A concentric circle model of multivesicular body cargo sorting

Nickerson, Daniel P.; Russell, Mark; Odorizzi, Greg

doi:10.1038/sj.embor.7401004

Cited by 85 publications

(107 citation statements)

References 35 publications

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“…However, mock-treated cytosol could rescue intralumenal vesicle formation within Tsg101-depleted late endosomes, and conversely mock-treated endosomes supported intralumenal budding in the presence of depleted cytosol ( Figure 8C). These observations fully support the notion that ESCRTs cycle between membrane and cytosol (Hurley and Emr, 2006;Nickerson et al, 2007;Williams and Urbe, 2007). But, they also indicate that Tsg101 siRNAs are unlikely to cause indirect or toxic effects, because both cytosol and endosome retain the competence to support in vitro budding after depletion, provided that they are incu- The ratio of HPTS fluorescence emission after excitation at 445 and 397 nm is represented as a function of pH.…”

Section: Role Of the Escrt-i Subunit Tsg101 In Intralumenal Vesicle Fsupporting

confidence: 63%

“…Although there is no doubt that Hrs and ESCRTs regulate sorting of ubiquitinated receptors into lumenal vesicles, the mechanism that concentrates receptors at the site of vesicle formation is not clear, involving perhaps a conveyor belt-like (Hurley and Emr, 2006) or concentric (Nickerson et al, 2007) subunit organization. Neither is it clear what controls the membrane deformation and budding processes (Williams and Urbe, 2007;Hanson et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

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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: Role Of the Escrt-i Subunit Tsg101 In Intralumenal Vesicle Fsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

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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“…The cause of the endosomal accumulation of overexpressed pHluorin-Vps27p remains unclear at present. It is known that ESCRTs shuttle between the endosomal membrane and the cytoplasm (3,48). Although ESCRT-III is dissociated from endosomal membrane by Vps4p (14), the dissociation of the ESCRT-0/I/II complexes might require additional unknown factors (3,48).…”

Section: Discussionmentioning

confidence: 99%

“…It is known that ESCRTs shuttle between the endosomal membrane and the cytoplasm (3,48). Although ESCRT-III is dissociated from endosomal membrane by Vps4p (14), the dissociation of the ESCRT-0/I/II complexes might require additional unknown factors (3,48). One possible explanation for the endosomal accumulation of Vps27p in nhx1⌬ cells is that an additional factor is involved in dissociation of Vps27p (ESCRT-0) from endosomal membranes.…”

Section: Discussionmentioning

confidence: 99%

The Endosomal Na+/H+ Exchanger Contributes to Multivesicular Body Formation by Regulating the Recruitment of ESCRT-0 Vps27p to the Endosomal Membrane

Mitsui

Koshimura

Yoshikawa

et al. 2011

Journal of Biological Chemistry

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Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosomelocalized Na ؉ /H ؉ exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.Multivesicular bodies (MVBs) 3 are a subset of late endosomes that contain internal vesicles, permitting precursor and plasma membrane proteins to reach the lysosome or vacuole lumen for degradation or maturation, respectively. In eukaryotic cells, the MVB pathway controls various important processes such as receptor down-regulation, antigen presentation, cytokinesis, retroviral budding, and autophagy (1-6). In the budding yeast Saccharomyces cerevisiae, previous studies identified a number of genes involved in vacuolar protein sorting (VPS) (7,8). These Vps proteins function at distinct steps of protein transport between the Golgi complex and the vacuole (7,8). A subset of Vps proteins, class E proteins, is involved in MVB formation. Most class E proteins are components of five distinct complexes; Vps27p-Hse1p (also called endosomal sorting complexes required for transport-0 (ESCRT-0), -I, -II, and -III and Vps4p. These complexes are required for the formation of internal vesicles and sorting of cargo proteins to the vesicles from the endosomal membrane (3, 9). Vps27p (ESCRT-0) recruits ESCRT-I, which in turn recruits and/or activates ESCRT-II and ESCRT-III at the endosome (3, 10). Recent in vitro studies have provided new insights into the precise role of each ESCRT complex in MVB formation (11-13). ESCRT-0 initiates the MVB sorting process by recruiting ESCRT-I to the endosomal membrane and concentrating ubiquitylated MVB cargos to the vesicle budding site (12). ESCRT-I and ESCRT-II are directly involved in membrane budding by stabilizing the bud necks, whereas ESCRT-III is responsible for the cleavage of bud necks (11-13). Finally, the AAA-type ATPase Vps4p dissociates the ESCRT-III complex from the endosome (14). The sequential activity of these complexes on the cytoplasmic surface of endosomes directs the budding and fission of vesicles into the lumen of the endosome and the sequestration of cargo proteins in the vesicle. Defects in ESCRT proteins prevent internal vesicle formation and result in exagg...

