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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

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SCRT-dependent cargo sorting at multivesicular endosomes

.B. Frankel, Anjon Audhya ∗

epartment of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, 440 Henry Mall, Madison, WI, 53706, USA

r t i c l e i n f o

rticle history:eceived 24 April 2017eceived in revised form 2 August 2017ccepted 5 August 2017vailable online xxx

a b s t r a c t

The endosomal sorting complex required for transport (ESCRT) machinery is composed of five multi-subunit protein complexes, which act cooperatively at specialized endosomes to facilitate the movementof specific cargoes from the limiting membrane into vesicles that bud into the endosome lumen. Over thepast decade, numerous proteins, lipids, and RNAs have been shown to be incorporated into intralumenalvesicles (ILVs), but the mechanisms by which these unique cargoes are captured are only now becoming

eywords:SCRT machineryultivesicular endosome

argo sortingntralumenal vesicle

better understood. Here, we discuss the potential roles that the ESCRT machinery plays during cargosorting at multivesicular endosomes (MVEs).

© 2017 Elsevier Ltd. All rights reserved.

biquitin

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Ubiquitin-mediated cargo recognition and sorting by the ESCRT machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Ubiquitin-independent cargo sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. Cargo transfer into ILVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005. Concluding perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Introduction

Eukaryotic cells have evolved elaborate mechanisms to sensenvironmental cues, rapidly initiate responses mediated by cellurface transmembrane receptors, and ultimately downregulateignaling via receptor sequestration within endosomal compart-

into cells and deposited into multivesicular endosomes (MVEs; alsoreferred to as multivesicular bodies/MVBs). Identification of MVEsat the ultrastructural level is made relatively simple by their char-acteristic morphology: membrane-bound organelles that harborsmall intralumenal vesicles (ILVs). To generate these unique mem-

Please cite this article in press as: E.B. Frankel, A. Audhya, ESCRT-depeBiol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.08.020

ents. This pathway was originally described several decades agon elegant electron microscopy-based studies using labeled epi-ermal growth factor (EGF) [1–4], which is internalized rapidly

Abbreviations: DUB, deubiquitinating enzyme; DUIM, double ubiquitin-nteracting motif; EGF, epidermal growth factor; ESCRT, endosomal sorting complexequired for transport; GLUE, GRAM-like ubiquitin-binding in Eap45; ILV, intralu-

enal vesicle; LBPA, lysobisphosphatidic acid; MVB, multivesicular body; MVE,ultivesicular endosome; PI3P, phosphatidylinositol 3-phosphate; SOUBA, solenoid

f overlapping ubiquitin associated motifs; TIRFM, total internal reflection fluores-ence microscopy; UBD, ubiquitin binding domain; UEV, ubiquitin E2 variant; UIM,biquitin-interacting motif; VHS, ‘Vps27, HRS, STAM’; Vps, vacuolar protein sorting.∗ Corresponding author.

E-mail address: audhya@wisc.edu (A. Audhya).

ttp://dx.doi.org/10.1016/j.semcdb.2017.08.020084-9521/© 2017 Elsevier Ltd. All rights reserved.

brane compartments, eukaryotes use a set of protein complexescollectively known as the Endosomal Sorting Complex Requiredfor Transport (ESCRT) machinery, which were defined originally ina series of genetic screens using yeast [5–10]. In the absence ofESCRT function, transmembrane cargoes are unable to efficientlyreach the lumen of the vacuole/lysosome, resulting in the forma-tion of an aberrant prevacuolar organelle in yeast termed the ‘classE compartment’. Since their initial discovery, the field has begunto reach consensus regarding the roles of the ESCRT complexes atthe endosome limiting membrane, which include cargo selection,membrane deformation to generate nascent, inward-budded vesi-

ndent cargo sorting at multivesicular endosomes, Semin Cell Dev

cles, cargo sequestration within these newly formed vesicles, andnarrowing of the vesicle bud neck to ultimately facilitate release ofILVs into the lumen of the endosome.

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ARTICLESCDB-2329; No. of Pages 7

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During the characterization of these processes, several modelargoes were identified as substrates of the ESCRT machinery. Per-aps most famously, post-translational modification of the EGFeceptor by ubiquitin was shown to play an integral role in itsndosomal sorting and degradation [11–14]. Together with othereminal discoveries linking ubiquitin to the ESCRT machinery atVEs, the trajectory for the field was set, and a unifying mechanism

hat supports the entry of numerous, unrelated cargoes into thendosome lumen was defined [2,15–18]. More recently, additionalargoes that do not rely on ubiquitin modification have also beenhown to enter ILVs, including proteins (both soluble and trans-embrane), specialized lipids, and nucleic acids, suggesting thatultiple modes of cargo recognition exist [19–24]. Furthermore,

he fate of MVEs has been expanded beyond lysosomal fusion andLV degradation. In particular, some MVEs are specialized to facili-ate the dissemination of cargoes to the extracellular environment,using with the plasma membrane to release their ILVs (exosomes)25].

The spatial distribution of MVEs can vary widely between cellypes, and probes specific to the organelle have been challengingo identify, as they regularly undergo fusion with lysosomes andther membrane compartments. Similarly, the size and contentf MVEs are not uniform and depend on cell cycle stage, nutri-nt availability, and other environmental conditions. Componentsf the ESCRT machinery may represent the best markers available,iven their intimate role in MVE biogenesis. In general, mammalianVEs tend to accumulate near the center of cells at steady state

26], in a manner dependent on dynein-mediated transport [27],hich positions them well to receive biosynthetic cargoes from

he perinuclear trans-Golgi network. However, a large number ofargoes also originate at the cell surface, entering the endosomalystem via clathrin-dependent and clathrin-independent endocy-osis, phagocytosis, and micropinocytosis [28–31]. Based on liveell total internal reflection microscopy (TIRFM), components ofhe ESCRT machinery have been observed at clathrin-coated struc-ures at or near the plasma membrane, suggesting that cargoes areorted rapidly upon cellular internalization [32]. Thus, MVE forma-ion likely initiates at multiple subcellular locations to sequesterargoes destined for degradation or exocytosis. Perhaps not sur-risingly, growth factor stimulation augments the rate of MVE

ormation, enabling a rapid response to limit cell signaling that isnitiated upon receptor binding [33–35].

Based on genetic and biochemical evidence, more than twoozen ESCRT proteins have been implicated in the deposition ofargoes into ILVs [36]. In many cases, structural and functional evi-ence points to mechanisms by which these factors participate inargo sorting. For example, early acting ESCRT complexes (ESCRT-, ESCRT-I, and ESCRT-II) harbor ubiquitin-binding domains, whichlay a key role in cargo selection [37]. However, it remains unclearow (and to what extent) substrates of the pathway, but not coreomponents of the ESCRT machinery, become selectively enrichedithin ILVs. Here, we will focus on this issue and discuss potentialodels to explain how ESCRT-mediated cargo sorting is efficiently

chieved.

. Ubiquitin-mediated cargo recognition and sorting by theSCRT machinery

Post-translational modification of integral membrane proteinsy ubiquitin serves as a key sorting signal for endocytosis andargeting to MVEs. In particular, members of the Cbl family of

Please cite this article in press as: E.B. Frankel, A. Audhya, ESCRT-depeBiol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.08.020

3 ubiquitin ligases have been tied to the internalization andSCRT-dependent degradation of dozens of activated cell sur-ace receptors [11,38–40]. Although the addition of a singlebiquitin moiety has been shown to be sufficient to enable ESCRT-

PRESSvelopmental Biology xxx (2017) xxx–xxx

mediated protein sorting at MVEs [41], many cargoes includingEGF receptor undergo polyubiquitin modification (predominantlyvia K63-linkages), which may enhance their rate of sequestra-tion within ILVs [18,42–44]. Several components of the ESCRTmachinery contain ubiquitin binding domains (UBDs), which actas receptors for ubiquitin-modified cargoes (Fig. 1). In general, theUBDs found on ESCRT subunits exhibit only modest affinity (Kdof ∼70–510 �M) for cargoes [45], but the avidity of these inter-actions may be sufficient to play a significant role in cargo captureand retention, especially when considering that integral membraneproteins are constrained to two dimensional movement within abilayer [46]. The two subunits of ESCRT-0 (Hrs and STAM) con-tain two UBDs each. The double ubiquitin interacting motif (DUIM)of Hrs can bind simultaneously to two ubiquitin molecules [47],each with an affinity of ∼120 �M [48], and both the UIM andVps27/Hrs/STAM (VHS) domains of STAM associate with ubiqui-tin [49], albeit with weaker affinities [48,50]. Importantly, uponassociation with membranes, ESCRT-0 exhibits the ability to gen-erate larger complexes, further increasing the number of UBDspresented [48,51]. Both fluorescence and immunogold electronmicroscopy-based studies in yeast and animal cells support theidea that ESCRT-0 self-associates at endosomal membranes, creat-ing subdomains that facilitate the retention of ubiquitin-modifiedcargoes through multiple low-affinity associations [52–54]. Addi-tionally, ESCRT-0 subdomains may be further stabilized by flatclathrin lattices that are recruited by the carboxyl-terminal clathrinbinding motif of Hrs [18,53,55]. However, a universal requirementfor clathrin assembly at MVEs during cargo sorting remains to bedemonstrated in all cell types. In the absence of clathrin heavychain, biosynthetic transport of lysosomal hydrolases into MVEsin yeast proceeds normally [56–58]. Additionally, subdomains ofESCRT-0 are capable of assembling stably in the absence of flatclathrin lattices in vitro [59]. One possibility is that clathrin assem-bly at MVEs increases the efficiency of cargo retention, whichmay be required in cases where cargo influx becomes elevated inresponse to developmental and extracellular cues.

In contrast to ESCRT-0, no evidence exists to suggest that theheterotetrameric ESCRT-I complex multimerizes on membranes[51]. Nevertheless, based on structural and functional studies, atleast two of its components can associate directly with ubiquitin.The core subunit Tsg101 possesses an amino-terminal, catalyti-cally inactive ubiquitin E2 variant (UEV) domain with weak affinityfor ubiquitin (Kd of ∼510 �M) [60]. Additionally, a subpopulationof ESCRT-I complexes contain the UBAP1 subunit, which har-bors a solenoid of overlapping ubiquitin associated motifs (SOUBAdomain) that binds mono- and di-ubiquitin with an affinity of∼70 �M [61]. Inhibition of UBAP1 leads to cargo-specific traffick-ing defects, indicating that it (and by extension, ESCRT-I) playsa role in ubiquitin-dependent sorting [62]. The ESCRT-II complexcontains a single subunit (Vps36) capable of binding to ubiquitinvia a GRAM-like ubiquitin-binding in Eap45 (GLUE) domain [63].Similar to other UBDs found in ESCRT subunits, the affinity of theGLUE domain for ubiquitin is modest (Kd of ∼330 �M) [63]. How-ever, ESCRT-II has been shown to multimerize on membranes in acholesterol dependent manner, which would elevate its avidity forubiquitin-modified cargoes [64]. Additionally, recent evidence inyeast highlighted a mutant form of ESCRT-II that is capable of par-tially suppressing phenotypes associated with the loss of ESCRT-0or ESCRT-I, implying a direct role for ESCRT-II in cargo sorting atMVEs [65].

Recruitment of ESCRT-III to endosomes is mediated largely byESCRT-II, via a direct interaction between the ESCRT-II subunit

ndent cargo sorting at multivesicular endosomes, Semin Cell Dev

Vps25 and the ESCRT-III subunit Vps20 [66]. However, additionalfactors have also been identified, which can bridge earlier act-ing ESCRT complexes with ESCRT-III. In particular, ALIX has beenshown to interact with both ESCRT-I (via Tsg101) and Vps32 iso-

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ig. 1. The early acting ESCRT machinery harbors multiple ubiquitin binding domaeveral components of ESCRT-0, ESCRT-I and ESCRT-II contain domains capable of l

orms [67–71]. Moreover, ALIX harbors a UBD (UBAN-like), whichay contribute to cargo sorting into ILVs [72–74]. Although defects

n cargo trafficking are relatively mild in the absence of ALIX func-ion, they become more pronounced when other components of theSCRT machinery are compromised [75–77], suggesting that ALIXontributes to the overall efficiency of cargo delivery into MVEs.

The ESCRT-III and Vps4 complexes lack the presence of UBDsnd their contribution to ubiquitin-dependent cargo sorting haseen challenging to reconcile. One compelling hypothesis is thatSCRT-III spiral filaments may encircle cargoes to physically restrictheir diffusion on MVEs [78,79], although direct evidence for such

model is currently lacking. The ability of ESCRT-III to corral car-oes may be particularly important considering that substrates ofhe ESCRT pathway are typically subject to deubiquitination prioro internalization into ILVs. In yeast, the ubiquitin peptidase Doa4as been demonstrated to associate with the ESCRT-associated pro-ein Bro1 (an orthologue of mammalian ALIX), Vps20 and Vps3280–83]. In particular, Vps20 binding to Doa4 appears to inhibitts deubiquitinase (DUB) activity, thereby delaying removal ofbiquitin from cargoes, perhaps until ESCRT-III spiral arrays canssemble properly [84]. In mammalian cells, several ESCRT-IIIubunits (CHMP1, CHMP2, CHMP3, and Ist1) associate with deu-iquitinating enzymes, including AMSH, which specifically cleaves63-linked ubiquitin chains [85–91]. However, unlike Doa4, therecise role of AMSH during cargo sorting remains to be clearlyefined.

Despite tremendous advances in our understanding ofbiquitin-dependent cargo sorting at MVEs, the key question ofow components of the ESCRT machinery function cooperativelyo ensure that specific cargoes are properly deposited into formingLVs remains unanswered. Although ESCRT-0 can bind to ESCRT-

(via a direct association between Hrs and Tsg101), and ESCRT-Ian associate with ESCRT-II (via a direct association between theSCRT-I subunit Vps28 and Vps36), localization studies have failedo demonstrate extensive co-localization between the complexesn endosomes [10,92–94]. Additionally, there exists little directvidence to support the idea that these complexes co-assemble toorm a higher order assembly in vivo. Instead, each complex mayarticipate independently in cargo binding and due to their lowffinities for ubiquitin, rapidly transfer cargoes to one another viaransient associations. Based on live cell imaging studies, the major-ty of ESCRT-0 is found on endosomes, while ESCRT-I and ESCRT-IIxhibit a more diffuse cytoplasmic distribution [95–98], consis-ent with biochemical data showing that ESCRT-I and ESCRT-II areoluble [99]. Thus, ESCRT-0 has been suspected to play the most sig-ificant role in ubiquitin-dependent cargo clustering at endosomes.

Please cite this article in press as: E.B. Frankel, A. Audhya, ESCRT-depeBiol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.08.020

onsistent with this idea, an auto-activated, mutant isoform of theeast ESCRT-III subunit Vps32 can largely bypass the requirementsor ESCRT-I and ESCRT-II function in cargo sorting, but not ESCRT-0

finity association with ubiquitin.

[100]. Additionally, in vitro studies suggest that ESCRT-0, but notESCRT-I or ESCRT-II, is capable of stably associating with ubiquitin-modified substrates on supported lipid bilayers [51]. Together, thecurrent evidence suggests a model in which ESCRT-0 functionsas the major sorting receptor for ubiquitin-modified substrates atMVEs, with ESCRT-I and ESCRT-II augmenting its actions to cre-ate subdomains enriched with select cargoes, and linking the earlyacting ESCRT machinery to the downstream ESCRT-III complex.

3. Ubiquitin-independent cargo sorting

In recent years, there have been numerous studies indicatingthat integral membrane proteins lacking ubiquitin modificationcan still be internalized into MVEs in an ESCRT-dependent fash-ion. The G protein-coupled protease-activated receptor-1 (PAR1)and the purinergic receptor P2Y1 both contain a YPX3L motif, towhich the ESCRT-associated protein ALIX binds [19,20]. Similarly,the interleukin-2 receptor � (IL-2R�) interacts directly with Hrs ina manner that does not require ubiquitin [22]. The mechanisms bywhich Hrs and ALIX translocate substrates into ILVs is unclear, buttheir internalization continues to require the downstream ESCRTmachinery (ESCRT-III and the Vps4 complex) [19,20].

There have also been reports of soluble cargoes entering ILVsfor degradation. In one case, selectivity of cargoes is achieved bythe chaperone hsc70, which associates with the limiting mem-brane of MVEs through a polybasic motif that interacts withphosphatidylserine [101]. Although unfolding of substrates isunnecessary for deposition into ILVs, both early (ESCRT-I) and lateacting (ESCRT-III and Vps4) components of the ESCRT machineryare required. Additionally, association of soluble cargoes with pro-teins heavily modified by ubiquitin, such as members of the Cosfamily of tetraspanin-like proteins, can lead to their internalizationwithin ILVs in an ESCRT-dependent manner [102].

Beyond the sorting of proteinaceous cargoes, several studieshave demonstrated that specific lipid species are preferentiallysorted into MVEs. In particular, the phospholipids lysobisphospha-tidic acid (LBPA) and phosphatidylinositol 3-phosphate (PI3P) areabundant on ILVs [23,103]. The ESCRT associated protein ALIX bindsdirectly to LBPA [21,104], and depletion studies suggest that ALIXpromotes LBPA enrichment specifically at sites of ILV formation[21]. In vitro, LBPA stimulates membrane deformation on syn-thetic liposomes [21], and it may behave similarly in cells to driveor stabilize the high curvature characteristic of ILVs in mammals(∼50–60 nm in diameter). PI3P similarly associates with compo-nents of the ESCRT machinery, including ESCRT-0 (via the Hrs FYVE

ndent cargo sorting at multivesicular endosomes, Semin Cell Dev

domain) [105] and ESCRT-II (via the Vps36 GLUE domain) [106].In particular, Hrs associates with PI3P with strong affinity [107],and the lipid likely facilitates the formation of ESCRT-0 subdomainson MVEs. Additionally, other ESCRT components, including several

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Fig. 2. Two speculative models to describe pathways for ESCRT-III spiral filament assembly.In one model (left), a subdomain formed by the early acting ESCRT machinery nucleates ESCRT-III filaments that spiral outward to surround cargoes destined for incorporationi dosomt mentsm

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nto ILVs. In this model, ESCRT-0, ESCRT-I and ESCRT-II must dissociate from the enhat the early acting ESCRT machinery forms a subdomain adjacent to ESCRT-III fila

ust be transferred between the early and late acting ESCRT complexes.

SCRT-III subunits, preferentially interact with acidic phospholipideadgroups like that found on PI3P [108]. However, given that PI3Pecomes enriched in ILVs, it remains unclear how components ofhe ESCRT machinery ultimately escape the degradative fate of a

ajor interacting lipid partner. In fact, contrary to studies suggest-ng that ESCRT components are usually recycled [10], proteomictudies of exosomes have consistently identified several compo-ents of the ESCRT machinery [109–111], indicating that recyclingf ESCRT subunits may not be as robust as once believed.

In recent years, exosomes have garnered wide interest, basedn their involvement in cell-to-cell communication and their roles

n immune system regulation, cancer biology, and nervous systemevelopment [112]. The analysis of purified exosomes has revealedhe presence of not only proteins and lipids, but several classesf RNAs, including mRNAs and miRNAs [113]. The mechanisms byhich specific RNAs accumulate in ILVs remain unclear. ESCRT-IIas shown previously to associate with the 3′ UTR of bicoid mRNA

ia the Vps36 GLUE domain to direct its localization in Drosophilaggs [114]. However, since neither ESCRT-I nor ESCRT-III inhibi-ion affected bicoid mRNA distribution, it was believed that theole of ESCRT-II in this process was independent of an endoso-

al sorting function [114]. More recent work suggested that theNA binding protein YBX1 acts as a molecular chaperone to pack-ge a subclass of miRNAs into ILVs [115]. Surprisingly, YBX1 is notembrane bound. Instead, it is largely associated with cytoplasmic

NA granules, which must intersect with MVEs to deliver specificiRNAs into ILVs [116]. It is possible that YBX1 is ubiquitin modi-

Please cite this article in press as: E.B. Frankel, A. Audhya, ESCRT-depeBiol (2017), http://dx.doi.org/10.1016/j.semcdb.2017.08.020

ed and/or capable of associating directly with components of theSCRT machinery or other ubiquitin-modified integral membraneroteins, but additional studies are required to gain a mechanisticnderstanding of its function.

al membrane to avoid engulfment into ILVs. An alternative model (right) suggests, which spiral inwards toward a preferred radius of curvature. In this case, cargoes

