REVIEW SUBJECT COLLECTION: ESCRT MACHINERY

Ubiquitin recognition in endocytic trafficking – with or withoutESCRT-0Niccolo Mosesso*, Marie-Kristin Nagel* and Erika Isono‡

ABSTRACTThe ability to sense and adapt to the constantly changing environmentis important for all organisms. Cell surface receptors and transportersare key for the fast response to extracellular stimuli and, thus, theirabundance on the plasma membrane has to be strictly controlled.Heteromeric endosomal sorting complexes required for transport(ESCRTs) are responsible for mediating the post-translationaldegradation of endocytosed plasma membrane proteins ineukaryotes and are essential both in animals and plants. ESCRTsbind and sort ubiquitylated cargoes for vacuolar degradation. Althoughmany components that comprise themulti-subunit ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III complexes are conserved in eukaryotes,plant and animal ESCRTs have diverged during the course ofevolution. Homologues of ESCRT-0, which recognises ubiquitylatedcargo, have emerged in metazoan and fungi but are not found inplants. Instead, the Arabidopsis genome encodes plant-specificubiquitin adaptors and a greater number of target of Myb protein 1(TOM1) homologues than in mammals. In this Review, we summariseand discuss recent findings on ubiquitin-binding proteins inArabidopsis that could have equivalent functions to ESCRT-0. Wefurther hypothesise that SH3 domain-containing proteins might serveas membrane curvature-sensing endophilin and amphiphysinhomologues during plant endocytosis.

KEYWORDS: Endocytosis, Endosomal transport, ESCRT, Ubiquitin-binding domains, Clathrin

IntroductionPlasma membrane-localised proteins, including various transportersand receptors, are essential for the regulation of growth anddevelopment as well as adaptation and the correct response tobiotic and abiotic stress factors (reviewed in He et al., 2018). Forthe organism to coordinate its growth and development with theenvironment, it is pivotal for it to regulate the abundance and activityof many of these plasma membrane proteins. The abundance ofproteins can be regulated at multiple levels including transcription,translation, protein targeting, post-translational modifications andprotein degradation (reviewed in Harper and Bennett, 2016; Noacket al., 2014). For rapid removal from the cell surface and subsequentdegradation, plasma membrane proteins are first endocytosed andthen transported to the lysosome or vacuole via the endosomaltrafficking pathway.Endocytosis of plasma membrane proteins is mediated mainly by

clathrin, which is conserved among eukaryotes (reviewed inReynolds et al., 2018; Robinson, 2015). In concert with several

adaptor proteins, clathrin cages – composed of clathrin trimers thatform symmetrical three-legged structures called triskelia – areassembled at the plasma membrane allowing the internalisation ofthe proteins in clathrin-coated vesicles (CCVs). Upon disassemblyof the clathrin cage, cargoes can be delivered to the endosomes.The endosomal transport of cargoes to the lysosome or vacuoledepends on post-translational modification with ubiquitin (Ub), orubiquitylation. Cargoes are passed on to and between conservedmembrane-bound protein complexes that recognise and bind the Ubchain on the cargo protein. In the end, cargo proteins are typicallysorted into intraluminal vesicles (ILVs) of multivesicular endosomes(MVEs) (reviewed in Paez Valencia et al., 2016). After fusion of theMVEs to the lysosomes or vacuoles, ILVs are digested by processingenzymes that reside in these compartments.

Ub is a small modifier protein that can be conjugated to targetproteins in different manners (reviewed in Komander and Rape,2012). In endocytic protein degradation, Ub chains linked throughits lysine residue 63 (K63) are reported to act as degradation signalsfor the targeted protein substrate (Lauwers et al., 2009). Allendosomal cargoes reported in Arabidopsis to date are K63-ubiquitylated (Dubeaux et al., 2018; Kasai et al., 2011; Leitner et al.,2012; Lu et al., 2011; Martins et al., 2015). In addition, a K63-Ubsensor (Vx3K0–GFP) localises to the plasma membrane, toendosomes and on the tonoplast in addition to at nuclear foci(Johnson and Vert, 2016), showing that the K63-linked Ubmodification is involved in endosomal degradation also in plants.

The transport of ubiquitylated endosomal cargo is mediated by theendosomal sorting complexes required for transport (ESCRTs),ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III (reviewed inSchöneberg et al., 2017). Essential in multicellular organisms,defects in ESCRTs cause severe developmental and growthphenotypes (reviewed in Hurley, 2015). ESCRT-0, ESCRT-I andESCRT-II each contain at least one Ub-binding- and one membrane-binding subunit that enables them to recognise and retain ubiquitylatedcargoes at endosomal membranes (Fig. 1). Mechanisms of ESCRT-dependent endosomal degradation in plants are summarised in recentreview articles (Gao et al., 2017; Isono and Kalinowska, 2017; PaezValencia et al., 2016; Romero-Barrios and Vert, 2018).

