ALG-2, and ESCRT-I.
The endosomal sorting complex required for transport (ESCRT) system comprises a series of protein complexes that play essential roles in multivesicular body (MVB) sorting of ubiquitylated membrane proteins, enveloped RNA virus budding, and cytokinesis in mammalian cells. The complex, named ESCRT-I, consists of four subunits (TSG101, VPS28, VPS37, and MVB12). There are four VPS37 isoforms. We have reported that ALIX (an ALG-2-interacting protein and accessory protein in the ESCRT system) is physically linked with TSG101 by ALG-2 in a Ca²⁺-dependent manner, but the role of ALG-2 as an adaptor protein for the ESCRT-I complex remains unknown. To characterize this adaptor function, initially we investigated the binding of ALG-2 to ESCRT-I complexes containing each one of the four different VPS37 isoforms by two approaches: first, Far-Western blot analysis with biotin-labeled ALG-2 probe, and second, a pulldown assay to determine the binding of the four recombinant ESCRT-I complexes to Strep-tagged ALG-2 after co-expression in HEK293T cells. VPS37B and VPS37C appeared to interact with ALG-2 in a stronger manner than TSG101 does. The results of in vitro binding assays using purified recombinant proteins indicated that ALG-2 functions as a Ca²⁺-dependent adaptor protein that bridges ALIX and ESCRT-I to form a ternary complex, ESCRT-I/ALIX/ALG-2.
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University.
Mayumi
M
Okumura
Angela M
AM
Katsuyama
Hideki
H
Shibata
Masatoshi
M
Maki
D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050801:Ternary Complex Factors
D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C097753:PDCD6 protein, human; C117020:PDCD6IP protein, human; D050801:Ternary Complex Factors; C502194:VPS37C protein, human; C541454:vacuolar protein sorting 37 homolog B, human