Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission

Abstract

The 12 related human ESCRT-III proteins form filaments that constrict membranes and mediate fission, including during cytokinetic abscission. The C-terminal tails of polymerized ESCRT-III subunits also bind proteins that contain Microtubule-Interacting and Trafficking (MIT) domains. MIT domains can interact with ESCRT-III tails in many different ways to create a complex binding code that is used to recruit essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 recombinant human MIT domains. We measured 228 pairwise interactions, quantified 60 positive interactions, and discovered 18 previously unreported interactions. We also report the crystal structure of the SPASTIN MIT domain in complex with the IST1 C-terminal tail. Three MIT enzymes were studied in detail and shown to: (1) localize to cytokinetic midbody membrane bridges through interactions with their specific ESCRT-III binding partners (SPASTIN-IST1, KATNA1-CHMP3, and CAPN7-IST1), (2) function in abscission (SPASTIN, KATNA1, and CAPN7), and (3) function in the ‘NoCut’ abscission checkpoint (SPASTIN and CAPN7). Our studies define the human MIT-ESCRT-III interactome, identify new factors and activities required for cytokinetic abscission and its regulation, and provide a platform for analyzing ESCRT-III and MIT cofactor interactions in all ESCRT-mediated processes.

The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

“…The tandem CAPN7 MIT domains (CAPN7(MIT) 2 ) bind a C-terminal region of IST1 that contains two distinct MIM elements (Figure 1A) (Agromayor et al ., 2009; Bajorek et al ., 2009; Osako et al ., 2010; Wenzel et al ., 2022). We quantified this interaction using fluorescence polarization anisotropy binding assays with purified, recombinant CAPN7(MIT) 2 and fluorescently labeled IST constructs that spanned either one or both of the MIM elements.…”

“…We quantified this interaction using fluorescence polarization anisotropy binding assays with purified, recombinant CAPN7(MIT) 2 and fluorescently labeled IST constructs that spanned either one or both of the MIM elements. These experiments showed that both IST1 MIMs contribute to binding (Figure 1B) (Osako et al ., 2010, Wenzel et al ., 2022). The double MIM IST1 316-366 construct bound CAPN7(MIT) 2 tightly (K D = 0.09 ± 0.01 µM), and the first IST1 MIM element (IST1 344-366 ) contributed most of the binding energy (K D = 1.8 ± 0.1 µM).…”

“…ESCRT-III filaments also recruit a variety of cofactors, including the VPS4 AAA+ ATPases, which dynamically remodel the filaments to drive midbody constriction (Elia et al ., 2012; Mierzwa et al ., 2017; Pfitzner et al ., 2020; Pfitzner et al ., 2021). Many of these cofactors contain MIT domains, which bind differentially to MIT-Interacting Motifs (MIMs) located near the C-termini of the different human ESCRT-III proteins (Hurley and Yang, 2008; Wenzel et al ., 2022).…”

“…Our recent quantitative survey of the human ESCRT-III-MIT interactome revealed a series of novel interactions between ESCRT-III subunits and MIT cofactors and implicated a subset of these cofactors in abscission and NoCut checkpoint maintenance (Wenzel et al ., 2022). One such cofactor was CAPN7 (Calpain-7), a ubiquitously expressed but poorly understood cysteine protease that had not previously been linked to abscission nor to the NoCut checkpoint.…”

“…One such cofactor was CAPN7 (Calpain-7), a ubiquitously expressed but poorly understood cysteine protease that had not previously been linked to abscission nor to the NoCut checkpoint. CAPN7 contains tandem MIT domains that can bind specifically to the ESCRT-III subunit IST1 (Osako et al ., 2010; Wenzel et al ., 2022). This interaction can activate CAPN7 autolysis and proteolytic activity towards non-physiological substrates (Osako et al ., 2010; Maemoto et al ., 2013), although authentic CAPN7 substrates are not yet known.…”