Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting.
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.
Yorikawa
Chiharu
C
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Shibata
Hideki
H
Waguri
Satoshi
S
Hatta
Kazumi
K
Horii
Mio
M
Katoh
Keiichi
K
Kobayashi
Toshihide
T
Uchiyama
Yasuo
Y
Maki
Masatoshi
M
D000595:Amino Acid Sequence; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002477:Cells; D004705:Endocytosis / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D005333:Fetus / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D025941:Protein Interaction Mapping; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment / Q000379:methods; D025801:Ubiquitin / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C478397:CHMP4B protein, human; C481528:CHMP6 protein, human; D002352:Carrier Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D010455:Peptides; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor