Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis

TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.     

UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting

Endosomal sorting complexes required for transport (ESCRTs) regulate several events involving membrane invagination, including multivesicular body (MVB) biogenesis, viral budding, and cytokinesis. In each case, upstream ESCRTs combine with additional factors, such as Bro1 proteins, to recruit ESCRT-III and the ATPase VPS4 in order to drive membrane scission. A clue to understanding how such diverse cellular processes might be controlled independently of each other has been the identification of ESCRT isoforms. Mammalian ESCRT-I comprises TSG101, VPS28, VPS37A-D, and MVB12A/B. These could generate several ESCRT-I complexes, each targeted to a different compartment and able to recruit distinct ESCRT-III proteins. Here we identify a novel ESCRT-I component, ubiquitin-associated protein 1 (UBAP1), which contains a region conserved in MVB12. UBAP1 binds the endosomal Bro1 protein His domain protein tyrosine phosphatase (HDPTP), but not Alix, a Bro1 protein involved in cytokinesis. UBAP1 is required for sorting EGFR to the MVB and for endosomal ubiquitin homeostasis, but not for cytokinesis. UBAP1 is part of a complex that contains a fraction of total cellular TSG101 and that also contains VPS37A but not VPS37C. Hence, the presence of UBAP1, in combination with VPS37A, defines an endosome-specific ESCRT-I complex.     

Identification of human MVB12 proteins as ESCRT-I subunits that function in HIV budding

Human ESCRT-I is a multiprotein complex that plays essential roles in HIV budding and endosomal protein sorting. All ESCRT-I complexes contain three common subunits (TSG101, VPS28, and VPS37), and a fourth subunit of yeast ESCRT-I was recently identified (Mvb12p). We now demonstrate that two related human proteins (MVB12A and MVB12B) constitute the fourth class of metazoan ESCRT-I subunits, despite lacking identifiable sequence homology to Mvb12p. Hydrodynamic studies indicate that soluble human ESCRT-I complexes contain one copy of each of the four subunit types. MVB12 subunits associate with the core region of the binary TSG101-VPS37 complex through conserved C-terminal sequence elements. Both MVB12 depletion and overexpression inhibit HIV-1 infectivity and induce unusual viral assembly defects, including aberrant virion morphologies and altered viral Gag protein processing. Taken together, these studies define the composition of human ESCRT-I complexes and indicate that the MVB12 subunits play a unique role in regulating ESCRT-mediated virus budding.     

The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.     

ALIX Is Recruited Temporarily into HIV-1 Budding Sites at the End of Gag Assembly

Polymerization of Gag on the inner leaflet of the plasma membrane drives the assembly of Human Immunodeficiency Virus 1 (HIV-1). Gag recruits components of the endosomal sorting complexes required for transport (ESCRT) to facilitate membrane fission and virion release. ESCRT assembly is initiated by recruitment of ALIX and TSG101/ESCRT-I, which bind directly to the viral Gag protein and then recruit the downstream ESCRT-III and VPS4 factors to complete the budding process. In contrast to previous models, we show that ALIX is recruited transiently at the end of Gag assembly, and that most ALIX molecules are recycled into the cytosol as the virus buds, although a subset remains within the virion. Our experiments imply that ALIX is recruited to the neck of the assembling virion and is mostly recycled after virion release.     

Structure and function of ALG-2, a penta-EF-hand calcium-dependent adaptor protein

