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

Abstract

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.

Membrane Fission Reactions of the Mammalian ESCRT Pathway

“…For example, the ESCRT-III protein CHMP1B binds the MIT domains of the AAA ATPase spastin (Figure 4e), which severs microtubules immediately prior to abscission (77). Additional MIT domain-containing proteins recruited by ESCRT-III subunits to function in abscission include the phospholipase D–like protein, MITD1 (CHMP1A, CHMP1B, CHMP2A, and IST1) (177, 178), spartin (SPG20) (IST1) (179), and the protease calpain 7 (IST1:CHMP1B complexes) (180). …”

Differential requirements of mammalian ESCRTs in multivesicular body formation, virus budding and cell division

“…Another MIT domain protein is calpain‐7, one of the cysteine proteases of the calpain superfamily. The MIT domain of calpain‐7 associates with CHMP1 and IST1 [63]. However, the role of calpain‐7 in cytokinesis is not fully understood.…”

Evolutionary and physical linkage between calpains and penta‐EF‐hand Ca2+‐binding proteins

“…However, binding of the MIT domains to CHMP1B exhibits a different mode of interaction. Calpain‐7 binds to the predicted second α‐helical region of CHMP1B as indicated by in vitro binding assays using purified recombinant proteins [30]. Calpain‐7, IST1 and CHMP1B form a ternary complex.…”