Structural and Biochemical Studies of ALIX/AIP1 and Its Role in Retrovirus Budding

Fisher, Robert D.; Chung, Hyo Young; Zhai, Qianting; Robinson, Howard; Sundquist, Wesley I.; Hill, Christopher P.

doi:10.1016/j.cell.2007.01.035

Cited by 292 publications

(617 citation statements)

References 43 publications

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“…Alix interacts with the ESCRT-I subunit Tsg101 during retrovirus budding at the plasma membrane (Strack et al, 2003;von Schwedler et al, 2003;Munshi et al, 2006;Fisher et al, 2007;Gottlinger, 2007) and cytokinesis (Carlton and MartinSerrano, 2007;Morita et al, 2007b). Similarly, we find that both proteins not only interact with each other, but also act together in the lumenal budding process.…”

Section: Discussionmentioning

confidence: 54%

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Falguières

Luyet

Bissig

et al. 2008

MBoC

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Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively. INTRODUCTIONIn eukaryotic cells, molecules of the plasma membrane, ligands and solutes are internalized into early endosomes, from where they can be recycled back to the plasma membrane, transported to the trans-Golgi network, or targeted to late endosomes and lysosomes (Gruenberg, 2001;Maxfield and McGraw, 2004;Mayor and Pagano, 2007). The latter pathway is followed in particular by ubiquitinated signaling receptors that need to be down-regulated. These receptors are incorporated into invaginations of the early endosomal membrane that form within their lumen (Hurley and Emr, 2006), thus giving rise to nascent multivesicular bodies (MVBs) or endosomal carrier vesicles (ECVs; Gruenberg and Stenmark, 2004;Piper and Katzmann, 2007), which function as intermediates between early and late endosomes. Eventually, intralumenal vesicles are delivered to lysosomes, where they are degraded together with their receptor cargo.Major progress has been made in our understanding of the molecular mechanisms that control the sorting of ubiquitinated receptors, in particular the epidermal growth factor (EGF) receptor (Hurley and Emr, 2006;Slagsvold et al., 2006;Piper and Katzmann, 2007;Williams and Urbe, 2007). These receptors interact via the ubiquitin moiety with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), which in turn binds clathrin and phosphatidyl-inositol-3-phosphate (PtdIns3P), thereby concentrating the receptor in early endosomal membrane domains (Raiborg et al., 2001;Urbe et al., 2003;Raiborg et al., ...

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Section: Discussionmentioning

confidence: 54%

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Falguières

Luyet

Bissig

et al. 2008

MBoC

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“…In both cases, ALIX must also bind the CHMP4 proteins, because ALIX point mutations that block CHMP4 binding inhibit HIV-1 budding (10,11) and abscission (18). Thus, ALIX can serve as an adaptor that recruits CHMP4/ ESCRT-III complexes to function at distinct biological membranes.…”

mentioning

confidence: 99%

“…The proline-rich region contains binding epitopes for a number of other cellular factors, including TSG101 (13,15,16), endophilins (21), and ALG-2 (38,39). The Bro1 domain contains binding sites for both HIV-1 NC (40) and the CHMP4 proteins (7)(8)(9)(10)(11). Mutations that inhibit CHMP4 binding cluster within an exposed hydrophobic patch on the concave surface of the Bro1 domain, which is thought to be the CHMP4 binding site (9)(10)(11).…”

mentioning

confidence: 99%

“…The different ESCRT-III proteins apparently adopt similar folds (6) and can copolymerize together on membranes, yet have evolved to interact differently with other ESCRT pathway components. For example, only the three human CHMP4 proteins (CHMP4A-C) can bind ALIX (yeast Bro1p), another protein in the ESCRT pathway (7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

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confidence: 99%

“…Crystal structures of different ALIX constructs (9,10,35) have revealed that the banana-shaped Bro1 domain is organized about a core of tetratricopeptide helical hairpins and that the V domain is composed of two extended helical arms that fold in the shape of the letter V. YP(X n )L sequence motifs within retroviral Gag proteins bind on the inner face of the second arm of the V domain (10,17,(35)(36)(37). The proline-rich region contains binding epitopes for a number of other cellular factors, including TSG101 (13,15,16), endophilins (21), and ALG-2 (38,39).…”

mentioning

confidence: 99%

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ALIX-CHMP4 interactions in the human ESCRT pathway

McCullough

Fisher

Whitby

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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214

252

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The ESCRT pathway facilitates membrane fission events during enveloped virus budding, multivesicular body formation, and cytokinesis. To promote HIV budding and cytokinesis, the ALIX protein must bind and recruit CHMP4 subunits of the ESCRT-III complex, which in turn participate in essential membrane remodeling functions. Here, we report that the Bro1 domain of ALIX binds specifically to C-terminal residues of the human CHMP4 proteins (CHMP4A-C). Crystal structures of the complexes reveal that the CHMP4 C-terminal peptides form amphipathic helices that bind across the conserved concave surface of ALIXBro1. ALIX-dependent HIV-1 budding is blocked by mutations in exposed ALIXBro1 residues that help contribute to the binding sites for three essential hydrophobic residues that are displayed on one side of the CHMP4 recognition helix (M/L/IxxLxxW). The homologous CHMP1-3 classes of ESCRT-III proteins also have C-terminal amphipathic helices, but, in those cases, the three hydrophobic residues are arrayed with L/I/MxxxLxxL spacing. Thus, the distinct patterns of hydrophobic residues provide a ''code'' that allows the different ESCRT-III subunits to bind different ESCRT pathway partners, with CHMP1-3 proteins binding MIT domain-containing proteins, such as VPS4 and Vta1/LIP5, and CHMP4 proteins binding Bro1 domaincontaining proteins, such as ALIX.cytokinesis ͉ ESCRT-III ͉ HIV ͉ mutivesicular body

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Bro1 family proteins harmonize cargo sorting with vesicle formation

2022

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The Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT-III polymers regulated by the AAA-ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT-associated Ub-binding protein, to coordinate ESCRT-III and Vps4-dependent ILV biogenesis with upstream events such as cargo recognition. BRO1 AS A CENTRAL PLAYER IN ESCRT FUNCTION Bro1 family proteins (Bro1, ALIX, and HD-PTP) possess a conserved domain architecture and interact with multiple factors that contribute to ESCRT functions: the N-terminal BOD binds to the ESCRT-III subunit Snf7/CHMP4, the V domain binds to proteins containing YPxL motifs (e.g., cargo adapter syntenin) and Ub as well as Vps4, and the C-terminal Proline-Rich Region (PRR) binds to the ESCRT-I subunit

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Structural and Biochemical Studies of ALIX/AIP1 and Its Role in Retrovirus Budding

Cited by 292 publications

References 43 publications

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

ALIX-CHMP4 interactions in the human ESCRT pathway

Bro1 family proteins harmonize cargo sorting with vesicle formation

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