ALG-2 (a gene product of PDCD6) is a 22-kD protein containing five serially repetitive EF-hand structures and belongs to the penta-EF-hand (PEF) family, including the subunits of typical calpains. ALG-2 is the most conserved protein among the PEF family members and its homologs are widely found in eukaryotes. X-ray crystal structures of various PEF proteins including ALG-2 have common features: presence of eight α-helices and dimer formation via paired EF5s that are positioned in anti-parallel orientation. ALG-2 forms a homodimer and a heterodimer with its closest paralog peflin. Like calmodulin, a well-known four-EF-hand protein, ALG-2 interacts with various proteins in a Ca²⁺-dependent fashion, but the binding motifs are completely different. With some exceptions, ALG-2-interacting proteins commonly contain Pro-rich regions, and ALG-2 recognizes at least two distinct Pro-containing motifs: PPYP(X)nYP (X, variable; n=4 in ALIX and PLSCR3) and PXPGF (represented by Sec31A). A shorter alternatively spliced isoform, lacking two residues and designated ALG-2^ΔGF122, does not bind ALIX but maintains binding capacity to Sec31A. X-ray crystal structural analyses have revealed that binding of calcium ions induces the configuration of the side chain of R125 so that it opens Pocket 1, which accepts PPYP, but Pocket 1 remains closed in the case of ALG-2^ΔGF122. ALG-2 dimer has two ligand-binding sites, each in a monomer molecule, and appears to function as a Ca²⁺-dependent adaptor protein to either stabilize a preformed complex or to bridge two proteins on scaffolds in systems of the endosomal sorting complex required for transport (ESCRT) and ER-to-Golgi transport.     

Penta-EF-hand protein ALG-2 functions as a Ca-dependent adaptor that bridges Alix and TSG101

Alix and TSG101, known to physically interact with each other, have Pro-rich regions that are bound by ALG-2 Ca²⁺-dependently. We investigated the role of ALG-2 in the Alix-TSG101 association by pulldown assays using Strep-tagged Alix and its various mutants. The ALG-2-binding site was required for the Ca²⁺-dependent pulldown of TSG101 using HEK293T cells, whereas the PSAP sequence, a binding motif for the UEV domain of TSG101, was dispensable. Alix-TSG101 association was not observed using ALG-2-knockdown cells but became detectable by addition of the purified recombinant ALG-2 protein in the assay mixtures. Exogenous expression of mGFP-fused ALG-2 also restored the pulldown capability of Strep-Alix, but an alternatively spliced shorter ALG-2 isoform and a dimerization-defective mutant were incompetent. Based on the X-ray crystal structure model showing the presence of one ligand-binding site in each molecule of an ALG-2 dimer, we propose that Ca²⁺-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein.     

Efficient cargo sorting by ESCRT-I and the subsequent release of ESCRT-I from multivesicular bodies requires the subunit Mvb12

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.     

Cellular ESCRT components are recruited to regulate the endocytic trafficking and RNA replication compartment assembly during classical swine fever virus infection

As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.     

Decoding the intrinsic mechanism that prohibits ALIX interaction with ESCRT and viral proteins

The adaptor protein ALIX [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] links retroviruses to ESCRT (endosomal sorting complex required for transport) machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 (charged multivesicular body protein 4) at the N-terminal Bro1 domain and retroviral Gag proteins at the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 (tumour susceptibility gene 101 protein)-docking site in the proline-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP (green fluorescent protein)-ALIX mutant with a constitutively open conformation, but not the wild-type protein, increases EIAV (equine infectious anaemia virus) budding from HEK (human embryonic kidney)-293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.     

Divergent pathways lead to ESCRT-III-catalyzed membrane fission

Endosomal sorting complexes required for transport (ESCRT) have been implicated in topologically similar but diverse cellular and pathological processes including multivesicular body (MVB) biogenesis, cytokinesis and enveloped virus budding. Although receptor sorting at the endosomal membrane producing MVBs employs the regulated assembly of ESCRT-0 followed by ESCRT-I, -II, -III and the vacuolar protein sorting (VPS)4 complex, other ESCRT-catalyzed processes require only a subset of complexes which commonly includes ESCRT-III and VPS4. Recent progress has shed light on the pathway of ESCRT assembly and highlights the separation of tasks of different ESCRT complexes and associated partners. The emerging picture suggests that among all ESCRT-catalyzed processes, divergent pathways lead to ESCRT-III assembly within the neck of a budding structure catalyzing membrane fission.     

