Plant ESCRT Complexes: Moving Beyond Endosomal Sorting

Review

Plant ESCRT Complexes: Moving Beyond Endosomal Sorting Caiji Gao,1,3 Xiaohong Zhuang,2,3 Jinbo Shen,2,3 and Liwen Jiang2,* The endosomal sorting complex required for transport (ESCRT) machinery is an ancient system that deforms membrane and severs membrane necks from the inside. Extensive evidence has accumulated to demonstrate the conserved functions of plant ESCRTs in multivesicular body (MVB) biogenesis and MVB-mediated membrane protein sorting. In addition, recent exciting findings have uncovered unique plant ESCRT components and point to emerging roles for plant ESCRTs in non-endosomal sorting events such as autophagy, cytokinesis, and viral replication. Plant-specific processes, such as abscisic acid (ABA) signaling and chloroplast turnover, provide further evidence for divergences in the functions of plant ESCRTs during evolution. We summarize the multiple roles and current working models for plant ESCRT machinery and speculate on future ESCRT studies in the plant field. Expanding Roles for the ESCRT Machinery in Eukaryotes The endosomal sorting complex required for transport (ESCRT, see Glossary) machinery is an evolutionarily-conserved, multi-subunit membrane remodeling complex, which plays essential and canonical roles in the biogenesis of the multivesicular body (MVB) and the sorting of ubiquitinated membrane proteins into their intraluminal vesicles (ILVs) for degradation upon the fusion of the MVB and the vacuole/lysosome [1–3]. Owing to its unique ability to form membrane constrictions and subsequent outward-directed scission, an increasing number of ESCRT functions at non-endosomal sites have been discovered in recent studies in yeast and mammalian cells (Figure 1A). These include processes occurring (i) at the plasma membrane (PM), such as PM repair by promoting budding and scission of PM-derived vesicles containing the lesion, cytokinetic abscission during the final step of cell division, ‘neuron pruning’ for the elimination of old synapses, virus budding, and exovesicle shedding from PM [4–8]; (ii) at the nuclear membrane, such as the clearance of defective nuclear pore complexes (NPCs) during interphase, nuclear envelope reformation during telophase, and nuclear envelope repair upon cell migration-induced nuclear rupture [9–13]; (iii) at the endoplasmic reticulum (ER), mitochondrial, or peroxisomal membranes, leading to the formation of the viral replication compartment (VRC) to facilitate plus-stranded RNA virus replication [14,15]; and (iv) in autophagosomes to affect autophagic degradation [16,17]. This involves a double-membrane compartment, called the compartment for unconventional protein secretion (CUPS), that mediates Acb1 secretion [18]. In addition, the ESCRT machinery is also involved in selective sorting of cytosolic proteins into the lumen of MVB or vacuole/lysosome, a process known as microautophagy in yeast and mammals (Figure 1A) [19,20]. A more recent study has even shown an intriguing role of ESCRTs in telomere length maintenance in yeast [21].

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Trends in Plant Science, November 2017, Vol. 22, No. 11 © 2017 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.tplants.2017.08.003

Trends ESCRT is an evolutionarily conserved machinery for membrane deformation and scission from the inner face of a membrane away from the cytoplasm. Plants encode most ESCRT isoforms in their genome, including ESCRT-I, -II, -III, and VPS4/SKD1, with the exception of the canonical ESCRT-0. TOL (TOM1-like) proteins were identified as upstream ESCRT factors that partially fulfill ESCRT-0 function in plants. Extensive evidence has accumulated to demonstrate the essential and conserved functions of ESCRTs in endosomal sorting in plants. Plant-specific ESCRT components have been identified. In addition, ESCRTs in plants are also involved in a variety of non-endosomal sorting events such as autophagosome maturation, chloroplast turnover, cytokinesis, and viral replication. Plant ESCRTs are also actively involved in hormone signaling and plant responses to biotic and abiotic stresses.

1 Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University (SCNU), Guangzhou 510631, China 2 Centre for Cell and Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong (CUHK), Shatin, New Territories, Hong Kong, China

3 These authors contributed equally to this work

*Correspondence: [email protected] (L. Jiang).

Figure 1. Overview and Comparison of ESCRT-Dependent Cellular Processes in Yeast, Mammals, and Plants. The ESCRT machinery (indicated with a brown circle) functions in diverse cellular contexts where it constricts membranes and severs narrow membrane necks from the inner face within the cytoplasm. The upper (A) and lower parts (B) demonstrate the cellular functions of ESCRTs in yeast, mammals, and in plants, respectively. Pathways that remain elusive are indicated with dashed arrows. Sites where ESCRTs perform functions through an unknown mechanism are indicated with brown questionmarks. Possible sites where plant ESCRTs might perform functions are indicated with black questionmarks. Abbreviations: CUPS, compartment for unconventional protein secretion; ILV, intraluminal vesicle; MVB, multivesicular body; PM, plasma membrane; RCB, rubisco-containing body. For a Figure360 author presentation of Figure 1, see the figure online at http://dx.doi.org/10.1016/j.tplants.2017.08. 003#mmc1