Besides their essential function in membrane trafficking, ESCRTsare also implicated in other cellular processes that depend on ESCRT-III activity (reviewed in Hurley, 2015; Schöneberg et al., 2017).ESCRT-III assembles at the internal face of membrane constrictionsites and mediates membrane scission. This process is essential forcytokinesis, mitotic nuclear envelope reformation and repair (Carltonand Martin-Serrano, 2007; Olmos et al., 2015; Vietri et al., 2015;Raab et al., 2016; Denais et al., 2016). Furthermore, the ability ofESCRT-III to act as a cellular scissor is also used by HIV and otherviruses that recruit the ESCRT machinery to promote their buddingfrom the host cells (Garrus et al., 2001; Martin-Serrano et al., 2003).

Studies in yeast have established that ESCRT-I and ESCRT-II arerequired for the assembly and the organisation of ESCRT-III

Department of Biology, University of Konstanz, D-78464 Konstanz, Germany.*These authors contributed equally to this work

‡Author for correspondence (erika.isono@uni-konstanz.de)

E.I., 0000-0002-3754-8964

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(Henne et al., 2012; Teis et al., 2010). In plants, ESCRT-I, ESCRT-II and ESCRT-III have been shown to be distributed along theendosomal pathway, with ESCRT-I and ESCRT-II mainly on earlyendosomes, and ESCRT-III mainly on late endosomes (Scheuringet al., 2011). In contrast, it was recently reported that all ESCRTs areinstead recruited, in a coordinated manner, to early endosomes inmammalian cells (Wenzel et al., 2018). Another study in yeast hasrevealed that ESCRT-III assembly can be triggered also by ESCRT-0 and the BCK1-like resistance to osmotic shock (Bro1) protein,thereby bypassing ESCRT-I and ESCRT-II (Tang et al., 2016).Whether the distribution and recruiting mechanisms of ESCRTs aredifferent in plants remains to be investigated.Once the cargoes are internalised in ILVs, ESCRT-III disassembly

is ensured by the activity of the AAA-ATPase vacuolar proteinsorting 4 (Vps4) (Babst et al., 1998). Vps4 exists in the cytosol asinactive monomers or dimers. Upon interaction with ESCRT-IIIsubunits and binding of its activator Vps twenty associated 1 (Vta1),

Vps4 assembles as hexamer on MVEs to promote ESCRT-IIIdisassembly (reviewed in McCullough et al., 2018). The plantorthologue of Vta1, LYST-INTERACTING PROTEIN 5 (LIP5), is apositive regulator of the Vps4 orthologue SUPPRESSOR OF K+

TRANSPORT GROWTH DEFECT 1 (SKD1) and is involved inILV formation and the constitutive degradation of the plasmamembrane-localised auxin efflux carriers PIN-FORMED 2 (PIN2)and PIN3 (Buono et al., 2016).

Although all eukaryotic organisms possess genes coding forESCRT subunits, the subunit composition and copy number of genesfor each subunit are surprisingly diverse (Leung et al., 2008;Wideman et al., 2014). Among the four ESCRTs, the two-subunitESCRT-0 is only found in opisthokonta and is absent in all otherorganisms, including plants (Leung et al., 2008; Wideman et al.,2014; Winter and Hauser, 2006). Furthermore, two subunits ofESCRT-I, multi-vesicular body sorting factor of 12 kDa (MVB12)and Ub-associated protein 1 (UBAP1) (Morita et al., 2007; Stefaniet al., 2011), are also absent in plants. The different composition ofESCRTs and ESCRT-interacting protein complexes in metazoan,fungi and plants suggest that the molecular mechanisms ofendocytosis and endosomal transport could differ in these organisms.

The opisthokonta-specific ESCRT-0 binds ubiquitylated cargoesthrough its Vps27–Hrs–STAM (VHS) domain, the Ub-interactingmotif (UIM) and the Src-homology 3 (SH3) domain and interactswith ESCRT-I, which then recruits further ESCRTs to sort thecargoes into the ILVs of the MVEs (Katzmann et al., 2003; Langeet al., 2012; Wenzel et al., 2018) (Table 1). The VHS domain bindspreferentially K63-linked Ub, which is a signal for ESCRT-dependent endosomal transport (Ren and Hurley, 2010). TheESCRT-0 subunit Vps27 (Hrs or HGS in metazoans) contains aFYVE domain through which it binds endosomal membranes.

ESCRT-0 knockout mutants in yeast show a typical class Ephenotype, in that they are defective in proper vacuolar sorting andaccumulate so-called class E compartments that are multilamellarpre-vacuolar structures (Babst et al., 1997; Raymond et al., 1992).Knockout of ESCRT-0 in metazoan causes lethality in earlydevelopmental stages and affects endosomal structures (Komadaand Soriano, 1999; Lloyd et al., 2002; Roudier et al., 2005).

