The ALG-2-interacting protein Alix associates with CHMP4b,a human homologue of yeast Snf7 that is involved in multivesicular body sorting

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-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 AlixDeltaC (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 (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.     

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.     

Brox, a novel farnesylated Bro1 domain-containing protein that associates with charged multivesicular body protein 4 (CHMP4)

Human Brox is a newly identified 46 kDa protein that has a Bro1 domain-like sequence and a C-terminal thioester-linkage site of isoprenoid lipid (CAAX motif) (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid). Mammalian Alix and its yeast ortholog, Bro1, are known to associate with charged multivesicular body protein 4 (CHMP4), a component of endosomal sorting complex required for transport III, via their Bro1 domains and to play roles in sorting of ubiquitinated cargoes. We investigated whether Brox has an authentic Bro1 domain on the basis of its capacity for interacting with CHMP4s. Both Strep Tactin binding sequence (Strep)-tagged wild-type Brox (Strep-Brox(WT)) and Strep-tagged farnesylation-defective mutant (Cys-->Ser mutation; Strep-Brox(C408S)) pulled down FLAG-tagged CHMP4b that was coexpressed in HEK293 cells. Treatment of cells with a farnesyltransferase inhibitor, FTI-277, caused an electrophoretic mobility shift of Strep-Brox(WT), and the mobility coincided with that of Strep-Brox(C408S). The inhibitor also caused a mobility shift of endogenous Brox detected by western blotting using polyclonal antibodies to Brox, suggesting farnesylation of Brox in vivo. Fluorescence microscopic analyses revealed that Strep-Brox(WT) exhibited accumulation in the perinuclear area and caused a punctate pattern of FLAG-CHMP4b that was constitutively expressed in HEK293 cells. On the other hand, Strep-Brox(C408S) showed a diffuse pattern throughout the cell, including the nucleus, and did not cause accumulation of FLAG-CHMP4b. Fluorescent signals of monomeric green fluorescent protein (mGFP)-fused Brox(WT) merged partly with those of Golgi markers and with those of abnormal endosomes induced by overexpression of a dominant negative mutant of AAA type ATPase SKD1/Vps4B in HeLa cells, but such colocalization was less efficient for mGFP-Brox(C408S). These results suggest a physiological significance of farnesylation of Brox in its subcellular distribution and efficient interaction with CHMP4s in vivo.     

Structural basis for autoinhibition of ESCRT-III CHMP3

Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family—cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.     

HD-PTP and Alix share some membrane-traffic related proteins that interact with their Bro1 domains or proline-rich regions

Mammalian Alix is a multifunctional adaptor protein involved in cell death, receptor endocytosis, endosomal protein sorting and cell adhesion by associating with various proteins such as ALG-2, CIN85/Rukl/SETA, endophilins, CHMP4s and TSG101. HD-PTP is a paralog of Alix and a putative protein tyrosine phosphatase (PTP) that contains a Bro1 domain, coiled-coils, a proline-rich region (PRR) in addition to a PTP domain. We investigated interactions between HD-PTP and Alix-binding proteins. In the yeast two-hybrid assay, HD-PTP showed positive interactions with CHMP4b/Shax1, TSG101, endophilin A1 and ALG-2 but not with either RabGAPLP or CIN85. We confirmed the interactions in a mammalian system by Strep-pulldown assays in which pulldown products from the lysates of HEK293T cells expressing either Strep-tagged HD-PTP alone or co-expressing with epitope-tagged proteins were analyzed by Western blotting using specific antibodies. While Alix associated with both ALG-2 and TSG101 in a Ca2+-dependent manner, HD-PTP interacted with ALG-2 Ca2+-dependently but with TSG101 Ca2+-independently.     