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Plants appear to encode most ESCRT isoforms in their genome, including ESCRT-I, -II, -III, and VPS4/SKD1 (vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT 1) subcomplexes, with the exception of the canonical ESCRT-0 subunits (Table 1 and Figure 2) [22]. In addition, the subunit interaction network among different ESCRT subcomplexes is largely conserved in plants [23,24]. In the past few years the study of ESCRT machinery in plants has flourished. Accumulated evidence demonstrates the essential roles of ESCRT paralogs in endosomal sorting in distinct tissues or developmental stages in plants (Table 1) [25–29]. In addition, several plant ESCRT subunits have also been reported to play non-canonical functions, such as in chloroplast turnover and autophagic degradation (Table 1 and Figure 1B) [30–32]. More interestingly, the identification of some plant unique ESCRT components such as FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) and POSITIVE REGULATOR OF SKD1 (PROS) provides evidence for evolutionary divergence of plant ESCRT proteins (Table 2) [33,34]. In this review we summarize the plant ESCRT components and outline the most recent advances regarding the canonical functions of ESCRT machinery in endosomal sorting in plants. In addition, we describe the non-endosomal functions of the plant ESCRT machinery. Finally, we attempt to identify possible new functions of the ESCRT machinery in the plant field.

Canonical Functions of ESCRT Machinery in Plant Endosomal Sorting ESCRT-0 In metazoa and fungi, MVB-mediated sorting of ubiquitinated membrane proteins (or cargoes) starts with cargo capture by ESCRT-0, which is a heteromeric multivalent ubiquitinbinding protein complex consisting of two subunits, VPS27/hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and Hse1 (Hbp STAM, EAST 1)/signal transducing adaptor molecule (STAM) [1,2]. Orthologs of ESCRT-0 subunits are absent in plants, instead, the Arabidopsis genome contains nine TOM1-like (TOL) proteins with conserved VHS (VPS27, HRS, STAM) domains followed by GAT (GGAs and TOM) domains and putative clathrinbinding motifs (Table 2 and Figure 2A) [35]. A further study has shown that some Arabidopsis TOLs can effectively bind to ubiquitin and regulate the internalization and vacuolar sorting of the auxin efflux facilitator PIN-FORMED 2 (PIN2) [35]. In addition to direct cargo recognition, other key features of ESCRT-0 functions such as interacting with clathrin for the clustering of ubiquitinated cargo into microdomains have been demonstrated [36]. Furthermore, the ability to physically bind to the ESCRT-I subunit leading to the recruitment of further ESCRT complexes followed by the initiation of ILV formation and cargo sorting has also been verified for ESCRT-0 in yeast and mammals [37,38]. Whether plant TOLs behave similarly and the functional link between TOLs and known plant ESCRT components remain to be explored experimentally. ESCRT-I In yeast and mammals, the ESCRT-I complex form an elongated heterotetramer of 18 nm in length which contains one copy each of the subunits VPS23/tumor susceptibility gene-101 (TSG101), VPS28, VPS37, and MVB sorting factor of 12 kDa (MVB12) [39]. The VPS23/ TSG101 has an N-terminal ubiquitin E2 variant (UEV) domain that is responsible for binding the ubiquitinated cargo, the late domain of HIV virus Gag protein, and VPS27/HRS of ESCRT-0; while the VPS28 has a C-terminal four helix bundle domain responsible for interaction with ESCRT-II [39]. Arabidopsis contains three ESCRT-I subunits VPS23, VPS28, and VPS37, but lacks the fourth subunit MVB12 (Table 1 and Figure 2A) [40]. Similarly to VPS23/TSG101, the Arabidopsis VPS23 is also able to bind to ubiquitin and to associate with VPS37 and VPS28 to form a putatively intact plant ESCRT-I complex [41]. Arabidopsis vps28-2 and vps37-1 mutants display compromised endosomal sorting of the pathogen-related receptor FLS2, and thus show impaired immune responses against bacterial pathogens [42].

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Glossary Autophagosome: a doublemembrane structure formed during macroautophagy for the sequestration of the cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles) and their subsequent delivery into the vacuole for degradation or recycling. Cytokinesis: a step during cell division in which the cytoplasm of a single eukaryotic cell divides into two daughter cells. In plants, a cell plate forms between the daughter nuclei through the fusion of membrane vesicles. Two daughter cells are produced when the centrifugally growing cell plate ultimately fuses with the existing plasma membrane. Endosomal sorting complexes required for transport (ESCRT): a machinery originally found to be responsible for the induction of a membrane budding process at the MVB that results in the formation of intraluminal vesicles inside the endosome. Intraluminal vesicles (ILVs): small vesicles formed inside the MVB by budding of portions of the endosome limiting membrane away from the cytosol and abscission of them into the lumen of the endosome. Multivesicular body (MVB): a specialized type of endosome that contains membrane-bound intraluminal vesicles that carrying soluble and membrane cargoes to be degraded upon fusion with vacuole or to be released into the extracellular space by fusion with the plasma membrane. Macroautophagy/autophagy: a self-degradation process in which cytoplasmic contents are enclosed into double-membrane structures, called autophagosomes, which fuse with the vacuole. Macroautophagy is induced under stress or nutritiondeficient conditions. Microautophagy: a type of autophagic pathway which is mediated by direct engulfment of the portion of cytoplasmic contents or cargo into endosomes or vacuoles/ lysosomes. Nuclear pore complex (NPC): a protein complex forming a channel in the nuclear envelope that mediates transport between the nucleoplasm and cytoplasm. Rubisco-containing body (RCB): a type of autophagic structure for