Given the indispensable role of ESCRT-0 in opisthokonta, wehere outline how other eukaryotes, especially multicellularorganisms that do not have this heterodimeric complex, recogniseubiquitylated endocytic cargoes. With a focus on the model plant

Endosome

Plasmamembrane

Cytoplasm

Cell wall

MVE

UbCME

ESCRT

-I

ESCRT-II

ESCRT-III

ESCRT-0 Ub receptors

Vacuole(lysosome)

Fig. 1. Overview of the ESCRT pathway. Plasma membrane-localisedreceptors and transporters have to be tightly regulated, which occurs partly byendosomal protein degradation. ESCRTs are multi-protein complexes thatbind endosomal membranes and ubiquitylated cargo proteins and sort theminto the ILVs of MVEs. The recognition, capture and transport of cargoesdepends on the Ub molecules on the cargo, typically linked through K63linkage. ESCRT-0 is a heterodimer that has evolved in opisthokonta but isabsent in other organisms, including plants. ESCRT-0 functions as a Ubreceptor in CME and is essential for the ESCRT-dependent proteindegradation pathway. Besides ESCRT-0, other Ub receptors are known tofunction together or in parallel to ESCRT-0.

Table 1. Number of homologues of ESCRT-I-interacting Ub-binding proteins

Ub-binding domain(s) A. thaliana Homo sapiens Saccharomyces cerevisiae

Hrs/Vps27 (ESCRT-0) VHSUIM

None 1 1

STAM/Hse (ESCRT-0) VHSUIMSH3

None 2 1

TOM1 VHSGAT

9 (Korbei et al., 2013) 3 None

GGA VHSGAT

None 3 2

ALIX V 1 (Kalinowska et al., 2015) 1 1

FYVE1 outside of FYVE domain, unclear 1 (van Leeuwen et al., 2004) None None

SH3Ps SH3 3 (Lam et al., 2001) None None

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Arabidopsis (Box 1), we will discuss Ub adaptors with conservedUb-binding entities that function in the endosomal pathway and thathave similar characteristics to ESCRT-0 of metazoan and fungi. Toconclude, we speculate that SH3 domain-containing proteins aregood candidates for endophilin and amphiphysin orthologues thatmediate membrane tubulation in Arabidopsis.

TOM1-like proteins are Ub adaptor proteins expandedin plantsTarget of Myb protein 1 (Tom1) proteins are widely conserved ineukaryotes and are thought to have appeared early in the evolutionas proteins that function in capturing ubiquitylated cargoes (Hermanet al., 2011; Wideman et al., 2014). A Tom1 orthologue is absent inSaccharomyces cerevisiae, but is present in the amoebaDictyostelium, where it acts to recruit the ESCRT-I proteintumour susceptibility gene 101 (DmTsg101, the yeast VPS23homologue) to sort ubiquitylated cargo proteins to MVEs (Blancet al., 2009). In mammals, Tom1 and its two homologues, Tom1L1and Tom1L2, represent a subfamily of the VHS domain-containingprotein family that includes the ESCRT-0 subunits, Hrs and STAM,and the Golgi-localised, γ-ear-containing, ARF-binding proteins

(GGA) homologues, which act as Ub receptors in metazoan andfungi but are absent in plants (reviewed in Shields and Piper, 2011)(Table 1).

Mammalian Tom1 proteins bind and recruit clathrin through theirC-terminal region (Katoh et al., 2006), and Ub through the Ub-binding VHS and GAT (GGA and TOM1) domains (Wang et al.,2010). Tom1 and Tom1L2 bind both K48- and K63-linked Ubchains with a preference for the K63 linkage (Nathan et al., 2013).Based on these findings, mammalian Tom1 proteins are proposed tobe mediating the sorting of ubiquitylated cargoes. Moreover, Tom1

Box 1. Arabidopsis as a model organism to studyendomembrane traffickingArabidopsis thaliana belongs to the family ofBrassicacea and is themostwidely used model plant in research labs. It was chosen as the firstflowering plant to be completely sequenced in 2000. The small genomesize of 135 megabases, which contains an estimated 26,000 genes thatare distributed on five chromosomes (Arabidopsis Genome, 2000),makes genetic analyses feasible. The short generation time of about 3months, light chamber-compatible growth height of 30–40 cm, ability toself-fertilise and high number of seeds per plant are all characteristicsthat make it an attractive organism for broad research areas in cellbiology, biochemistry, genetics and developmental biology. Moreover,Arabidopsis is geographically widely distributed, enabling comparisonsof and studies on natural variations (reviewed in Koornneef and Meinke,2010). A simple floral-dip transformation method using Agrobacteriatumefaciens (recently renamed as Rhizobium radiobacter) wasestablished in 1998 (Clough and Bent, 1998) and led the generation ofa number of T-DNA-insertion collections that are curated and availablefrom seed stock centres.

