CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway.
All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1-CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG-CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG-CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b-GFP or GFP-Vps4B(E235Q), a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP-CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP-CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting pathway.
Horii
Mio
M
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Shibata
Hideki
H
Kobayashi
Ryota
R
Katoh
Keiichi
K
Yorikawa
Chiharu
C
Yasuda
Jiro
J
Maki
Masatoshi
M
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
C481529:CHMP4A protein, human; C478397:CHMP4B protein, human; C514910:CHMP7 protein, human; D002352:Carrier Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins