Novel Interactions of ESCRT-III with LIP5 and VPS4 and their Implications for ESCRT-III Disassembly

Abstract

The AAA؉ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/ hVps2-1, and CHMP3/hVps24 but not CHMP4A/hSnf7-1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with "MIT interacting motifs" (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

“…Thus, LIP5(MIT) 2 can bind MIM1 elements from multiple ESCRT-III proteins but apparently cannot bind MIM2 elements. Our data generally agree well with a previous study (50), except that the equilibrium dissociation constant we measured for the CHMP1B constructs (ϳ3 M) was higher than that in the previous report (EC 50 ϭ 25 nM). In that case, however, the tighter binding measurement may have been enhanced by avidity effects because the experiment was configured so that dimeric LIP5 was binding to immobilized CHMP1B.…”

“…5. In excellent agreement with a previous report (50), full-length LIP5 bound CHMP5(139 -195) with a dissociation constant of 2.0 Ϯ 0.2 nM, whereas a control LIP5(260 -307) construct corresponding to the C-terminal VSL domain alone bound only very weakly, if at all (K D Ͼ 10 M). Analytical ultracentrifugation analyses confirmed that the LIP5 protein formed a tight dimer, as expected based upon structural studies of the dimeric C-terminal VSL domain (48, 58) (data not shown).…”

“…Novel CHMP5 Element Binds the Second LIP5 MIT Module-A previous study showed that LIP5 binds CHMP5 with unusually high affinity (50), suggesting that the interaction with this ESCRT-III family member may be unique. The minimal LIP5-binding site was mapped to CHMP5 residues 149 -173 (50), and we therefore initially mapped and quantified LIP5 binding using a CHMP5 fragment that spanned this region (CHMP5 (139 -195)).…”

“…CHMP1 (yeast Vps46p/Did2p) and IST1 (Ist1p) bind one another and help recruit VPS4 to sites of action (12, 13, 33, 39 -43). LIP5/Vta1p stimulates VPS4 oligomerization and ATPase activity in vitro and in vivo (28,34,40,(43)(44)(45)(46)(47)(48)(49) and makes a particularly high affinity interaction with CHMP5 (yeast Vps60p) (44,50). These two complexes appear to act synergistically because they exhibit synthetic multivesicular body sorting phenotypes in Saccharomyces cerevisiae (51,52).…”

“…The 12 different human ESCRT-III subunits contain distinct classes of MIMs that can be displayed singly (as in CHMP5) (34,50) or in tandem (as in IST1) (42) and can even bind differently to different MIT domains (as in CHMP1B) (36,54). Moreover, humans express more than a dozen different MIT domain-containing proteins (14,(55)(56)(57), whose MIT domains can be arrayed either singly (as in VPS4 enzymes) or in tandem (as in LIP5) (58).…”