Membrane trafficking pathways, including vacuolar protein transport,endosomal and autophagic protein degradation, play essential roles inmany different aspects of plant physiology and therefore intensivestudies have been conducted in the past decades. An advantage ofArabidopsis is that cell biological, biochemical and genetic analyses arepossible in one organism. Application of live-cell imaging techniques,such as light sheet microscopy, total internal reflection fluorescence(TIRF) and super-resolution microscopy, have enabled detailed cellbiological analyses of membrane trafficking processes. Electrontomography is another very useful method that enables thevisualisation of membrane compartments. In addition, increasedsensitivity of proteomic analysis has contributed to the unravelling ofprotein networks regulating individual processes. At the same time,advances in genome editing and new generation sequencing techniqueshave drastically shortened the time-intensive process to establish newmutant alleles.

Studies using state-of-the-art techniques revealed many details on theunderlying mechanisms of plant membrane transport (reviewed in PaezValencia et al., 2016). Although many conserved trafficking regulatorshave been found to be as important in plants as in other organisms, at thesame time, plant-specific regulators have also been identified (Gao et al.,2014; Kolb et al., 2015; Nagel et al., 2017; Reyes et al., 2014; Shen et al.,2018), providing a glimpse into the intriguing adaptation and evolution ofthis pathway.

SH3

SH3(SH3P2)

GAT(TOL1)GATGATGAT

D E

B C

TOL1

V

BAR

PRD

GAT

VHS

Membrane

Bro1

A

ALIX SH3P2

V-domain(ALIX)

VHS(TOL1)

Fig. 2. Predicted models of Arabidopsis Ub-binding domains inendosomal trafficking. (A) Schematic overview of Ub adaptors withstructurally characterised Ub-binding domains. Both conserved and plant-specific Ub-adaptors use the sameUb-binding domains for ubiquitylated cargorecognition. (B) The VHS domain of TOL1 was modelled on Protein Data Bank(PDB) template 1ELK (the VHS domain of human TOM1). TOLs bind Ub andfunction in the Ub-dependent endosomal degradation pathway. (C) GATdomain of TOL1, modelled on PDB 1WRD (crystal structure of the GAT domainof human Tom1). (D) ALIX V-shaped domain, modelled on PDB 2R02 (crystalstructure of the human ALIX). Arabidopsis ALIX is an ESCRT-interactingprotein that binds Ub in vitro through its V domain. (E) SH3 domain of SH3P2,modelled on PDB template 2JT4 (solution structure of the SH3 domain of yeastSla1). All models were generated with SWISS-MODEL (Waterhouse et al.,2018) and the figure was obtained from the resulting .pdb files using PyMOLsoftware.

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proteins interact with Toll-interacting protein (Tollip) that binds Uband endosomal membranes and is involved in protein sorting anddegradation (Yamakami et al., 2003). Tom1L1 can also be recruitedon endosomes interacting with Tollip, Hrs or TSG101 (Puertollano,2005).The TOM1 subfamily has expanded in plants, as there are nine

Arabidopsis TOM1-LIKE PROTEINs (TOLs) (Winter and Hauser,2006) (Table 1). TOLs, like Tom1, have VHS and GAT domains(Fig. 2A–C) that are also present in ESCRT-0 and GGAs and areimportant domains for capturing ubiquitylated cargoes. TOLs bindUb in vitro and localise to the plasma membrane and on earlyendosomal structures (Korbei et al., 2013).Arabidopsis TOLs function redundantly, as only higher-order

mutant combinations show defects in endosomal cargo traffickingand severe defects in plant development (Korbei et al., 2013).Although it has not been investigated yet, spatio-temporalregulation of the nine TOLs, including the possible differentialexpression patterns in different cell types, organs or during differentdevelopmental stages might fine-tune the function of TOLs as a Ubadaptor in trafficking processes. To date, it has not been investigatedwhether TOLs, like their orthologues in other organisms, interactwith the ESCRT machinery and other factors involved in theendosomal transport pathway. It is also yet to be established whereTOLs recognise ubiquitylated targets and whether they contribute toclathrin recruitment.