ESCRT-II ESCRT-II has a pivotal role in linking the upstream ubiquitin-binding ESCRT complexes to the downstream ESCRT-III complex. Similarly to mammal and yeast ESCRT-II, the plant ESCRT-II is also composed of three subunits VPS22, VPS25, and VPS36 (Table 1 and Figure 2A), but the functions of Arabidopsis VPS22 and VPS25 remain to be established. Knockout of OsVPS22 in rice seems to cause seedling lethality and severe reduction in shoot and root growth; however, whether endosomal sorting is affected in osvps22 mutants remains unknown [43]. A more recent study shows that VPS36 might form an ESCRT-II complex with VPS22 and VPS25 in Arabidopsis [27]. In addition, Arabidopsis VPS36 shows ubiquitin-binding activity and regulates MVB biogenesis as well as the endosomal sorting of several membrane cargoes into the vacuole for degradation [27].

delivering chloroplast stroma proteins into the vacuole for degradation. Viral replication compartment (VRC): a membranous compartment formed in host cells upon infection by some RNA viruses which serves as a specialized compartment to protect their RNA transcription and replication activities.

ESCRT-III In yeast, binding of the ESCRT-II subunit VPS25 to VPS20 initiates the membrane recruitment and activation of the ESCRT-III complex, which drives membrane invagination and scission of ILVs [44]. VPS20 further recruits sucrose non-fermenting 7 (SNF7) which undergoes homooligomerization followed by capping with VPS24 and VPS2 to complete ESCRT-III assembly [44]. Three accessory proteins, Did2/CHMP1 (Doa4-independent degradation 2/charged multivesicular body protein 1), VPS60/CHMP5, and increased salt tolerance 1 (IST1), modulate the association of ESCRT-III and the VPS4/SKD1 complex, as well as the activity of

Figure 2. Models of ESCRT-Dependent Protein Sorting Pathways in Plants. (A) An updated working model of ESCRTs in the plant endosorting pathway. Ubiquitinated membrane cargoes are captured by endosome-localized ESCRT-0-like proteins, TOLs. The cargoes are subsequently transferred to ESCRT-I and II via the ubiquitin-binding proteins VPS23, FREE1, and VPS36. Through the interaction between VPS25 and VPS20, ESCRT-II further activates the ESCRT-III complex which constricts membranes and severs membrane buds to form intraluminal vesicles (ILVs). Before sorting into ILVs, the ubiquitin is removed from the cargo by the deubiquitinating enzyme AMSH3, which is recruited to endosomal membrane by AtBRO1/ALIX. The ESCRT-III accessory proteins help to recruit the VPS4/SKD1–LIP5 complex which dissociates the ESCRT-III complex from the endosomal membrane. (B) AtBRO1/ALIX is also engaged in the ESCRT-I complex via a direct interaction with VPS23. An incorporation of AtBRO1/ALIX in ESCRT-I may function to remove ubiquitin from the ubiquitinated membrane cargoes that need to be recycled from the late endosome before further sorting into ILVs. (C) FREE1 is also possibly incorporated into ESCRT-III via association with SNF7, although the exact function of FREE1 in the ESCRTing pathway remains unclear.


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Table 1. ESCRT Components and Their Functions in Plantsa Regulators

Yeast (Sc)

Human (Hs)

Plant (AGI of At)

ESCRT-0

VPS27

HRS

N.I.

Hse1

STAM1,2

N.I.

VPS23

TSG101

VPS28

hVPS28

ESCRT-I

VPS37

VPS37 A, B, C, D

Functional annotation in plants

Refs

VPS23A/ELC (At3g12400)

Endosomal sorting, viral replication, cytokinesis

[15,41,65,75]

VPS23B (At5g13860)

Viral replication

[15,75]

VPS28-2 (At4g05000)

Endosomal sorting, immune response against bacterial pathogens

[42]

VPS37-1 (At3g53120)

Endosomal sorting, immune response against bacterial pathogens

[42]

VPS28-1 (At4g21560)

VPS37-2 (At2g36680)

ESCRT-II

ESCRT-III and accessory proteins

MVB12

hMVB12A, B

N.I.