Structural Study of the HD-PTP Bro1 Domain in a Complex with the Core Region of STAM2, a Subunit of ESCRT-0

EGFR is a key player in cell proliferation and survival signaling, and its sorting into MVBs for eventual lysosomal degradation is controlled by the coordination of multiple ESCRT complexes on the endosomal membrane. HD-PTP is a cytosolic protein tyrosine phosphatase, and is associated with EGFR trafficking by interacting with the ESCRT-0 protein STAM2 and the ESCRT-III protein CHMP4B via its N-terminal Bro1 domain. Intriguingly, the homologous domain of two other human Bro1 domain-containing proteins, Alix and Brox, binds CHMP4B but not STAM2, despite their high structural similarity. To elucidate this binding specificity, we determined the complex structure of the HD-PTP Bro1 domain bound to the STAM2 core region. STAM2 binds to the hydrophobic concave pocket of the HD-PTP Bro1 domain, as CHMP4B does to the pocket of Alix, Brox, or HD-PTP but in the opposite direction. Critically, Thr145 of HD-PTP, corresponding to Lys151 of Alix and Arg145 of Brox, is revealed to be a determinant residue enabling this protein to bind STAM2, as the Alix- or Brox-mimicking mutations of this residue blocks the intermolecular interaction. This work therefore provides the structural basis for how HD-PTP recognizes the ESCRT-0 component to control EGFR sorting.     

A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5

Disassembly of the endosomal sorting complex required for transport (ESCRT) machinery from biological membranes is a critical final step in cellular processes that require the ESCRT function. This reaction is catalyzed by VPS4, an AAA-ATPase whose activity is tightly regulated by a host of proteins, including LIP5 and the ESCRT-III proteins. Here, we present structural and functional analyses of molecular interactions between human VPS4, LIP5, and the ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly inhibits the stimulation. Both of these observations are distinct from what was previously described for homologous yeast proteins. The crystal structure of LIP5NTD in complex with the MIT (microtubule-interacting and transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, was determined at 1 Å resolution. It reveals an ESCRT-III binding induced moderate conformational change in LIP5NTD, which results from insertion of a conserved CHMP5 tyrosine residue (Tyr¹⁸²) at the core of LIP5NTD structure. Mutation of Tyr¹⁸² partially relieves the inhibition displayed by CHMP5. Together, these results suggest a novel mechanism of VPS4 regulation in metazoans, where CHMP5 functions as a negative allosteric switch to control LIP5-mediated stimulation of VPS4.     

Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central “V” domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.     

CHMP4B, a novel gene for autosomal dominant cataracts linked to chromosome 20q

Cataracts are a clinically diverse and genetically heterogeneous disorder of the crystalline lens and a leading cause of visual impairment. Here we report linkage of autosomal dominant "progressive childhood posterior subcapsular" cataracts segregating in a white family to short tandem repeat (STR) markers D20S847 (LOD score [Z] 5.50 at recombination fraction [theta] 0.0) and D20S195 (Z=3.65 at theta =0.0) on 20q, and identify a refined disease interval (rs2057262-(3.8 Mb)-rs1291139) by use of single-nucleotide polymorphism (SNP) markers. Mutation profiling of positional-candidate genes detected a heterozygous transversion (c.386A-->T) in exon 3 of the gene for chromatin modifying protein-4B (CHMP4B) that was predicted to result in the nonconservative substitution of a valine residue for a phylogenetically conserved aspartic acid residue at codon 129 (p.D129V). In addition, we have detected a heterozygous transition (c.481G-->A) in exon 3 of CHMP4B cosegregating with autosomal dominant posterior polar cataracts in a Japanese family that was predicted to result in the missense substitution of lysine for a conserved glutamic acid residue at codon 161 (p.E161K). Transfection studies of cultured cells revealed that a truncated form of recombinant D129V-CHMP4B had a different subcellular distribution than wild type and an increased capacity to inhibit release of virus-like particles from the cell surface, consistent with deleterious gain-of-function effects. These data provide the first evidence that CHMP4B, which encodes a key component of the endosome sorting complex required for the transport-III (ESCRT-III) system of mammalian cells, plays a vital role in the maintenance of lens transparency.     

A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex

In Saccharomyces cerevisiae 6 closely related proteins (Did2p, Vps2p, Vps24p, Vps32p, Vps60p, Vps20p) form part of the extended ESCRT III complex. This complex is required for the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins. In contrast the human genome encodes 10 homologous proteins (CHMP1A (approved gene symbol PCOLN3), 1B, 2A, 2B, 3 (approved gene symbol VPS24), 4A, 4B, 4C, 5, and 6). In this study we have performed a series of protein interaction experiments to generate a more comprehensive picture of the human CHMP protein-interaction network. Our results describe novel interactions between known components of the human ESCRT III complex and identify a range of putative binding partners, which may indicate new ways in which the function of human CHMP proteins may be regulated. In particular, we show that two further MIT domain-containing proteins (AMSH/STAMBP and LOC129531) interact with multiple components of the human ESCRT III complex.     

