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Updated: May 11, 2026

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans
Published on: May 23, 2020
Mammalian ESCRT-III-related protein IST1 has a distinctive met-pro repeat sequence that is essential for interaction
Mayumi Okumura1, Takeshi Takahashi, Hideki Shibata
1Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
Abstract:
ALG-2 is an EF-hand-type Ca(2+)-binding protein that interacts with a variety of intracellular proteins that possess Pro-rich regions (PRRs) in mammalian cells. IST1 is an endosomal sorting complex required for transport (ESCRT)-III-related charged multivesicular body protein (CHMP)-like protein, but unlike other ESCRT-III proteins, mammalian IST1 has a PRR and a distinctive sequence of Met-Pro repeats. We found that ALG-2 binds to IST1 by Far-Western analysis using biotinylated ALG-2 as probe, and that the Met-Pro repeat sequence is essential for interaction. The results of pulldown assays using Strep-tagged ALG-2 and lysates of cells expressing GFP-fused IST1 proteins indicated that the binding of ALG-2 to IST1 is Ca(2+)-dependent, and that it is enhanced by co-expression with CHMP1 proteins. Moreover, pulldown assays using various mutants of GST-ALG-2 revealed that the ability of IST1 to bind to mutants is different from those of known ALG-2-interacting proteins, suggesting that IST1 binds to ALG-2 by a different mode of recognition.
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