VPS22

EAP30

VPS22 (At4g27040)

VPS25

EAP20

VPS25 (At4g19003)

VPS36

EAP45

VPS36 (At5g04920)

MVB biogenesis, endosomal sorting

[27]

VPS20

CHMP6

VPS20-1 (At5g63880)

MVB biogenesis, endosomal sorting

[45]

SNF7-1 (At2g19830)

MVB biogenesis, endosomal sorting, viral replication

[15,45]

SNF7-2 (At4g29160)

Viral replication

[15]

VPS24-1 (At5g22950)

MVB biogenesis, endosomal sorting, viral replication

[15,45]

MVB biogenesis, endosomal sorting, autophagic degradation

[30,45]

MVB biogenesis, endosomal sorting, chlorophagy

[26,31]

ISTL1 (At1g34220)

MVB biogenesis, plant immunity, and cell death

[52]

VPS20-2 (At5g09260) SNF7 (VPS32)

CHMP4A, B, C

VPS24

CHMP3

VPS24-2 (At3g45000) VPS2

CHMP2A, B

VPS2-1 (At2g06530) VPS2-2 (At5g44560) VPS2-3 (At1g03950)

Did2

VPS60

CHMP1.5, CHMP1A, CHMP1B CHMP5

CHMP1A (At1g73030) CHMP1B (At1g17730) VPS60-1 (At3g10640) VPS60-2 (At5g04850)

IST1

VPS4 and accessory proteins

Other ESCRT-related proteins

990

Cmp7

CHMP7

CHMP7 (At3g62080)

Senescence, abiotic stress responses

[48]

VPS4

VPS4/SKD1

VPS4/SKD1 (AT2G27600)

MVB biogenesis, endosomal sorting, viral replication, plant immunity, cytokinesis

[15,25,49,50]

VTA1

LIP5

LIP5 (AT4g26750)

MVB biogenesis, endosomal sorting, plant basal immunity, abiotic stress responses

[28,51,52]

BRO1

ALIX

BRO1/ALIX (AT1G15130)

MVB and vacuole biogenesis, endosomal sorting, viral replication

[15,57–59,75]


Table 1. (continued) Regulators

Yeast (Sc)

Human (Hs)

Plant (AGI of At)


Refs

Autophagic degradation, plant immune response to mildew infection

[30]

Vacuole biogenesis, endosomal sorting, autophagic degradation

[46,56]

Bro1L1 (AT1G17940) Bro1L2 (AT5g14020) AMSH

Doa4

AMSH1 (AT1G48790)

AMSH2 (AT1G10600) AMSH3 (At4g16144)

a

Abbreviations: AGI, Arabidopsis gene identifier number; At, Arabidopsis thaliana; Hs, Homo sapiens; N.I., not identified; Sc, Saccharomyces cerevisiae.

VPS4/SKD1 [1]. The less well characterized ESCRT-III associated protein CHMP7 was recently shown to interact with the inner nuclear membrane protein LEM2 (lap/emerin/man domain protein 2); thereafter they cooperate to recruit ESCRT-III complexes to holes in the nuclear membrane for the regeneration of a sealed nuclear envelope during mitotic exit in yeast and mammals [11,13]. Arabidopsis contains all the isoforms of ESCRT-III subunits (Table 1 and Figure 2A). The induced expression of dominant negative forms of either ESCRT-III core subunit results in defects in MVB biogenesis and vacuolar degradation of membrane proteins in Arabidopsis cells [45]. The Arabidopsis vps2.1 mutant and the double homozygous chmp1a chmp1b mutant plants are embryo/seedling lethal [26,46]. In addition, transgenic Arabidopsis plants with overexpression of the VPS2.1–GFP or the ESCRT-III dominant negative mutant also display severe dwarf or lethal phenotypes [30,45], pointing to an essential role of the ESCRT-III complex in postembryonic development in plants. The Arabidopsis CHMP1 proteins interact with the VPS4/SKD1–LIP5 (lyst-interacting protein 5) complex to regulate MVB biogenesis and vacuolar sorting of auxin transporters [26]. In addition, SUPERNUMERARY ALEURONE

Table 2. Unique ESCRT Components and Functions in Plantsa Regulators

Yeast (Sc)

Human (Hs)

Plant (AGI of At)

Proteins show functional analogies to ESCRT-0

GGA1, 2

GGA1, 2, 3

N.I.

TOM1 TOM1L1 TOM1L2 TOM1L3

TOL1 (At5g16880) TOL2 (At1g06210)


Refs

TOL-2, -3, -5, -6, -9 are involved in endosomal sorting of PIN2 and plant development

[35]

TOL3 (At1g21380) TOL4 (At1g76970) TOL5 (At5g63640) TOL6 (At2g38410) TOL7 (At5g01760) TOL8 (At3g08790) TOL9 (At4g32760)

Plant-specific ESCRT components

a

N.I.

N.I.

FREE1/FYVE1 (At1g20110)

MVB and vacuole biogenesis, endosomal sorting of integral membrane proteins such as PIN2 and IRT1, endosomal sorting of the non-integral membrane proteins that are ABA receptors, autophagic degradation

[32,34,60,64]

N.I.

N.I.

PROS (At4g24370)

Regulate SKD1 ATPase activity and plant cell expansion

[33]

Abbreviations: AGI, Arabidopsis gene identifier number; At, Arabidopsis thaliana; Hs, Homo sapiens; N.I, not identified; Sc, Saccharomyces cerevisiae.