ALIX is a scaffold protein with Ub-binding affinityMammalian ALG-2-interacting protein X (ALIX; also known asPDCD6IP) and its yeast counterpart Bro1 or Vps31 are ESCRT-I-and ESCRT-III-binding proteins that bind Ub and function in theendosomal trafficking pathway (reviewed in Bissig and Gruenberg,2014). The yeast Bro1 is important for the recruitment of thedeubiquitylating enzyme (DUB) degradation of alpha 4 (Doa4) toendosomes, a protein that is important for recycling of Ub andendosomal protein degradation (Dupre and Haguenauer-Tsapis,2001; Luhtala and Odorizzi, 2004; Nikko and André, 2007).Furthermore, Bro1 has been proposed to function in parallel toESCRT-0 as a Ub receptor and to mediate ESCRT-III assembly byactivating the ESCRT-III subunit Snf7 (Pashkova et al., 2013; Tanget al., 2016).Arabidopsis has one ALIX homologue, which has the same

domain structure as the mammalian ALIX and yeast Bro1 (Table 1).The structure of these proteins comprises an N-terminal Bro1domain, a V-shaped domain and a C-terminal proline-rich domain(PRD) (Cardona-Lopez et al., 2015; Kalinowska et al., 2015).Although the sequence identity between orthologues is only ∼20%,the organisation of their protein domains is identical. In all organismstested so far, including plants, the Bro1 domain is essential for theinteraction with ESCRT-III (Cardona-Lopez et al., 2015; Fisheret al., 2007; Kim et al., 2005). Although ALIX proteins do not have atypical Ub-binding domain, the V domain binds mono-Ub, K63-linked di-Ub and K63-linked tetra-Ub (Dowlatshahi et al., 2012;Kalinowska et al., 2015; Keren-Kaplan et al., 2013; Pashkova et al.,2013) (Fig. 2A,D). The C-terminal PRD of ALIX and Bro1 interactswith E3 Ub ligases and is necessary to bind and recruit Doa4 in yeast(Nikko and André, 2007). The PRD of ALIX is the binding site forESCRT-I, whereas the PRD is dispensable for Bro1–ESCRT-Iinteraction (Nikko and André, 2007; von Schwedler et al., 2003).In mammals, an additional Bro1 domain-containing protein, His

domain phosphotyrosine phosphatase (HD-PTP, also known asPTPN23), has been reported to be involved in ESCRT-dependentintracellular trafficking events (Ali et al., 2013; Gahloth et al.,

2016). Besides ALIX, four Bro1 domain-containing proteins can befound in the Arabidopsis genome (Shen et al., 2018). Although asecond Bro1 domain-containing protein named BRO1-DOMAINPROTEIN AS FYVE1/FREE1 SUPPRESSOR (BRAF) has beenshown to be involved in plant endosomal protein degradation, thereis no obvious homolog of HD-PTP in the Arabidopsis genome(Shen et al., 2018).

Arabidopsis ALIX localises to endosomes and is essential forplant growth and development (Cardona-Lopez et al., 2015;Kalinowska et al., 2015). In addition to binding K63-linked Ub,the ESCRT-I subunit VPS23 and the ESCRT-III subunit SNF7,Arabidopsis ALIX interacts and recruits the DUB-associatedmolecule with the SH3 domain of signal transduction adaptormolecule (STAM) 3 (AMSH3), a metalloproteinase DUB that isunrelated to Doa4 (Kalinowska et al., 2015). This is particularlyinteresting, since the ESCRT-0 subunit STAM is known to interactwith and activate AMSH (also known as STAMBP) in mammals(McCullough et al., 2006; Tanaka et al., 1999). Whether, in additionto STAM, mammalian ALIX is also involved in AMSH regulationhas yet to be established.

One of the biochemical properties of ESCRT-0 is to bindphospholipids. Mammalian ALIX has been implicated in viralinfection processes in that it binds the MVE membrane in a Ca2+-dependent manner (Bissig et al., 2013). This lipid-binding module ofALIX recognises and binds an atypical membrane lipidlysobisphosphatidic acid (LBPA) that is specific to late endosomesin mammals. The LBPA insertion loop in human ALIX is not presentin yeast, nor in plants, which is in accordance with the fact that theyare not known to produce LBPA as endomembrane lipids(Bohdanowicz and Grinstein, 2013). Whether plant ALIXfunctions as an alternative to ESCRT-0 and whether and how itbinds membrane lipids will be an interesting topic for future studies.

Altogether, by binding Ub and ESCRT-I, ALIX proteins, likeTom1 orthologues, could serve as functional equivalents ofESCRT-0 in capturing ubiquitylated cargoes at the endosomesand transfer them to the ESCRT machinery.