Identification of human VPS37C, a component of endosomal sorting complex required for transport-I important for viral budding

Endosomal sorting complex required for transport-I (ESCRT-I) is one of three defined protein complexes in the class E vacuolar protein sorting (VPS) pathway required for the sorting of ubiquitinated transmembrane proteins into internal vesicles of multivesicular bodies. In yeast, ESCRT-I is composed of three proteins, VSP23, VPS28, and VPS37, whereas in mammals only Tsg101(VPS23) and VPS28 were originally identified as ESCRT-I components. Using yeast two-hybrid screens, we identified one of a family of human proteins (VPS37C) as a Tsg101-binding protein. VPS37C can form a ternary complex with Tsg101 and VPS28 by binding to a domain situated toward the carboxyl terminus of Tsg101 and binds to another class E VPS factor, namely Hrs. In addition, VPS37C is recruited to aberrant endosomes induced by overexpression of Tsg101, Hrs, or dominant negative form of the class E VPS ATPase, VPS4. Enveloped viruses that encode PTAP motifs to facilitate budding exploit ESCRT-I as an interface with the class E VPS pathway, and accordingly, VPS37C is recruited to the plasma membrane along with Tsg101 by human immunodeficiency virus, type 1 (HIV-1) Gag. Moreover, direct fusion of VPS37C to HIV-1 Gag obviates the requirement for a PTAP motif to induce virion release. Depletion of VPS37C from cells does not inhibit murine leukemia virus budding, which is not mediated by ESCRT-I, however, if murine leukemia virus budding is engineered to be ESCRT-I-dependent, then it is inhibited by VPS37C depletion, and this inhibition is accentuated if VPS37B is simultaneously depleted. Thus, this study identifies VPS37C as a functional component of mammalian ESCRT-I.     

Human ESCRT-II complex and its role in human immunodeficiency virus type 1 release

The budding of many enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1), requires some of the same cellular machinery as vesicle formation at the multivesicular body (MVB). In Saccharomyces cerevisiae, the ESCRT-II complex performs a central role in MVB protein sorting and vesicle formation, as it is recruited by the upstream ESCRT-I complex and nucleates assembly of the downstream ESCRT-III complex. Here, we report that the three subunits of human ESCRT-II, EAP20, EAP30, and EAP45, have a number of properties in common with their yeast orthologs. Specifically, EAP45 bound ubiquitin via its N-terminal GRAM-like ubiquitin-binding in EAP45 (GLUE) domain, both EAP45 and EAP30 bound the C-terminal domain of TSG101/ESCRT-I, and EAP20 bound the N-terminal half of CHMP6/ESCRT-III. Consistent with its expected role in MVB vesicle formation, (i) human ESCRT-II localized to endosomal membranes in a VPS4-dependent fashion and (ii) depletion of EAP20/ESCRT-II and CHMP6/ESCRT-III inhibited lysosomal targeting and downregulation of the epidermal growth factor receptor, albeit to a lesser extent than depletion of TSG101/ESCRT-I. Nevertheless, HIV-1 release and infectivity were not reduced by efficient small interfering RNA depletion of EAP20/ESCRT-II or CHMP6/ESCRT-III. These observations indicate that there are probably multiple pathways for protein sorting/MVB vesicle formation in human cells and that HIV-1 does not utilize an ESCRT-II-dependent pathway to leave the cell.     

NEDD4L overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking PTAP and YPXL late domains

The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6(Gag) bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101- and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.     