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LAYER1 (SAL1), a CHMP1 homolog in maize, regulates the endosomal sorting and degradation of the plasma membrane-localized receptor kinase CRINKLY4 to control the development of aleurone cell layers in endosperm [47]. A recent study shows that overexpression of the BnCHMP7 from rapeseed (Brassica napus) in Arabidopsis causes early senescence and hypersensitivity to dark treatment of the transgenic plants [48], but the exact molecular and cellular function of CHMP7 remains unclear in plants. VPS4/SKD1–LIP5 Complex The AAA ATPase VPS4/SKD1 hydrolyzes ATP to dissociate the ESCRT-III complex from the endosomal membrane to the cytoplasm for recycling (Figure 2A) [1]. Vps twenty-associated 1 (VTA1)/LIP5 functions as a positive regulator for VPS4/SKD1 activity. In addition, VPS4/SKD1 interacts with the accessory ESCRT-III subunits that further modulate its activity. Disruption of VPS4/SKD1 activity via gene depletion or expression of a dominant negative form of VPS4/SKD1 in plants results in severe defects in development or the immune response to bacterial and fungal pathogens, accompanied by aberrant MVB and vacuole biogenesis [25,49,50]. The Arabidopsis VPS4/SKD1 positive regulator LIP5 is involved in plant biotic and abiotic stress responses through affecting the degradation of ubiquitinated protein aggregates or by regulating the transport of defense-related molecules via MVBs or exosome-like paramural vesicles, respectively (Figure 1B) [51,52]. A more recent study further demonstrates that LIP5 genetically and physically interacts with increased salt tolerance 1-LIKE1 (ISTL1), a protein predicted to be the Arabidopsis homolog of yeast IST1, to regulate MVB biogenesis and MVB-mediated sorting of membrane proteins [28]. Deubiquitination of Cargo Before release into the ILVs, cargoes are generally deubiquitinated by the ESCRT-associated deubiquitinating enzymes (DUBs). However, cargo deubiquitination does not seem to be mandatory for the vacuolar degradation because the membrane proteins with translationally fused ubiquitin that cannot be cleaved by the DUBs still reach the vacuolar lumen via an ESCRT-dependent pathway [53–55]. In yeast and mammals, Doa4 (degradation of alpha 4) and AMSH (associated molecular with SH3 domain of STAM) are the DUBs that associate with ESCRT components and are responsible for removing the ubiquitin from cargo molecules [1]. Three AMSH proteins, AMSH 1–3, are present in Arabidopsis (Table 1). AMSH1 possesses DUB activity toward lysine (K)63-linked, but not K48-linked, ubiquitin chains, whereas AMSH3 can hydrolyze both K48- and K63-linked ubiquitin chains [30,56]. In addition, AMSH1 interacts with the ESCRT-III subunit VPS2.1 and is involved in autophagy to control plant senescence and the immune response upon pathogen infection [30], whereas AMSH3 interacts with the ESCRT-III subunits VPS2.1 and VPS24.1 and is essential for intracellular transport and vacuole biogenesis [46,56]. Recent studies have further shown that the recruitment of AMSH3 to endosomes in Arabidopsis is mediated by a direct interaction with the ESCRT-related protein AtBRO1 (Arabidopsis BRO1-like domain containing protein 1)/ALIX (apoptosis-linked gene-2 interacting protein X) (Figure 2A), which associates with both ESCRT-1 and ESCRT-III components to regulate MVB and vacuole biogenesis as well as the vacuolar sorting of various membrane proteins [57–59]. Unique Plant ESCRT Components: PROS and FREE1 Plants have evolved unique ESCRT components which display limited protein sequence similarity to the yeast and mammalian ESCRT components but interact with known plant ESCRT subunits to regulate ESCRT-dependent cellular activities in plants. One good example of plant-specific ESCRT components is PROS, which was originally identified as a VPS4/SKD1-interacting partner in a yeast two-hybrid screen using Arabidopsis SKD1 as bait (Table 2) [33]. Intriguingly, PROS can only be identified in flowering plants, is ubiquitously expressed in Arabidopsis, and is required for cell expansion and plant growth [33]. In addition to

