Endocytic and autophagic pathways crosstalk in plants

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ScienceDirect Endocytic and autophagic pathways crosstalk in plants Xiaohong Zhuang1,3, Yong Cui1,3, Caiji Gao1 and Liwen Jiang1,2 The vacuole is the central site for both storage and metabolism in plant cells and mediates multiple cellular events during plant development and growth. Cargo proteins are usually sequestered into membrane-bound organelles and delivered into the vacuole upon membrane fusion. Two major organelles are responsible for the recognition and transport of cargos targeted to the vacuole: the single-membrane multivesicular body (MVB) or prevacuolar compartment (PVC) and the double-membrane autophagosome. Here, we will highlight recent discoveries about MVB/PVC-mediated and autophagosome-mediated protein trafficking and degradation, and will pay special attention to a possible interplay between the endocytic and autophagic pathways in regulating vacuolar degradation in plants. Addresses 1 School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China 2 CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518057, China

(PVC) by different sorting machineries [3–5] (Figure 1). The endosomal sorting complexes required for transport (ESCRT) machinery is one well-known representative, which mainly functions in the endocytic pathway to sort membrane proteins internalized at the PM for degradation in the vacuole [6]. Macroautophagy (hereafter as autophagy) is another evolutionary conserved process for cellular bulk degradation and nutrient sequestration (Figure 1). Autophagy is induced during starvation or other stress conditions but also occurs at a basal level for routine turnover of unwanted or damaged intracellular constituents (including proteins and organelles) [7]. During autophagy, cargo is sequestered into a double membrane structure called an autophagosome, which subsequently fuses with the endosome/ vacuole to become the autophagic body and finally breakdown in the vacuole [7,8].

Edited by Hiroo Fukuda and Zhenbiao Yang

In this review, we summarize and highlight the recent advances in our understanding of vacuolar degradation as mediated by two MVB/PVC-related regulators (ESCRTs and Rab GTPases) as well as by autophagy. We then discuss the possible interplay between the conventional endocytic-based membrane trafficking system to the vacuole and autophagy in regulating vacuolar degradation in plants.

http://dx.doi.org/10.1016/j.pbi.2015.08.010

Part 1: conserved and unique aspects of ESCRTs and Rab GTPases in MVB/PVC– vacuole trafficking in plants

Corresponding author: Jiang, Liwen ([email protected]) 3 These authors contributed equally to this work.

Current Opinion in Plant Biology 2015, 28:39–47 This review comes from a themed issue on Cell biology

1369-5266/# 2015 Elsevier Ltd. All rights reserved.

A set of evolutionarily conserved molecular machinery has been reported to maintain and regulate membrane trafficking in plant cells. In the following we will highlight the recent findings related to the conserved and unique functions of ESCRTs and Rab GTPases in MVB/PVC– vacuole trafficking in plants, as summarized in Table 1a.

Introduction Cells use sophisticated mechanisms to target materials to different places or compartments through tightly controlled processes, which play pivotal roles in basic cellular activities as well as development and environmental responses. Usually, vacuoles are considered as the terminus of catabolic pathways in plant cells after membrane-bound materials are sorted through the endomembrane trafficking system [1,2]. In addition to the conserved lytic function shared with non-plant cells, the plant vacuole harbors distinctive features including the storage of proteins and sugars, the balance of cell volume, and defense responses. Before cargos are delivered into the vacuole, sorting events are active in the trans-Golgi network (TGN) and further in the multivesicular body (MVB)/prevacuolar compartment www.sciencedirect.com

ESCRT-dependent formation of intraluminal vesicles of MVBs/PVCs In eukaryotic organisms, the conserved ESCRT machinery is responsible for sequestering ubiquitinated membrane cargo molecules into the intraluminal vesicles (ILVs) of MVBs/PVCs for subsequent delivery into the lumen of the vacuole. In yeast and mammalian cells, the core ESCRT complex consists of five distinct parts, known as ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III and the vacuolar protein sorting4 (VPS4) complex [6]. Ubiquitinated cargoes are initially recognized by ESCRT-0 and passed from one subcomplex to the next. In plant cells, several putative homologs of ESCRT components have been identified, including ESCRT-I, ESCRT-II, ESCRT-III and VPS4 Current Opinion in Plant Biology 2015, 28:39–47

40 Cell biology

Figure 1

PM (i)

TGN

Vacuole (ii)

MVB/PVC

(iii) Golgi

Autophagosome FREE1 SH3P2 Rab7 Rab5

ER

MON1-CCZ1 Current Opinion in Plant Biology

Schematic model of membrane trafficking pathways for vacuolar degradation. (i) The vacuolar trafficking pathway: Rab5 recruits the MON1-CCZ1 complex and then this complex activates Rab7 to mediate MVB/PVC maturation; (ii) Plant-unique vacuolar trafficking pathway from the TGN to the vacuole: at least two other plant-unique routes exist: one depends on Rab5 only, the other one depends on AP-3, but not Rab5 or Rab7; (iii) Autophagosome–vacuole pathway: SH3P2 is recruited onto the autophagosome membrane to regulate autophagosome formation, and finally autophagosomes fuse with the vacuole to deliver the cargo into the vacuole for degradation.

complex [9–11,12,13,14]. Consistent with other organisms the ESCRT III complex was also found to regulate internal vesicle formation of MVBs/PVCs in plants [10]. The expression of several dominant negative mutants of ESCRT-III subunits (VPS20.1-1-112, SNF7.1 [L22W],