Structural basis for budding by the ESCRT-III factor CHMP3

The vacuolar protein sorting machinery regulates multivesicular body biogenesis and is selectively recruited by enveloped viruses to support budding. Here we report the crystal structure of the human ESCRT-III protein CHMP3 at 2.8 A resolution. The core structure of CHMP3 folds into a flat helical arrangement that assembles into a lattice, mainly via two different dimerization modes, and unilaterally exposes a highly basic surface. The C terminus, the target for Vps4-induced ESCRT disassembly, extends from the opposite side of the membrane targeting region. Mutations within the basic and dimerization regions hinder bilayer interaction in vivo and reverse the dominant-negative effect of a truncated CHMP3 fusion protein on HIV-1 budding. Thus, the final steps in the budding process may include CHMP protein polymerization and lattice formation on membranes by employing different bilayer-recognizing surfaces, a function shared by all CHMP family members.     

CC2D1A is a regulator of ESCRT-III CHMP4B

Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function.     

Identification of Rab GTPase-activating protein-like protein (RabGAPLP) as a novel Alix/AIP1-interacting protein

Alix/AIP1 is a multifunctional adaptor protein involved in endocytosis, cell adhesion, and cell death. By yeast two-hybrid screening we identified a novel Alix/AIP1-interacting protein named Rab GTPase-activating protein-like protein (RabGAPLP). Interaction between Alix and RabGAPLP was confirmed by pull-down assays using fusion proteins of either glutathione-S-transferase (GST) or chitin-binding domain (CBD) and lysates of cultured mammalian cells expressing the respective proteins. Partial colocalization of FLAG-tagged RabGAPLP and green fluorescent protein (GFP)-fused Alix was observed at cell edges and filopodia-like structures by fluorescence confocal laser scanning microscopic analysis. The identity of RabGAPLP to merlin-associated protein (MAP), one of the interacting partners of neurofibromatosis type 2 (NF2) tumor suppressor gene product (merlin), implies cross-talk of membrane traffic and cell adhesion.     

The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.     

The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells

We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release.     

Regulation of CHMP4/ESCRT-III function in human immunodeficiency virus type 1 budding by CC2D1A

The detachment of human immunodeficiency type 1 (HIV-1) virions depends on CHPM4 family members, which are late-acting components of the ESCRT pathway that mediate the cleavage of bud necks from the cytosolic side. We now show that in human cells, CHMP4 proteins are to a considerable extent bound to two high-molecular-weight proteins that we have identified as CC2D1A and CC2D1B. Both proteins bind to the core domain of CHMP4B, which has a strong propensity to polymerize and to inhibit HIV-1 budding. Further mapping showed that CC2D1A binds to an N-terminal hairpin within the CHMP4 core that has been implicated in polymerization. Consistent with a model in which CC2D1A and CC2D1B regulate CHMP4 polymerization, the overexpression of CC2D1A inhibited both the release of wild-type HIV-1 and the CHMP4-dependent rescue of an HIV-1 L domain mutant by exogenous ALIX. Furthermore, small interfering RNA against CC2D1A or CC2D1B increased HIV-1 budding under certain conditions. CC2D1A and CC2D1B possess four Drosophila melanogaster 14 (DM14) domains, and we demonstrate that these constitute novel CHMP4 binding modules. The DM14 domain that bound most avidly to CHMP4B was by itself sufficient to inhibit the function of ALIX in HIV-1 budding, indicating that the inhibition occurred through CHMP4 sequestration. However, N-terminal fragments of CC2D1A that did not interact with CHMP4B nevertheless retained a significant level of inhibitory activity. Thus, CC2D1A may also affect HIV-1 budding in a CHMP4-independent manner.     

Calpain-7 binds to CHMP1B at its second α-helical region and forms a ternary complex with IST1

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.