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VPS4/SKD1, PROS is also able to interact with LIP5 to positively regulate VPS4/SKD1 ATPase activity [33]. Another clear piece of evidence supporting the variation of unique plant ESCRT components comes from recent studies of FREE1, the orthologs of which can be widely found in eudicots, all encoded by a single gene (Table 2) [34]. FREE1 binds to phosphatidylinositol-3phosphate and ubiquitin, and specifically interacts with the ESCRT-I subunit VPS23 via the PTAP-like tetrapeptide motifs [34]. The Arabidopsis free1 mutant is seedling lethal and defective in MVB biogenesis and endosomal sorting of membrane proteins [34,60,61]. In addition, FREE1 also interacts with the IRON-REGULATED TRANSPORTER 1 (IRT1) to control the polar localization of IRT1, providing a molecular mechanism for the involvement of ESCRT component in IRT1-dependent metal transport and metal homeostasis in plants [62]. Plant ESCRTs Participate in Sorting of Non-Integral Membrane Proteins In addition to the canonical and essential function in MVB sorting of integral membrane cargoes, recent studies have revealed that the plant ESCRT components are also involved in endosomal sorting of non-integral membrane proteins. In plants, the soluble ABA receptors PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) are subjected to ubiquitination by CULLIN4-RING E3 ubiquitin ligase and degradation by the 26S proteasome [63]. Moreover, recent studies have demonstrated that a proportion of ubiquitinated ABA receptors can also be recognized by the Arabidopsis ESCRT components FREE1 and VPS23A for sorting into endosomal compartments and the vacuole for degradation (Tables 1,2) [64,65]. Knockdown of FREE1 or depletion of VPS23A leads to increased accumulation of ubiquitinated PYL4 and thereby an enhanced response of the mutant plants to ABA [64,65]. The loading signal of PYR/PYL/RCAR into the ESCRT machinery is presumably the ubiquitination of the receptors by the RING FINGER OF SEED LONGEVITY1 (RSL1), a single subunit RING-type E3 ubiquitin ligase containing a C-terminal transmembrane domain for targeting to the PM [64,66]. In addition, the membrane association of PYR/PYL/RCAR through interaction with either the PM-localized C2-domain ABA-related (CAR) proteins or the E3 ligase RSL1 may be a prerequisite for ESCRT-dependent degradation of these ABA receptors [66,67]. The ESCRT-dependent vacuolar degradation of PYR/PYL/RCAR thus represents an excellent example of participation of the plant ESCRT machinery in endosomal sorting of non-integral membrane cargo.

Non-Canonical Functions of the Plant ESCRT Machinery ESCRT in Plant Autophagy and Chloroplast Turnover The involvement of the ESCRT machinery in autophagy (refer to as macroautophagy unless otherwise indicated) was originally suggested by studies on nematodes, flies, and mammals where ESCRT dysfunction causes an accumulation of autophagosomes either because of induction of autophagic flux or due to inhibition of autophagosomal maturation [16]. Because autophagosomes may fuse with MVBs during their maturation, it is not surprising that the ESCRT machinery is required during autophagy. However, recent studies in Arabidopsis have shed new light on the connection between ESCRTing and autophagic pathways (Tables 1,2) [68,69]. The first indication of ESCRT function in plant autophagy comes from a study of the Arabidopsis ESCRT-III-associated deubiquitinating enzyme AMSH3, depletion of which causes the accumulation of autophagosomes presumably due to malfunction of vacuole in the amsh3 mutant [46,56]. Later, knockdown of AMSH1 or overexpression of its interaction partner VPS2.1–GFP, which is an ESCRT-III subunit and functions as a dominant negative mutant upon overexpression as a form of C-terminal GFP fusion, were shown to cause an accumulation of autophagosomes and a defect in autophagic degradation in mutant plants [30]. Consequently, both mutants display early senescence and hypersensitivity to carbon starvation resembling the phenotypes of previously reported autophagy mutants [30]. Unexpectedly, the newly identified plant-specific ESCRT component FREE1 has been also shown to directly interact with SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), which


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functions together with the autophagic machinery in autophagosome formation in Arabidopsis [32,70]. FREE1 depletion leads to a defect in autophagosome–vacuole fusion and consequently the accumulation of autophagosomes [32]. The interaction of FREE1 and SH3P2 provides molecular evidence for the direct involvement of the ESCRT component in plant autophagy. Autophagy has been shown to play an active role in regulating the degradation of chloroplast proteins and even whole photodamaged chloroplasts [71–73]. Interestingly, a recent study suggests that the ESCRT machinery is also engaged in this process. The Arabidopsis ESCRT-III accessory protein CHMP1, which plays crucial roles in MVB biogenesis and vacuolar sorting of membrane proteins, has also been implicated in the autophagic clearance of chloroplast proteins (Table 1) [26,31]. In contrast to the defect of autophagosome–vacuole fusion upon impairment of other ESCRT subunits as discussed above, depletion of CHMP1 does not abolish bulk autophagic degradation, but specifically affects phagophore closure and loading of chloroplast proteins, and consequently causes the accumulation of plastid proteins in the cytoplasmic bodies that morphologically resemble Rubisco-containing bodies (RCBs) (Figure 1B) [31]. It will be interesting to see whether such defects in chlorophagy are specific to chmp1 as a divergent function of the ESCRT subunit in plants, or whether these are also found in other plant ESCRT mutants. ESCRT in Plant Viral Replication In mammals, membrane enveloped viruses usually hijack the host ESCRT machinery to facilitate their outward budding from the PM of infected cells, a membrane remodeling process that is topologically equivalent to the budding of ILVs from the limiting membrane of MVBs [6]. Plant viruses do not adopt this type of budding to spread because of the presence of the cell wall, but instead move between host cells through plasmodesmata. Upon infection, plusstranded RNA viruses such as tombusviruses utilize the membrane surfaces of various host organelles, such as the ER, chloroplast, and mitochondria, to induce the formation of specialized VRCs to facilitate robust virus replication inside the host plant cells (Figure 1) [74]. In the past few years, the group of Nagy has demonstrated that transient coexpression of dominant negative mutants of Arabidopsis ESCRT components such as VPS24-DN(1–152) or VPS4/SKD1(K178A), which are created by deletion of an autoinhibition domain or a mutation which blocks the ATPase activity, leads to a dramatic reduction of tombusvirus replication in tobacco leaves [15,75,76]. Because the tombusvirus replicase complexes are assembled less precisely in those plants expressing the dominant negative ESCRT-III mutants, the minus-strands of replication RNAs are more accessible to targeted ribonucleases and thus display increased sensitivity to cleavage by the host RNAi machinery [15,75]. In another study, carnation Italian ringspot virus (CIRV), a tombusvirus that replicates at mitochondrial membranes, also recruits the ESCRT subunit VPS23 to mitochondria in tobacco BY-2 cells [77]. Overall, these studies point to a crucial role for plant ESCRT proteins in the precision of viral replicase assembly. In contrast to ILV budding into MVBs and retroviral budding from the PM, the budded membrane-bound VRCs are not severed from the membrane of host organelles (Figure 1). Thus, an interesting question is how the same ESCRT machinery prevents membrane scission in the formation of VRCs. ESCRT in Plant Cytokinesis In mammalian cytokinesis, ESCRT components including the ESCRT-III subunits, VPS4, TSG101, and ALIX are recruited to the midbody ring via an interaction with the midbodylocalized microtubule-bundling protein CEP55 to conduct the final membrane abscission step, a membrane remodeling process that topologically resembles the curvatures needed in the budding of ILVs and endocytic virus vesicles [78]. Plant cytokinesis is initiated by the transport of membrane vesicles to the cell plate, which is formed in the center and radially expands

