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PMID: 15511219 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

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

The Biochemical journal ·Vol. 387 ·No. Pt 1 ·2005-04-01 ·Pages 17-26

Yorikawa C, Shibata H, Waguri S, Hatta K, Horii M, Katoh K, Kobayashi T, Uchiyama Y, Maki M

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.

MeSH Terms
Amino Acid Sequence Biological Transport/physiology Carrier Proteins/metabolism Cell Line, Tumor Cells Endocytosis/physiology Endosomal Sorting Complexes Required for Transport Endosomes/chemistry,metabolism Epidermal Growth Factor/metabolism Fetus/chemistry,metabolism Gene Expression Regulation/genetics Green Fluorescent Proteins/genetics HeLa Cells/chemistry,metabolism Humans Kidney/chemistry,cytology,embryology,metabolism Lysosomes/chemistry,metabolism Membrane Proteins/chemistry,genetics,metabolism Molecular Sequence Data Myristic Acid/metabolism Peptides/metabolism Protein Interaction Mapping Receptors, Transferrin/metabolism Recombinant Fusion Proteins/genetics Sequence Alignment/methods Ubiquitin/metabolism Vesicular Transport Proteins/chemistry,genetics,metabolism
Chemicals
CHMP4B protein, human CHMP6 protein, human Carrier Proteins Endosomal Sorting Complexes Required for Transport Membrane Proteins Peptides Receptors, Transferrin Recombinant Fusion Proteins Ubiquitin Vesicular Transport Proteins Myristic Acid Green Fluorescent Proteins Epidermal Growth Factor
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Yorikawa Chiharu
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Shibata Hideki
Waguri Satoshi
Hatta Kazumi
Horii Mio
Katoh Keiichi
Kobayashi Toshihide
Uchiyama Yasuo
Maki Masatoshi
References (40)
40 references, click to expand
  1. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport.
    Nat Cell Biol. 1999 Jun;1(2):113-8 PMID: 10559883
  2. Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein.
    Cell Death Differ. 1999 Feb;6(2):124-9 PMID: 10200558
  3. Role of membrane organization and membrane domains in endocytic lipid trafficking.
    Traffic. 2000 Mar;1(3):203-11 PMID: 11208103
  4. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I.
    Cell. 2001 Jul 27;106(2):145-55 PMID: 11511343
  5. CHMP1 functions as a member of a newly defined family of vesicle trafficking proteins.
    J Cell Sci. 2001 Jul;114(Pt 13):2395-404 PMID: 11559748
  6. Signal transduction and endocytosis: close encounters of many kinds.
    Nat Rev Mol Cell Biol. 2002 Aug;3(8):600-14 PMID: 12154371
  7. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting.
    Dev Cell. 2002 Aug;3(2):271-82 PMID: 12194857
  8. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body.
    Dev Cell. 2002 Aug;3(2):283-9 PMID: 12194858
  9. Insights from yeast endosomes.
    Curr Opin Cell Biol. 2002 Aug;14(4):454-62 PMID: 12383796
  10. Structures, functions and molecular evolution of the penta-EF-hand Ca2+-binding proteins.
    Biochim Biophys Acta. 2002 Nov 4;1600(1-2):51-60 PMID: 12445459
  11. Receptor downregulation and multivesicular-body sorting.
    Nat Rev Mol Cell Biol. 2002 Dec;3(12):893-905 PMID: 12461556
  12. Mechanisms of enveloped RNA virus budding.
    Trends Cell Biol. 2002 Dec;12(12):569-79 PMID: 12495845
  13. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation.
    Nat Cell Biol. 2003 May;5(5):461-6 PMID: 12717448
  14. When ubiquitin meets ubiquitin receptors: a signalling connection.
    Nat Rev Mol Cell Biol. 2003 Jun;4(6):491-7 PMID: 12778128
  15. Protein sorting into multivesicular endosomes.
    Curr Opin Cell Biol. 2003 Aug;15(4):446-55 PMID: 12892785
  16. Vps27 recruits ESCRT machinery to endosomes during MVB sorting.