MVB-12, a fourth subunit of metazoan ESCRT-I, functions in receptor downregulation

After ligand binding and endocytosis, cell surface receptors can continue to signal from endosomal compartments until sequestered from the cytoplasm. An important mechanism for receptor downregulation in vivo is via the inward budding of receptors into intralumenal vesicles to form specialized endosomes called multivesicular bodies (MVBs) that subsequently fuse with lysosomes, degrading their cargo. This process requires four heterooligomeric protein complexes collectively termed the ESCRT machinery. In yeast, ESCRT-I is a heterotetrameric complex comprised of three conserved subunits and a fourth subunit for which identifiable metazoan homologs were lacking. Using C. elegans, we identify MVB-12, a fourth metazoan ESCRT-I subunit. Depletion of MVB-12 slows the kinetics of receptor downregulation in vivo, but to a lesser extent than inhibition of other ESCRT-I subunits. Consistent with these findings, targeting of MVB-12 to membranes requires the other ESCRT-I subunits, but MVB-12 is not required to target the remaining ESCRT-I components. Both endogenous and recombinant ESCRT-I are stable complexes with a 1:1:1:1 subunit stoichiometry. MVB-12 has two human homologs that co-localize and co-immunoprecipitate with the ESCRT-I component TSG101. Thus, MVB-12 is a conserved core component of metazoan ESCRT-I that regulates its activity during MVB biogenesis.     

植物ESCRT复合体的功能研究进展

真核细胞中,功能高度保守的内体蛋白分选转运装置ESCRT在胞吞途径和蛋白分泌途径中均扮演重要角色。植物细胞中,该装置包含ESCRT-Ⅰ、ESCRT-Ⅱ、ESCRT-Ⅲ和VPS4/SKD1复合体4个亚基,但缺乏ESCRT-0亚基。ESCRT的每个亚基均由多个蛋白构成。目前,针对ESCRT的研究已经证实,其在泛素化的膜蛋白进入多囊泡体/液泡前体(MVB/PVC)内腔过程中发挥重要调控作用;同时在自噬途径以及应对环境胁迫等方面也具有重要的调节功能。该文首先介绍了植物中ESCRT复合体的组成及生物学功能,然后总结了植物中特有ESCRT复合体组分蛋白的最新研究进展,最后探讨了有关ESCRT复合体研究中尚未解决的重要科学问题。     

Biochemical Analyses of Human IST1 and Its Function in Cytokinesis

The newly described yeast endosomal sorting complexes required for transport (ESCRT) protein increased sodium tolerance-1 (Ist1p) binds the late-acting ESCRT proteins Did2p/charged MVB protein (CHMP) 1 and Vps4p and exhibits synthetic vacuolar protein sorting defects when combined with mutations in the Vta1p/LIP5–Vps60p/CHMP5 complex. Here, we report that human IST1 also functions in the ESCRT pathway and is required for efficient abscission during HeLa cell cytokinesis. IST1 binding interactions with VPS4, CHMP1, LIP5, and ESCRT-I were characterized, and the IST1–VPS4 interaction was investigated in detail. Mutational and NMR spectroscopic studies revealed that the IST1 terminus contains two distinct MIT interacting motifs (MIM1 and MIM2) that wrap around and bind in different groves of the MIT helical bundle. IST1, CHMP1, and VPS4 were recruited to the midbodies of dividing cells, and depleting either IST1 or CHMP1 proteins blocked VPS4 recruitment and abscission. In contrast, IST1 depletion did not inhibit human immunodeficiency virus-1 budding. Thus, IST1 and CHMP1 act together to recruit and modulate specific VPS4 activities required during the final stages of cell division.     

Evolutionary and physical linkage between calpains and penta-EF-hand Ca2+-binding proteins

The name calpain was historically given to a protease that is activated by Ca(2+) and whose primary structure contains a Ca(2+)-binding penta-EF-hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2-domain-like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain-like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain-7, indicating that the calpain-7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N-terminal region of calpain-7 has a tandem repeat of microtubule-interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca(2+)-binding proteins including ALG-2 that associates with ALIX (an ESCRT-III accessory protein) and TSG101 (an ESCRT-I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein-protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system.     

