Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca2؉ -binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human Alix⌬C (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b

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

ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIP1 (ALG-2-interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport I) and ESCRT-III respectively. In the present study, we investigated the association between ALG-2 and ESCRT-I. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] were shown to associate with GST-ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101. Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpressed a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD1/Vps4B (GFP-SKD1(E235Q)), ALG-2 exhibited a punctate distribution at the perinuclear area and co-localized with GFP-SKD1(E235Q) to aberrant endosomes. This punctate distribution of ALG-2 was markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 did not co-localize with GFP-SKD1(E235Q). Our findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix.

No abstract

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.