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towards the periphery [79]. Although all the ESCRT components that are actively required for cytokinesis are present in the Arabidopsis genome, thus far the function of the ESCRT machinery in plant cytokinesis has not been well established. The first and also the best characterization of ESCRT machinery in plant cytokinesis came originally from the phenotypic analysis of the Arabidopsis elch mutant with abnormal trichome morphogenesis. ELC encodes a functional Arabidopsis VPS23A protein, and the elch mutant displays a multinucleated phenotype in various cell types including trichomes, epidermal pavement cells, and hypocotyl cells [41]. How ELC/VPS23A functions in cell division in plants remains unclear, although it has been suggested that ELC regulates cytokinesis through the microtubule cytoskeleton [41]. Another piece of evidence in favor of the plant ESCRT machinery operating in cytokinesis comes from a study of VPS4/SKD1 in Arabidopsis. Shahriari et al. found that transgenic Arabidopsis plants expressing dominant negative mutants of VPS4/SKD1 under the control of the trichome-specific GLABRA2 promoter display a pronounced increase of the trichomes with multiple nuclei and more branches [49]. Future investigations to search for the linkage between the ESCRT machinery and the regulators resident on the cell plate in plants are certainly required.

Links Between the Localizations and Functions of Plant ESCRTs: A Complicated Issue In the ESCRTing pathway, ESCRT-0 subunits can recognize clathrin-coated vesicles to cluster cargo, ESCRT-I and -II sequester cargo and induce membrane deformation, and ESCRT-III finally mediates membrane scission [1]. Thus, it is reasonable to assume that loading of upstream ESCRT components occurs at an earlier stage in endosomal sorting, whereas recruitment of downstream ESCRT subunits occurs in the late endosomal compartment, thereby creating a differential distribution of ESCRT subunits along the endosomal sorting route. Subcellular localizations of plant ESCRT subunits seem to well support the above scenario. Fluorescently tagged plant upstream ESCRT component TOL6 shows localizations to the PM and the trans-Golgi network (TGN), an equivalent of the early endosome in plant cells [35]. Immunogold colocalization analysis using high-pressure frozen and freeze-substituted Arabidopsis roots demonstrates that endogenous VPS28, an ESCRT-I subunit, mainly localizes to the Golgi apparatus and the TGN, but not to the MVB [80]. The ESCRT-II subunit VPS22 mainly localizes to TGN, whereas the ESCRT-III subunits localize principally to subdomains of MVBs [45,80]. The differential distributions of plant ESCRT subunits indicate their sequential recruitment and release from membranes. However, the predominant MVB localization of VPS23, an ESCRT-I subunit, revealed by fluorescent tag fusions in several studies seems to contradict the above concept [23,34,41]. Plausible explanations might be the artifact of overexpressed fusion proteins or the broader localizations as a result of diversified functions of some plant ESCRT subunits. Careful examination by immunogold labeling with specific antibodies is needed to clarify these contradictions. Another complex issue is the divergent roles of certain plant ESCRT subunit, as exemplified by FREE1 and AtBRO1/ALIX. FREE1 was originally characterized as specifically interacting with VPS23 via the PTAP-like tetrapeptide motifs to be incorporated into the ESCRT-I complex (Figure 2) [34]. In other studies, however, FREE1 was shown to interact with SNF7 to be incorporated into the ESCRT-III complex (Figure 2) [60,64]. In all of these studies, FREE1 shows a predominant localization to the MVB, even though the exact function of FREE1 in the ESCRTing pathway remains elusive [34,60,62,64]. Another example is AtBRO1/ALIX, the GFP fusion of which localizes to MVB [57,58]. In a recent study, AtBRO1/ALIX shows an interaction with the ESCRT-III subunit SNF7 to regulate the endosomal localization of the deubiquitinating enzyme AMSH3 (Figure 2) [57]. In another study, AtBRO1/ALIX associates with VPS23 and FREE1 to be incorporated into ESCRT-I complex (Figure 2) [58]. Incorporation of AtBRO1/ALIX in ESCRT-I may function to remove ubiquitin from the ubiquitinated cargoes


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that need to be recycled from late endosome before further sorting into ILVs (Figure 2). These studies indicate that FREE1 and AtBRO1/ALIX may function to bridge ESCRT-I and ESCRT-III complexes in plants, but whether these two proteins bind simultaneously to both ESCRT-I and -III complexes in parallel remains unclear.

