Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting

Abstract

CHMP6 (charged multivesicular body protein 6) is a human orthologue of yeast Vps (vacuolar protein sorting) 20, a component of ESCRT (endosomal sorting complex required for transport)-III. Various CHMP6 orthologues in organisms ranging from yeast to humans contain the N-myristoylation consensus sequence at each N-terminus. Metabolic labelling of HEK-293 (human embryonic kidney) cells showed the incorporation of [3H]myristate into CHMP6 fused C-terminally to GFP (green fluorescent protein) (CHMP6–GFP). Interactions of CHMP6 with another ESCRT-III component CHMP4b/Shax [Snf7 (sucrose non-fermenting 7) homologue associated with Alix] 1, one of three paralogues of human Vps32/Snf7, and with EAP20 (ELL-associated protein 20), a human counterpart of yeast Vps25 and component of ESCRT-II, were observed by co-immunoprecipitation of epitope-tagged proteins expressed in HEK-293 cells. The in vitro pull-down assays using their recombinant proteins purified from Escherichia coli demonstrated direct physical interactions which were mediated by the N-terminal basic half of CHMP6. Overexpressed CHMP6-GFP in HeLa cells exhibited a punctate distribution throughout the cytoplasm especially in the perinuclear area, as revealed by fluorescence microscopic analysis. Accumulation of LBPA (lysobisphosphatidic acid), a major phospholipid in internal vesicles of an MVB (multivesicular body), was observed in the CHMP6–GFP-localizing area. FLAG-tagged EAP20 distributed diffusely, but exhibited a punctate distribution on co-expression with CHMP6–GFP. Overexpression of CHMP6–GFP caused reduction of transferrin receptors on the plasma membrane surface, but caused their accumulation in the cytoplasm. Ubiquitinated proteins and endocytosed EGF continuously accumulated in CHMP6–GFP-expressing cells. These results suggest that CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes and regulates cargo sorting

Control of Bro1-Domain Protein Rim20 Localization by External pH, ESCRT Machinery, and theSaccharomyces cerevisiaeRim101 Pathway

“…The recent analysis of Bro1-domain structure makes it clear that all Bro1-domain proteins may interact with Snf7 or its orthologues (Kim et al, 2005). Like Snf7 itself (Kranz et al, 2001;Babst et al, 2002), the characterized mammalian Snf7 orthologues have been localized to the endosomal surface and implicated in endosome trafficking (Katoh et al, 2003;Peck et al, 2004;Yorikawa et al, 2005). With that as a backdrop, our finding that Rim20 is localized to Snf7-containing endosomes is entirely expected.…”

Interaction of the Mammalian Endosomal Sorting Complex Required for Transport (ESCRT) III Protein hSnf7-1 with Itself, Membranes, and the AAA+ ATPase SKD1

“…One possibility is that hSnf7-1 could be post-translationally modified with a hydrophobic moiety such as the myristoylation involved in bringing Vps20 (CHMP6 in mammalian systems) to the membrane (15,55). There are, however, no consensus sequences for lipid modification in hSnf7-1.…”

Cytokinetic Abscission: Phosphoinositides and ESCRTs Direct the Final Cut

“…This model of abscission involves an element of interaction between ESCRT-III components and the membrane. ESCRT-III proteins exhibit a preference for acidic membranes in in vitro binding studies (Muziol et al, 2006) and have been associated with PtdIns3P or cholesterol rich membranes during multivesicular body formation and viral budding, in which analogous ESCRT-III-dependent membrane scission events occur (Lin et al, 2005;Whitley et al, 2003;Yorikawa et al, 2005).…”