Endosome-associated complex,ESCRT-II,recruits transport machinery for protein sorting at the multivesicular body

Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study characterizes ESCRT-II, a soluble approximately 155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. We propose that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome.     

Burning cellular bridges: Two pathways to the big breakup

During cytokinetic abscission, the endosomal sorting complex required for transport (ESCRT) proteins are recruited to the midbody and direct the severing of the intercellular bridge. In this issue, Christ et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507009) demonstrate that two separate but redundant pathways exist to recruit ESCRT-III proteins to the midbody.Over the past 140 years, eukaryotic cell division has been extensively studied and is now understood to be an elaborate, tightly regulated set of events that culminates in the formation of two distinct daughter cells. The M phase of animal cells is characterized by a profound structural reorganization, regulated by a cohort of mitotic kinases and performed by mitosis-specific cytoskeletal structures, including the spindle apparatus and the cytokinetic midbody (Scholey et al., 2003). The completion of cytokinesis, called abscission, involves the severing of the intercellular bridge on both sides of the midbody. In 2007, two landmark studies demonstrated that several ESCRT proteins localize to the midbody and are required for the completion of cytokinesis (Carlton and Martin-Serrano, 2007; Morita et al., 2007). Aside from abscission, the ESCRTs participate in the formation of multivesicular endosomes (MVEs), function in plasma membrane repair, and participate in numerous other cellular processes (Katzmann et al., 2002; Morita and Sundquist, 2004; Hurley, 2015).The canonical model for ESCRT function at MVEs involves the hierarchical recruitment of ESCRT proteins in four unique complexes: ESCRT-0 through ESCRT-III. The ESCRTs cluster cargos and deform membrane, and current models suggest that ESCRT-III subunits polymerize to form filaments that spiral down into the neck of a nascent intralumenal vesicle (Schuh and Audhya, 2014). With the assistance of the VPS4 AAA ATPase, ESCRT filaments are remodeled to facilitate vesicle fission (Shen et al., 2014). Though MVE maturation utilizes all four ESCRT complexes, cytokinetic abscission has been previously thought to require only ESCRT-I, ESCRT-III, and the ESCRT-associated ALG2-interacting factor ALIX (Morita et al., 2010). ALIX interacts with both the ESCRT-I protein TSG101 and all three ESCRT-III CHMP4 isoforms and has been postulated to act as an ESCRT-II bypass for linking ESCRT-I and ESCRT-III in abscission (Schuh and Audhya, 2014). However, the precise mechanism underlying the recruitment of the ESCRT-III complex to the midbody during cytokinesis has remained ambiguous.In this issue, Christ et al. address how the ESCRT-III component CHMP4B (Vps32 in other metazoan systems) is recruited to the midbody and demonstrate the necessity of the ESCRT-II complex in this process. They observed that recruitment of CHMP4B to the midbody was abrogated when they codepleted ALIX and the ESCRT-I component TSG101 in cultured HeLa cells, but that CHMP4B did accumulate when only one of these components was depleted. These data indicate that CHMP4B can be recruited to the midbody via TSG101 or ALIX, but that the two proteins are unlikely to perform this function as a complex, suggesting that CHMP4B recruitment to the midbody involves two independent pathways.After immunofluorescence staining of fixed cells, Christ et al. (2016) found that the endogenous ESCRT-III protein CHMP6 and the ESCRT-II protein EAP20 (VPS20 and VPS25 in other systems, respectively) localize to the midbody, consistent with a previous overexpression study (Thoresen et al., 2014). They additionally performed several depletion experiments to establish that ESCRT-II recruits CHMP6 without affecting TSG101 localization, demonstrating that CHMP6 acts downstream of ESCRT-I and ESCRT-II. This shows that ESCRT-I recruits ESCRT-III to the cytokinetic