The Elusive Functions of Plant ESCRT Machinery In comparison to the well-characterized functions of the ESCRT machinery in membrane remodeling processes in mammals and yeast, the ESCRT functions so far identified in plant cellular activities are fairly limited, raising the question of whether additional ESCRT-based membrane remodeling functions also occur in plants. Physiological roles for ESCRT-III in nuclear envelope and PM remodeling have relatively been well documented in yeast and mammals (Figure 1A). The genes encoding for all the ESCRT-III components are present in Arabidopsis (Table 1). In addition, the nuclear envelope in plant cells also breaks down during the cell cycle, and needs to be reformed during telophase, and plants always need to face the challenge of PM damage following pathogen infection or UV irradiation. It will be interesting to dissect the roles of ESCRTs in nuclear envelope reformation during plant cell mitosis and to explore the potential functions of ESCRT machinery in maintaining PM integrity in plant cells. In yeast and mammalian cells, ESCRT proteins predominantly display cytosolic or endosomal localizations [1,2], and some ESCRT components can be transiently recruited to specific PM domains to perform functions such as cytokinetic abscission or the scission of PM-derived vesicles (Figure 1A) [4]. Intriguingly, a functional GFP fusion of plant ESCRT-II subunit VPS36–GFP, which can successfully complement the lethal phenotype of Arabidopsis vps36 mutant, was shown to be predominantly localized to the PM [23,27]. It would be interesting to explore whether such a distribution is required for the possible involvement of plant ESCRTs in PM-related cellular activities such as endocytosis or cytokinesis. Besides the commonly used genetic and cell biological tools, ectopic expression of plant ESCRT genes in yeast or mammalian cells with depletion or knockdown of the corresponding orthologous genes may be a powerful approach to dissect the unknown functions of plant ESCRTs.

Concluding Remarks and Future Perspectives Research into ESCRT functions has undergone a significant revolution in recent years. It seems that the ESCRT machinery participates in all the membrane budding and scission processes that topologically protrude out of the cytoplasm. As compared to the tremendously diverse roles of ESCRTs in mammals and yeast, the reported functions of ESCRT machinery in plants remain limited. The structure and organization of the ESCRT subunits have been exhaustively studied with the aid of new high-resolution microscopy techniques, for example, cryo-electron microscopy, electron tomography, electron cryotomography, and correlative light electron microscopy [81]. It is notable that some ESCRT domains are so divergent in sequence that they can only be identified by 3D structure determination. In addition, in vitro reconstitution of the ordered assembly of ESCRT subunits has also provided insightful information for dissecting ESCRT assembly and biological roles [82,83]. With the applications of these new techniques and the discoveries of plant-unique ESCRT components, future studies on plants will be necessary to explore additional unique functions of the plant ESCRT machinery and to visualize the plant ESCRT machinery in high resolution. We can expect new breakthroughs in understanding the multiple functions of ESCRTs that are already moving beyond endosomal sorting in plants (see Outstanding questions). Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (31671467), the China 1000-Talents Plan for young researchers (C83025), and the SCNU start-up funds (S80833 and S81030) to C.G., as well as from the Natural Science Foundation of China (NSFC; 31470294 and 31670179), the Research Grants Council of Hong Kong

(G-CUHK402/15, CUHK466613,

14130716, 14102417, CUHK2/CRF/11G, C4011-14R,

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C4012-16E,

Outstanding Questions What factors/adaptors are needed for ESCRT recruitment in these diverse cellular processes? For example, which protein(s) recruit ESCRTs to the cell plate during plant cytokinesis? In addition to ubiquitin, do any other signals initiate the loading and activation of the plant ESCRT machinery? What type of membrane is required for ESCRT recruitment in plants? Is there any specificity for plant-unique structures such as the chloroplast? Are there other sites where ESCRTs perform non-endosomal sorting functions in plant cells? For example, do plant ESCRTs perform functions in nuclear envelope reformation during plant cell division? Why do plants have abundant ESCRT paralogs that outnumber those in other eukaryotes? Do the paralogs of a specific ESCRT component perform redundant or different functions in an organelle/tissue-specific manner? Do combinations of different ESCRT subunit isoforms fulfill distinct ESCRT pathway functions in plants? Do parallel ESCRT pathways exist in plant cells?

HKUST12/CRF/13G, and AoE/M-05/12), the CUHK Research Committee, and the Shenzhen Peacock Project (KQTD201101) to L.J.

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