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.

Figure 1. Schematic representation of various expression constructs used in the present study

Full-length deletion or point mutation constructs of CHMP6, CHMP4b or EAP20 were prepared as fusion proteins of GFP, FLAG, GST, Trx–His and MBP. The numbers denote the amino acid positions in each construct. The coiled-coil regions (CC) are indicated by grey boxes. CHMP6^G2A–GFP is the point mutant of critical residue 2 (substitution of alanine for glycine). Regions similar to WH (winged helix) domains of the yeast ESCRT-II components, whose structures were elucidated by X-ray crystalography [37,38], are indicated by closed boxes for EAP20.

Figure 2. N-myristoylation of human CHMP6

(A) N-terminal amino acid sequences of human CHMP6 and its orthologues. A schematic representation of human CHMP6 is shown at the top. The vertical broken line divides the protein into two highly charged regions rich in basic and acidic residues respectively. Two coiled-coil regions (CC) predicted by using a program on the Internet (http://www.ch.embnet.org/software/COILS_form.html; window 21; values greater than 0.5) are indicated by grey boxes. The amino acid sequences were aligned using the default setting of ClustalX 1.83, a multiple sequence alignment program (http://www-igbmc.u-strasbg.fr/BioInfo/ClustalX/Top.html). A bar above the human sequence indicates a segment that matches an N-myristoylation pattern (PROSITE code PDOC00008) consisting of six residues where glycine is N-myristoylated. Organism names are abbreviated and sequences were taken either from GenBank® (accession numbers indicated in parentheses) or from each genome project home page for respective organisms (accession numbers indicated in brackets) as follows: Hs, Homo sapiens (BC010108); Mm, Mus musculus (XP_126613); Fr, Fugu rubripes [SINFRUP00000076922]; Dm, Drosophila melanogaster (NP_726213.1); Ce, Caenorhabditis elegans (NP_490762.1); At, Arabidopsis thaliana (AAM62458.1); Dd, Dictyostelium discoideum [DDB0187234]; Sc, Saccharomyces cerevisiae (NP_013794). (B) Metabolic labelling of CHMP6 with [³H]myristic acid. HEK-293 cells transfected with pGFP, pGFP-CHMP6 or pGFP-CHMP6^G2A were cultured for 16 h with [³H]myristic acid. GFP-fusion proteins were immunoprecipitated with GFP antiserum and analysed by SDS/PAGE, followed by immunoblotting with anti-GFP mAb (left-hand panel, unlabelled) or fluorography (right-hand panel, [³H]myristate-labelled). Similar results were obtained in two independent experiments, and a representative Figure is shown. Arrows indicate CHMP6–GFP and CHMP6^G2A–GFP, and an arrowhead indicates GFP. Molecular mass sizes are indicated in kDa.

Figure 3. Dissection of CHMP6 interaction with CHMP4b and EAP20

(A) HEK-293 cells were co-transfected with pFLAG-CHMP4b and pGFP, pCHMP6-GFP, pCHMP6NT-GFP or pCHMP6CT-GFP. After 24 h, cells were lysed, and the 10000 g supernatants (Input) were immunoprecipitated (IP) with GFP antiserum. The supernatants and immunoprecipitates were analysed by Western blotting with anti-FLAG (two upper panels) and anti-GFP (α-GFP; lower panel) mAbs. (B) HEK-293 cells were co-transfected with pFLAG-EAP20 and pGFP, pCHMP6-GFP, pCHMP6NT-GFP or pCHMP6CT-GFP, and processed for immunoprecipitation followed by Western blotting as described in (A). Molecular mass sizes are indicated in kDa.

Figure 4. Direct interaction of CHMP6 with CHMP4b and EAP20

(A) CHMP4b-binding assay by GST pull-down. Recombinant protein of Trx–His–CHMP4b (400 pmol) was incubated with 200 pmol of GST or one of the GST-fusion proteins (full-length CHMP6 or truncated mutants) in 200 μl of binding buffer as described in the Materials and methods section, and glutathione–Sepharose beads were added. After the beads had been pelleted by centrifugation and washed, pulled-down proteins were analysed by SDS/PAGE and visualized by CBB staining. (B) EAP20-binding assay by GST pull-down. Recombinant protein of MBP–EAP20 (480 pmol) was incubated with GST (480 pmol) or one of the GST–CHMP6 proteins (240 pmol). (C) EAP20-binding assay by MBP pull-down. One of the GST–CHMP6 fusion proteins (480 pmol) was incubated with amylose resin that carried MBP–EAP20 (approx. 200 pmol). The beads were pelleted by centrifugation, and pulled-down proteins were analysed by SDS/PAGE and visualized by CBB staining. Molecular mass sizes are indicated in kDa.

Figure 5. Co-localization of CHMP6–GFP with FLAG–CHMP4b and FLAG–EAP20

HeLa cells transfected independently with pCHMP6-GFP (A), pFLAG-CHMP4b (B) or pFLAG-EAP20 (C), or co-transfected with pCHMP6-GFP and pFLAG-CHMP4b (D, E) or with pCHMP6-GFP and pFLAG-EAP20 (F, G) were visualized with a confocal microscope either directly (A, D and F) or by indirect immunofluorescence analyses using anti-FLAG mAb and Cy3-labelled goat anti-mouse IgG (B, C, E and G). Scale bars, 10 μm.

Figure 6. Effects of CHMP6–GFP overexpression on the distribution of LBPA, TfRs and ubiquitinated proteins

(A–F, J–L) HeLa cells transfected with pCHMP6-GFP were subjected to immunofluorescence confocal microscopy using monoclonal antibodies against LBPA (A–C), against TfR (D–F) and against ubiquitinated proteins (J–L). (G–I) HeLa cells transfected with pCHMP6-GFP were incubated at 4 °C for 30 min in the presence of Rh–Tf, washed, and fixed. (A, D, G and J) CHMP6–GFP; (B) LBPA; (E) TfR; (H) Rh–Tf binding to TfR on the plasma membrane; (K) ubiquitinated proteins; (C, F, I and L) merged images. Scale bars, 10 μm.

Figure 7. Effects of CHMP6–GFP overexpression on the fate of endocytosed EGF

HeLa cells transfected with pCHMP6-GFP (A–H) were incubated at 4 °C for 1 h in the presence of Rh–EGF, washed, and then incubated at 37 °C for 30 min (A–D) or 5 h (E–H). (A and E) CHMP6–GFP; (B and F) Rh–EGF; (C and G) merged images; (D and H) differential interference microscopic images. Asterisks indicate untransfected cells. Scale bars, 10 μm.