4. Cargo transfer into ILVs

With several lines of evidence supporting the idea that theearly acting ESCRT complexes promote initial cargo sorting andclustering on MVEs and the late acting components restrict cargodiffusion prior to their incorporation into ILVs, a major unresolvedissue is the mechanism by which cargoes are efficiently transferredbetween these ESCRT complexes. Two main models have emergedto explain this phenomenon. In the concentric circle model, cargoesare initially retained within a subdomain established by a combina-tion of ESCRT-0, ESCRT-I and ESCRT-II [78]. Subsequent nucleationof ESCRT-III complexes by ESCRT-II results in the formation of aflat spiral polymer that surrounds cargoes, which rapidly adoptsan energetically unfavorable conformation, leading to mechani-cal tension on the membrane [117,118]. Ultimately, the polymeris forced to convert into a three-dimensional helix, deforming themembrane to generate a nascent vesicle, which harbors the car-goes found at its core. To prevent internalization of the early actingESCRT machinery in this model, each complex must be releasedfrom the membrane prior to bud formation (Fig. 2). Currently, thereexist no data to suggest how displacement of ESCRT-0, ESCRT-I, andESCRT-II from the limiting MVE membrane is achieved. Nonethe-less, there are several testable predictions based on this model.First, the early acting ESCRT machinery should not form stable,long-lived subdomains, but instead generate transient patches onMVEs that turnover with each cycle of ILV formation. Second, theearly acting ESCRT complexes should at least transiently co-localize

ndent cargo sorting at multivesicular endosomes, Semin Cell Dev

with late acting ESCRT components on MVEs. Finally, ESCRT-III spi-ral filaments should grow in an outward manner to generate thenecessary compression forces on membranes. Although the livecell imaging studies necessary to address the first two predictions

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ave yet to be reported, multiple groups have examined the thirdn vitro. In both cases, ESCRT-III polymers, consisting largely ofps32 subunits, were found to exhibit a growing diameter, from25 nm at their center to several micrometers at their periph-ry, consistent with outward polymerization [117–119]. These datare supported by other studies indicating that Vps32 polymersxhibit a preference for a high radius of curvature [120]. However,t remains unclear how other ESCRT-III subunits may influence thenitial orientation of polymer assembly. If the nucleation processed to the formation of inward spiraling filaments, the ESCRT-IIIomplex could assemble adjacent to a subdomain composed of thearly acting ESCRT complexes (Fig. 2). In this scenario, it is easy toecognize how ESCRT components would avoid engulfment, but itecomes more challenging to comprehend how cargoes are trans-erred between the early acting ESCRT machinery and a neighboringSCRT-III polymer. One possibility is that cargoes are only tran-iently retained by ESCRT-0, ESCRT-I and ESCRT-II, consistent withheir modest affinity for ubiquitin sorting signals. This would enableargoes to diffuse into juxtaposed subdomains that become encir-led by ESCRT-III spiral filaments, targeting them into ILVs. Highesolution imaging of native ESCRT complexes will be necessary toesolve these conflicting models.

. Concluding perspectives

In the majority of intracellular protein sorting reactions, recep-ors play an integral function in concentrating or retaining specificargoes within budding transport carriers. For example, duringOPII-mediated transport, members of the Sec24 or Tango1 cargoeceptor families play intimate roles in directing specific substratesut of the endoplasmic reticulum [121,122]. However, the ESCRTachinery acts at the neck of nascent vesicles. While potentially

ufficient to restrict cargo escape, this distribution diminishes thebility to prevent the non-specific inclusion of bulk cytoplasmicroteins and nucleic acids within ILVs. Nonetheless, the contentsf ILVs appear to be highly selective, suggesting the existence ofechanisms to enrich for specific cargoes. Notably, other regions

f the endosomal membrane are also active in cargo sorting.pecifically, retromer activity and other recycling pathways simul-aneously regulate the content of endosomal membranes [123].nly through the cooperative actions of multiple cargo sorting

ystems is ILV content appropriately maintained. In the future,ontinuing improvements to live cell imaging technologies andRISPR-mediated genome editing approaches to label endogenousSCRT components will enable us to gain a better understanding ofhe dynamic actions of the ESCRT machinery at endosomes, whichhould address many of the outstanding questions that remain inhis field.

cknowledgements

This work was supported in part by a grant from the NIHGM088151 to AA). We thank Marisa Otegui and members of theudhya lab for suggestions and critically reading this manuscript.

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