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“…19, November 2008 4697 or vacuole for degradation via the MVB pathway (Hicke and Dunn, 2003;Piper and Luzio, 2007), and a previous report concluded that Ftr1, but not Fet3, is ubiquitylated in response to iron shock (Felice et al, 2005). In yeast and mammalian cells, the ESCRTs are required to incorporate ubiquitylated cargo proteins into the vesicles budding into the lumen of MVBs (Babst, 2005;Nickerson et al, 2007;Piper and Katzmann, 2007;Saksena et al, 2007). Several ESCRT proteins bind ubiquitin, most notably the Vps27-Hse1 MVB pathway cargo receptor (sometimes referred to as "ESCRT-0") that mediates entry of proteins destined for degradation into this pathway (Bilodeau et al, 2002;Shih et al, 2002).…”

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confidence: 99%

Opposing Activities of the Snx3-Retromer Complex and ESCRT Proteins Mediate Regulated Cargo Sorting at a Common Endosome

Strochlic

Schmiedekamp

Lee

et al. 2008

MBoC

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Endocytosed proteins are either delivered to the lysosome to be degraded or are exported from the endosomal system and delivered to other organelles. Sorting of the Saccharomyces cerevisiae reductive iron transporter, composed of the Fet3 and Ftr1 proteins, in the endosomal system is regulated by available iron; in iron-starved cells, Fet3-Ftr1 is sorted by Snx3/Grd19 and retromer into a recycling pathway that delivers it back to the plasma membrane, but when starved cells are exposed to iron, Fet3-Ftr1 is targeted to the lysosome-like vacuole and is degraded. We report that iron-induced endocytosis of Fet3-Ftr1 is independent of Fet3-Ftr1 ubiquitylation, and after endocytosis, degradation of Fet3-Ftr1 is mediated by the multivesicular body (MVB) sorting pathway. In mutant cells lacking any component of the ESCRT protein-dependent MVB sorting machinery, the Rsp5 ubiquitin ligase, or in wild-type cells expressing Fet3-Ftr1 lacking cytosolic lysyl ubiquitin acceptor sites, Fet3-Ftr1 is constitutively sorted into the recycling pathway independent of iron status. In the presence and absence of iron, Fet3-Ftr1 transits an endosomal compartment where a subunit of the MVB sorting receptor (Vps27), Snx3/Grd19, and retromer proteins colocalize. We propose that this endosome is where Rsp5 ubiquitylates Fet3-Ftr1 and where the recycling and degradative pathways diverge. INTRODUCTIONThe endocytic pathway is initiated with the internalization of protein and lipid cargo from the plasma membrane. After endocytosis, internalized molecules transit through the endosomal system, a network of interconnected organelles that process and sort cargo molecules back to the plasma membrane, to other organelles, or to lysosomes for degradation. In mammalian cells, early endosomes are pleiomorphic organelles composed of vacuolar domains that contain lumenal vesicles and tubular domains that lead into and emanate from the vacuolar portion. This distinct architecture has functional significance. The tubular elements of the organelle are regions where the majority of internalized plasma membrane proteins and lipids are exported and recycled from the endosomal system via bulk flow and cargo-specific processes (Mayor et al., 1993;Maxfield and McGraw, 2004;Bonifacino and Rojas, 2006). Cargo proteins that are not exported are delivered to lysosomes and many of these are degraded. This occurs via an active, signal-mediated event in which these proteins are targeted to the vacuolar region of the endosome where they are incorporated into vesicles that bud from the limiting membrane into the lumen of the organelle (Piper and Katzmann, 2007). During endosome maturation lumenal vesicles accrue until fusion of the late endosome with the lysosome results in degradation of these vesicles and their contents. A central sorting function of the endosomal system is thus to segregate cargo to be exported from the endosome from cargo to be degraded via lysosomal proteolysis.The core components of the recycling (retrograde) and degradative sorting pathways have be...

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A concentric circle model of multivesicular body cargo sorting

Cited by 85 publications

References 35 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

The Endosomal Na+/H+ Exchanger Contributes to Multivesicular Body Formation by Regulating the Recruitment of ESCRT-0 Vps27p to the Endosomal Membrane

Opposing Activities of the Snx3-Retromer Complex and ESCRT Proteins Mediate Regulated Cargo Sorting at a Common Endosome

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