midbody the same way it does at the MVE.Christ et al. (2016) also show that CHMP4B can still be recruited normally when the ESCRT-II component EAP30 (VPS22 in other systems) is depleted, but not when EAP30 is codepleted with ALIX, strongly suggesting that ALIX-dependent accumulation of CHMP4B does not involve CHMP6 and, more generally, that there are two pathways that can each recruit CHMP4B to the midbody: an ESCRT-I–ESCRT-II–CHMP6 pathway and an ALIX-dependent pathway. It will be important for future work to consider the partial redundancy between these two pathways when assaying the dispensability of early acting ESCRT complexes in cellular processes.In addition, Christ et al. (2016) observed that ALIX depletion led to furrow regression and binucleation in dividing cells with chromatin spanning the intercellular bridge, the same phenotype observed in cells expressing a CHMP4C construct lacking the ALIX interaction domain. Further, they showed that CHMP4C localization to the midbody is abrogated after ALIX depletion but is unaffected by TSG101 knockdowns, strongly implicating ALIX in CHMP4C recruitment independently of ESCRT-I.Our overall understanding of the regulation of abscission still remains elementary (Fig. 1). In addition to the roles of the ESCRT machinery, the chromosomal passenger complex (CPC) regulates the timing of cytokinesis and abscission via interactions with the Polo-like kinase PLK1, the mitotic kinesin-like protein MKLP1, and CEP55, a key component of the midbody that associates directly with both ESCRT-I and ALIX (Schuh and Audhya, 2014). One current model is that the CPC promotes the formation of a ternary complex consisting of CHMP4C, ANCHR, and VPS4 and prevents premature action by VPS4 in response to chromatin trapped in the midbody (Thoresen et al., 2014). It has also been suggested that CHMP4C phosphorylation by the enzymatic core of the CPC, the Aurora B kinase, directs CHMP4C localization to the midbody and its retention of VPS4 (Carlton et al., 2012). With the new findings by Christ et al. (2016), the relationship between Aurora B–mediated phosphorylation of CHMP4C and its ability to bind ALIX must now be further explored. Additionally, because ALIX appears to be the primary factor that recruits CHMP4C to the midbody, it may represent a novel therapeutic target for activation or bypass of the NoCut abscission checkpoint.Open in a separate windowFigure 1.Model for the recruitment of CHMP4B and CHMP4C to the midbody and their roles in regulating the timing of abscission. PLK-1 phosphorylation of CEP55 inhibits its binding to MKLP1. At the end of anaphase, PLK1 is degraded and MKLP1 recruits CEP55 to the midbody. CEP55 recruits TSG101 and ALIX to the midbody, and Christ et al. (2016) demonstrate that there are two pathways that lead to the subsequent recruitment of CHMP4B: one through ESCRT-I–ESCRT-II–CHMP6 and the second directly through ALIX. ALIX also recruits CHMP4C, which, upon phosphorylation by the CPC, is hypothesized to form a ternary complex with ANCHR and VPS4. Formation of this complex prevents VPS4 from facilitating the completion of abscission until all chromatin is cleared from the intercellular bridge.In contrast to the necessity of ALIX during cytokinetic abscission, its role during MVE formation and ubiquitin-dependent cargo degradation remains debatable. Depletion studies suggest that ALIX is dispensable for the lysosomal sorting of several cargoes (Bowers et al., 2006). However, ALIX is capable of targeting to late endosomal membranes through its interaction with lysobisphosphatidic acid, and some data suggest that ALIX can promote ESCRT-III filament assembly at MVEs (Matsuo et al., 2004; Pires et al., 2009; Bissig and Gruenberg, 2014). In the future, it will be essential to elucidate the mechanisms by which ALIX and CHMP6 direct the nucleation of CHMP4B/ESCRT-III spiral filaments and to determine whether the membrane landscapes of the MVE and the cytokinetic bridge differ in a manner that promotes one pathway over the other. As cryoelectron microscopy–based approaches in cells and reconstituted systems advance, the answer to these questions may become more accessible.