The FYVE domain-containing protein FYVE1To recruit ubiquitylated cargoes to the endosomal membranes,phospholipid-binding domains, such as pleckstrin homology (PH),phox homology (PX) and Fab1, YOTB, Vac1 and EEA1 (FYVE)domains are essential (reviewed in Lemmon, 2008). The Zn2+-binding FYVE domain present in the ESCRT-0 subunit Hrs (Vps27in yeast) specifically interacts with PI3P, a phospholipid present inendosomal membranes (Burd and Emr, 1998; Gaullier et al., 1998).The FYVE domain of both Hrs and Vps27 is necessary for therecruitment of ESCRT-0 to the endosomal membrane (Katzmannet al., 2003; Raiborg et al., 2001). Interestingly, the Arabidopsisgenome encodes 16 FYVE domain-containing proteins (vanLeeuwen et al., 2004; Wywial and Singh, 2010). Among them,FYVE1 [also known as FYVE DOMAIN PROTEIN REQUIREDFOR ENDOSOMAL SORTING (FREE)1] localises on endosomesand is involved in the control of vacuole biogenesis and membraneprotein localisation as well as endosomal and autophagic proteindegradation (Barberon et al., 2014; Belda-Palazon et al., 2016; Gaoet al., 2014, 2015; Kolb et al., 2015). Recent studies have shownthat, by binding to transcription factors involved in abscisic acidsignalling, FYVE1 inhibits their DNA-binding abilities, thussuggesting a dual function of FYVE1 on endosomes and in thenucleus (Li et al., 2019).

FYVE1 binds PI3P through its FYVE domain and colocaliseswith late endosome markers (Barberon et al., 2014; Gao et al., 2014;

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Kolb et al., 2015). This is in accordance with the fact that althoughPI3P is present in early endosomes in animals (Gaullier et al., 2000),it is a constituent of late endosomes in plants (Simon et al., 2014). Incontrast to Vps27, none of the 16 FYVE domain-containingproteins in Arabidopsis, including FYVE1, have a VHS domain.Although a canonical Ub-binding motif cannot be detected, FYVE1binds Ub and interacts with the ESCRT-I subunit VPS23 (Table 1)(Gao et al., 2014). Whether and how FYVE1 coordinates thetransfer of ubiquitylated cargoes with the ESCRT machinery onendosomes has still to be elucidated.As FYVE1 interacts with Ub, binds PI3P and localises to

endosomes and interacts with ESCRT-I, it possesses major bindingcharacteristics of ESCRT-0 and thus could fulfil ESCRT-0functions in plants.

SH3 domain-containing proteins are Ub-binding proteinsThe ESCRT-0 heterodimer contains STAM (Hse in yeast), a proteinwith multiple Ub-binding domains; an N-terminal VHS domain, aUIM and an SH3 domain. A subset of SH3 domain-containingproteins including STAM binds UBs (Lange et al., 2012;Stamenova et al., 2007). The SH3 domain also acts as aninteraction surface with other proteins, mostly with PRDs. Incontrast to mammals, which have up to 300 SH3 domain-containingproteins (Zarrinpar et al., 2003), our in silico search identified onlyfive SH3 domain-containing proteins in Arabidopsis. All five areplant specific, and none has a VHS domain or a UIM similar to Hseor STAM. Four of these proteins – in addition to the SH3 domain –have a Bin/amphiphysin/Rvs (BAR) domain at the N-terminus(Gadeyne et al., 2014; Zhuang et al., 2013). One is TPLATE-ASSOCIATED SH3 DOMAIN-CONTAINING PROTEIN(TASH), which is a plant-specific subunit of the TPLATEcomplex, which acts in concert with adaptor protein 2 (AP2) inclathrin-mediated endocytosis (CME) (Gadeyne et al., 2014; Hirstet al., 2014).The other three proteins, SH3P1, SH3P2 and SH3P3 (together

SH3Ps), are homologous to each other and are proposed to beinvolved in clathrin-mediated processes (Lam et al., 2001). The SH3domain of SH3P2 (Fig. 2A,E) preferentially binds K63-linked tetra-Ub, and interacts with the ESCRT-I subunit VPS23, the endosome-associated DUB AMSH3 in vitro and also with the autophagsomecomponent ATG8 (Nagel et al., 2017; Zhuang et al., 2013).Interestingly, human AMSH was first identified as an interactor ofthe SH3 domain of the ESCRT-0 subunit STAM and was latershown to be an ESCRT-associated DUB (McCullough et al., 2004;Tanaka et al., 1999). In addition to an SH3 domain, STAM has aUIM and activates AMSH in a UIM-dependent manner (Davieset al., 2013; McCullough et al., 2006). However, the addition ofSH3P2 did not impact the in vitro DUB activity of ArabidopsisAMSH (Nagel et al., 2017), which could be attributed to the factthat, unlike STAM, SH3P2 does not possess a UIM. Whether andhow SH3P2 is regulating AMSH has still to be investigated.Both ESCRT-0 subunits contain clathrin-binding domains and

are essential for clathrin recruitment to the endosomal membrane(McCullough et al., 2006; Raiborg et al., 2001). ESCRT-0 inC. elegans is recruited to the plasma membrane, where it bindsubiquitylated cargoes by interacting with plasma membrane-localised adaptor complexes (Mayers et al., 2013). Plant SH3P2localises to the plasma membrane, associates with CCVs,colocalises with clathrin light chain-labelled structures and co-immunoprecipitated with clathrin heavy chains (Nagel et al., 2017).In contrast to SH3P2, it has yet to be established whetherArabidopsis ALIX, FYVE1 and TOLs function on clathrin-

positive membranes, although in silico analysis has revealed thatsix of the nine TOLs contain putative clathrin-binding motifs(Korbei et al., 2013).