    J Cell Biol. 2003 Aug 4;162(3):413-23 PMID: 12900393
  17. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding.
    Cell. 2003 Sep 19;114(6):689-99 PMID: 14505569
  18. The protein network of HIV budding.
    Cell. 2003 Sep 19;114(6):701-13 PMID: 14505570
  19. The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting.
    J Biol Chem. 2003 Oct 3;278(40):39104-13 PMID: 12860994
  20. Identification of mammalian Vps24p as an effector of phosphatidylinositol 3,5-bisphosphate-dependent endosome compartmentalization.
    J Biol Chem. 2003 Oct 3;278(40):38786-95 PMID: 12878588
  21. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins.
    Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12414-9 PMID: 14519844
  22. Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome.
    J Cell Biol. 2003 Oct 27;163(2):237-43 PMID: 14581452
  23. Distinct monoubiquitin signals in receptor endocytosis.
    Trends Biochem Sci. 2003 Nov;28(11):598-603 PMID: 14607090
  24. Hrs function: viruses provide the clue.
    Trends Cell Biol. 2003 Dec;13(12):603-6 PMID: 14624836
  25. Permease recycling and ubiquitination status reveal a particular role for Bro1 in the multivesicular body pathway.
    J Biol Chem. 2003 Dec 12;278(50):50732-43 PMID: 14523026
  26. CHMP4b is a major binding partner of the ALG-2-interacting protein Alix among the three CHMP4 isoforms.
    Arch Biochem Biophys. 2004 Jan 1;421(1):159-65 PMID: 14678797
  27. Structure and function of human Vps20 and Snf7 proteins.
    Biochem J. 2004 Feb 1;377(Pt 3):693-700 PMID: 14583093
  28. Role of LBPA and Alix in multivesicular liposome formation and endosome organization.
    Science. 2004 Jan 23;303(5657):531-4 PMID: 14739459
  29. The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1.
    J Biochem. 2004 Jan;135(1):117-28 PMID: 14999017
  30. Endocytic recycling.
    Nat Rev Mol Cell Biol. 2004 Feb;5(2):121-32 PMID: 15040445
  31. The biogenesis of multivesicular endosomes.
    Nat Rev Mol Cell Biol. 2004 Apr;5(4):317-23 PMID: 15071556
  32. Protein-protein interactions of ESCRT complexes in the yeast Saccharomyces cerevisiae.
    Traffic. 2004 Mar;5(3):194-210 PMID: 15086794
  33. Structure of the ESCRT-II endosomal trafficking complex.
    Nature. 2004 Sep 9;431(7005):221-5 PMID: 15329733
  34. ESCRT-II, an endosome-associated complex required for protein sorting: crystal structure and interactions with ESCRT-III and membranes.
    Dev Cell. 2004 Oct;7(4):559-69 PMID: 15469844
  35. Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface.
    J Biol Chem. 1995 May 5;270(18):10999-1003 PMID: 7738042
  36. Portrait of a myristoyl switch protein.
    Curr Opin Struct Biol. 1996 Aug;6(4):432-8 PMID: 8794166
  37. A lipid associated with the antiphospholipid syndrome regulates endosome structure and function.
    Nature. 1998 Mar 12;392(6672):193-7 PMID: 9515966
  38. A role for Saccharomyces cerevisiae fatty acid activation protein 4 in regulating protein N-myristoylation during entry into stationary phase.
    J Biol Chem. 1998 Oct 2;273(40):25864-74 PMID: 9748261
  39. Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction.
    J Biol Chem. 1999 Jan 15;274(3):1533-40 PMID: 9880530
  40. The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells.
    Mol Biol Cell. 2000 Feb;11(2):747-63 PMID: 10679028
Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
1470-8728
DOI
10.1042/BJ20041227
Published
2005-04-01
Pages
17-26
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1134928
Subset
IM
External Links
PubMed source DOI: 10.1042/BJ20041227 PMC Full Text View journal details
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