Structure/Function Analysis of Four Core ESCRT‐III Proteins Reveals Common Regulatory Role for Extreme C‐Terminal Domain

Abstract

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins -hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/ CHMP6, and hSnf7-1/CHMP4a -to each other, studying the effects of deleting predicted a-helical domains on their behavior in transfected cells. Surprisingly, removing $40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/ CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA1 adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default 'closed' state and an activated 'open' state under control of sequences at their C-terminus and associated factors.

Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission

“…2 A-D). Moreover, under these conditions the fluorescently tagged ESCRT proteins show their expected interphase cytosolic localization (8,20,21), and there is no increase in multinucleated cells, indicating that cytokinesis is not disrupted (Fig. S2).…”

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

“…Nevertheless, there again appear to be significant differences between the analogous metazoan and yeast interactions because the primary Vta1p interaction site was mapped to Vps60p helix 4 (34), whereas the LIP5 interaction site spans CHMP5 helices 5 and 6, and CHMP5 helix 4 does not contact LIP5(MIT) 2 (Figs. 6 -8) (53). In most ESCRT-III proteins, the fifth helix and surrounding regions can fold back onto the core domain and make autoinhibitory interactions (12,53,75,76).…”

“…Vta1p is required for Vps60p endosomal localization, but the converse is not true, and indeed, Vps60p antagonizes Vta1p endosomal localization (34). In mammalian cells, however, CHMP5 and LIP5 form stable complexes in the cytoplasm, and the two proteins are therefore likely to be recruited together in this case (53). CHMP5 activities appear to be more important for some ESCRT-dependent processes than others because siRNA depletion of CHMP5 inhibits multivesicular body sorting, but does not inhibit HIV-1 budding (44).…”

Cytokinesis in Animal Cells

“…This ensures that Cep55 and, in turn, the ESCRT proteins accumulate at the midbody ring at a very late stage in cytokinesis. Furthermore, the CPC has been suggested to regulate abscission through the interaction of its subunit Borealin with all three ESCRT-III Snf7 components, CHMP4A, CHMP4B, and CHMP4C, and Aurora B phosphorylation of the carboxy-terminal tail of CHMP4C (Capalbo et al 2012;Carlton et al 2012), a region known to regulate CHMP4C's ability to polymerize and associate with membranes (Shim et al 2007). The exact nature of this regulation, however, has been debated and two different models have been proposed.…”