In addition to the plasma membrane, SH3P2 is also localised tothe growing cell plate and is involved in cell plate formation (Ahnet al., 2017). During cell plate formation, active trafficking events,including CME, occur at the developing cell plate (reviewed in VanDamme et al., 2008). Whether trafficking events at the cell platerequire Ub adaptors and whether SH3P2 serves as such an adaptor isnot yet known. As described in the following section, to date,SH3P2 has only been implicated in membrane tubulation at the cellplate.

Taken together, SH3P2 is an plasma membrane- and endosome-localised Ub-binding protein that is associated with CCVs, ESCRT-I and with the DUB AMSH3, thereby sharing interactors withESCRT-0 and thus, similar to ESCRT-0, could function as a Ubadaptor in the ESCRT pathway.

Endophilin and amphiphysin in plant endocytosisAlthough SH3P2 shows biochemical and cell biologicalcharacteristics that are typical of ESCRT-0, recent studies suggestthat it might function as an orthologue of yet another protein family.

Human amphiphysins and endophilins belong to the BAR domainprotein family. Amphiphysins and endophilins are implicated inCME and share a similar domain organisation to SH3P2: a BARdomain at the N-terminus and an SH3 domain at the C-terminus(Fig. 3A). BAR domains bind membranes upon dimerisation and caninducemembrane curvature and tubulation (Carman andDominguez,2018). The BAR domain of SH3P2 binds phospholipids, tubulatesmembranes in vitro and localises to the constricted regions of anexpanding cell plate (Ahn et al., 2017).

Arabidopsis SH3P1, SH3P2 and SH3P3 share only 17%, 19%and 16% amino acid identity with human endophilin A1,respectively (Fig. 3B). Similar to endophilin, SH3P2 and SH3P3have a short linker region between the BAR- and SH3 domains[SH3P1, 108 amino acids (aa); SH3P2, 38 aa; SH3P3, 17 aa] incontrast to mammalian amphiphysin, which has a linker length of upto 400 aa (Fig. 3A). Human amphiphysin-1 contains a binding motiffor clathrin (sequence LLDLD) and a clathrin–AP-2 bindingdomain (CLAP) (sequence PWDLW) in the linker region (Zhangand Zelhof, 2002) (Fig. 3A). Our sequence analysis did not revealclear clathrin-binding motifs in Arabidopsis SH3Ps, although theyassociated with clathrin-containing structures (Lam et al., 2001;Nagel et al., 2017). Endophilin has also been implicated in clathrin-independent endocytosis (CIE) (Boucrot et al., 2015; Renard et al.,2015). Despite indications for the existence of CIE in plants(Bandmann and Homann, 2012), the molecular mechanismsdriving CIE have yet to be investigated.

Both endophilin and amphiphysin interact with the PRD of theGTPase dynamin through their SH3 domain and recruit it to thebudding vesicle neck, which is an essential step during CCVbudding (Sundborger et al., 2011; Takeda et al., 2018; Wigge et al.,1997). Arabidopsis DYNAMIN RELATED PROTEIN 2b(DRP2B) is recruited to clathrin foci at the plasma membrane(Fujimoto et al., 2010). Arabidopsis SH3Ps interact with DRP2Aand with DRP1A (Ahn et al., 2017; Lam et al., 2002), and couldpotentially be involved in the recruitment of dynamins. Althoughfurther in-depth studies are required, SH3Ps are probably not strictlyrequired for dynamin function, as, in contrast to DRP2-encodinggenes that are essential in Arabidopsis (Backues et al., 2010), thesh3p triple mutant does not show apparent developmentalphenotypes nor is it impaired in the uptake of the endocytic tracer

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dye FM4-64 (Nagel et al., 2017). Whether SH3Ps fulfil dualfunctions at the neck of the budding CCV and also on endosomes asUb adaptors for the ESCRT pathway, or whether they are morespecialised in one of the tasks has to be further investigated.Although plants do not possess the ESCRT-0 heterodimer,

domains that are present in opisthokont ESCRT-0 subunits can befound in plant-specific proteins that function together with theESCRTmachinery. As discussed above, the Ub- and ESCRT-bindingproteins TOLs, ALIX, FYVE1 and SH3Ps function in similar proteinnetworks to ESCRT-0 in opisthokonta and thus present an overallsimilar ESCRT pathway in plants, fungi and metazoan. Thedifferences in individual proteins between organisms might serve tofine-tune the ESCRT pathway specific to the need of the organisms,thereby enabling rapid and flexible adaptation to environmentalchanges of plants.

