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Calpain-7 binds to CHMP1B at its second α-helical region and forms a ternary complex with IST1

Yuki Maemoto1, Yohei Osako, Emi Goto

  • 1Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.

Journal of Biochemistry
|May 28, 2011
PubMed

Insights

The endosomal sorting complex required for transport (ESCRT) system proteins CHMP1B and IST1 enhance calpain-7 autolysis. Coexpression of these proteins promotes calpain-7 localization to cellular membranes, suggesting a role in protein transport.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Protein Interactions

Background:

  • Intracellular proteins in the endosomal sorting complex required for transport (ESCRT) system, such as charged multivesicular body proteins (CHMPs), possess microtubule interacting and transport (MIT) domains.
  • These MIT domains bind to ESCRT-III proteins via MIT-interacting motifs (MIMs) located in the C-terminal regions.

Purpose of the Study:

  • To investigate the interaction between calpain-7 and ESCRT-III proteins CHMP1B and IST1.
  • To determine the effect of CHMP1B and IST1 coexpression on calpain-7 autolytic activity and subcellular localization.

Main Methods:

  • Co-immunoprecipitation assays to study protein-protein interactions.
  • In vitro binding assays to map interaction sites.
  • Subcellular fractionation to analyze protein localization.
  • HEK293T cell coexpression systems.

Main Results:

  • Coexpression of CHMP1B enhanced mGFP-fused calpain-7 autolysis, with further activation observed upon additional IST1 coexpression.
  • Calpain-7's tandem MIT domains (CL7MIT) interacted with CHMP1B's second α-helical region, not its canonical MIM1 region.
  • Coexpression of IST1 or CHMP1B strengthened the interactions between CL7MIT and CHMP1B/IST1, indicating ternary complex formation.
  • Concomitant overexpression of CHMP1B and IST1 increased calpain-7 presence in membrane/organelle fractions.

Conclusions:

  • CHMP1B and IST1 enhance calpain-7 autolysis in a cooperative manner.
  • The interaction between calpain-7 and CHMP1B involves regions beyond the canonical MIM1 motif.
  • Calpain-7, IST1, and CHMP1B form a ternary complex.
  • Coexpression of these ESCRT-III proteins influences calpain-7 subcellular localization towards membrane compartments.

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