Conclusions and perspectivesAlthough endomembrane trafficking has diversified in the courseof evolution, the protein or lipid interaction domains that areinvolved in this pathway are conserved and appear in differentcombinations. Similar to ESCRT-0 components Vps27 and Hse,TOLs, ALIX, FYVE1 and SH3Ps all localise to endomembranecompartments, interact with Ub and either interact with orhave conserved motifs for the interaction with ESCRT-I (Fig. 4).To date, there is no evidence of a protein heterodimersimilar to ESCRT-0 known to function in plant endocytictrafficking. Almost nothing is known about the transcriptional,translational and post-translational regulation of plant Ubadaptors. Whether these proteins are expressed in the same celltypes, developmental stage or environmental conditions awaitsfuture in-depth analyses.

Endophilin-A1Endophilin-A2Endophilin-A3

SH3P2SH3P3

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Endophilin-A1Endophilin-A2Endophilin-A3

SH3P2SH3P3

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B

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440

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H. sapiens

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Fig. 3. Comparison between SH3Ps and the N-BAR protein family of endophilin and amphiphysin. There is similarity in the domain organisation, but not ahigh similarity in aa sequences for SH3Ps, endophilins and amphiphysin. (A) Schematic representation of Arabidopsis SH3Ps, yeast Rvs167, human endophilin-A1 and amphiphysin. Boxed regions show domains and grey lines show linker regions. Positions of the clathrin-binding site (CBS) and the clathrin–AP2-bindingmotif (CLAP) are indicated by the aa numbers. Scale bar: 100 aa. (B) Sequence alignment of Arabidopsis SH3P1, SH3P2, SH3P3 and human endophilin-A1,endophilin-A2 and endophilin-A3. The sequences of SH3P1, SH3P2, SH3P3 share only 16.8%, 18.5% and 15.7% pairwise identity with endophilin-A1. Thealignment was generated with a ClustalW algorithm using BLOSUM 62. Red, 100% similarity; grey, 80–100% similarity. The grey bar below the alignmentindicates the BAR domain and the blue bar indicates the SH3 domain.

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Another yet poorly explored process in plant endocytosis is CIE.In mammals, among others, endophilins, caveolins, and flotillins areimportant in clathrin-independent endocytosis events [reviewed in(Ferreira and Boucrot, 2018)]. There are three homologues of flotillinin Arabidopsis that localise on the plasma membrane; however, noneof them has been well characterised to date (Junkova et al., 2018).Homologues of caveolin do not exist in plants and, as discussedbefore, a true functional orthologue of endophilins has not beenidentified yet, though SH3Ps could represent possible candidates.Even highly conserved components such as ESCRT-III have

functional divergence in different organisms. For example, in contrastto what is found in yeast, mutants defective in the ESCRT-pathway inArabidopsis do not typically accumulate stacked membranes calledclass-E compartments but rather show clustered or enlarged MVEs(Haas et al., 2007; Kalinowska et al., 2015). PlantMVEs, upon closerultrastructural examination, do not show typical ILVs, but oftengenerate concatenated vesicles (Buono et al., 2017). Thus, studyingthe regulation of endocytosis and membrane trafficking in plants notonly gives insights into the evolutional flexibility of this pathway, but

could also reveal insights into the molecular mechanisms ofendocytic trafficking processes in other species. In recent years,more and more proteomics, transcriptomics and protein interactiondata were generated and sophisticated light and electron microscopytechniques have become available for research. By using these tools,future research will unravel the molecular network supportingendocytosis and endosome-mediated transport pathways andelucidate the intriguing evolutional history of organisms to use andadapt protein domains to serve complex cellular processes.

Competing interestsThe authors declare no competing or financial interests.

FundingThe work in the authors’ laboratory is supported by funds from the DeutscheForschungsgemeinschaft (German Science Foundation; DFG) (SFB924/A06 andSFB969/C08).

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K. J., Johnson, K. A., Zhuang, X. H., Liang, Z. et al. (2017). SH3P2 plays a

CCV

Plasma membrane

Cytoplasm

Cell wall

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CME

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Endosome

MVE

CME

lysosome

Endosome

CIE

TOLs?

FYVE1TOM1

TOM1Ls

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ESCRT-I

ESCRT-I

AnimalsPlants

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Ub UbUb adaptormembrane tubulation?

Ub adaptorMVE biogenesis

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TOM1L1

ALIX

ESCRT-IIIESCRT-II

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ESCRT-III ESCRT-II

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