Table 1 Representative regulators in MVB/PVC-mediated and autophagosome-mediated vacuolar degradation a. Conventional trafficking regulators in MVB–vacuole pathway ESCRTs FREE1 Gao et al. [12,13] FYVE1 Kolb et al. [14] ESCRTIII Cai et al. [10] AMSH3 Katsiarimpa et al. [16]; Isono et al. [15] Rabs,GEFs

Rab7, MON1/CCZ1 Rab7, SAND Rab7, SAND/CCZ1

Cui et al. [19] Singh et al. [17] Ebine et al. [18]

b. Conventional regulators that involved in autophagy pathway ESCRTs FREE1 Gao et al. [13] CHAM1 Spitzer et al. [37] VPS2 Katsiarimpa et al. [53] AMSH1 Katsiarimpa et al. [53] Rabs,SNAREs RabG3b Kwon et al. [57] VTI11, VTI12 Sanmartin et al. [58] Retromers VPS35 Munch et al. [64] Exocysts EXO80B1 Kulich et al. [48] Others SH3P2 Zhuang et al. [31] ATI1 Honig et al. [45]; Michaeli et al. [46] TSPO Vanhee et al. [43]; Hachez et al. [44]

Current Opinion in Plant Biology 2015, 28:39–47

VPS24.1-1-152 and VPS2.1-1-179) has been shown to reduce the number of ILVs in PVCs/MVBs and to affect membrane protein degradation [10]. Similarly, overexpression of SKD1 (E232Q), a dominant negative VPS4 homolog mutant, also induced PVC/MVB enlargement, which contained a reduced number of internal vesicles [9]. In plant cells, orthologs of ESCRT-0 subunits and the ESCRT-I component Mvb12 are missing, which indicates that plants have evolved unique ways to recognize ubiquitinated cargo and assemble the ESCRT complex to mediate ILV formation in MVBs/PVCs. Recent reports have identified FREE1 (FYVE domain protein required for endosomal sorting 1) as a unique plant ESCRT component, which binds ubiquitin and specifically interacts with Vps23 (a component of the ESCRT-I complex) allowing it to be incorporated into the ESCRT-I complex. Consequently, in free1 mutants, endocytosed plasma membrane proteins destined for degradation were trapped at the tonoplast, instead of gaining access to the vacuole lumen [12,13,14]. The FREE1 mutation also resulted in defects in the formation of ILVs in MVBs. The above evidence indicates that FREE1 may serve as a novel ESCRT component for MVB/PVC biogenesis and vacuolar sorting of membrane proteins in plants. In addition, another new player AMSH3 (Associated Molecule with the SH3 Domain of STAM 3), which interacts with ESCRTIII subunits VPS2.1 and VPS24.1, also plays an essential role in the degradation of ubiquitinated membrane proteins in plant cells [15,16]. Due to its defects in ESCRT mediated intracellular trafficking, AMSH3 mutation or expression of a dominant-negative AMSH3 (AXA) mutant www.sciencedirect.com

Endocytosis and autophagy crosstalk Zhuang et al. 41

causes the accumulation of ubiquitinated proteins. The above evidence demonstrates that while plants also possess the ESCRT machinery for the formation of ILVs in MVBs/ PVCs, they have acquired in the course of evolution some plant-specific components.

Rab GTPase in MVB/PVC maturation In plant cells, the specificity of MVB/PVC–vacuole fusion is thought to be mediated by two Rab GTPases: Rab5 and Rab7, which localize on MVBs/PVCs and tonoplast respectively. Recent studies pointed out that both Rab5 and Rab7 play conserved and unique roles in regulating vacuolar protein trafficking [17,18,19,20]. In Arabidopsis, MVB/PVC localized Rab5 first recruits a MON1 (SAND)-CCZ1 heterodimer, which also serves as a Rab7 guanine nucleotide exchange factor (GEF). Rab5 is then replaced by Rab7 to form a Rab7-positive MVB/PVC. After the maturation, MVBs/PVCs fuse with vacuoles to deliver their contents to vacuole lumen for degradation [17,19]. This process therefore differs from one in yeast, in which the MON1-CCZ1 complex mediates the maturation of Rab5-positive early endosomes to Rab7positive late endosomes. Thus, while a Rab5–Rab7 conversion is evolutionary conserved, in plants this step occurs later in post-Golgi trafficking to the vacuole. In addition to a route to the vacuole requiring the sequential action of Rab5 and Rab7, other routes have also been identified in plant cells. For example, the vacuolar transport of the SNARE protein, SYP22 has been shown to be Rab5-dependent/Rab7-independent, while the correct targeting of another tonoplast localized SNARE VAMP713 needs neither Rab5 nor Rab7, but is dependent on the AP3 complex [18]. These evidences conclude that in plants the conserved Rab GTPases play some unique roles in vacuolar trafficking pathway compared with the ones in non-plant organisms.

Part 2: machinery for the autophagy–vacuole pathway: ATG proteins and non-ATG proteins Autophagy typically starts with a preautophagosomal structure (PAS), which then grows and expands into a double membrane structure termed the autophagosome to deliver degradable cargo into the vacuole [21]. The identification of several conserved autophagy genes in plants has led to the notion that autophagy also plays an important role in numerous plant physiological and pathophysiological processes, such as cell development, senescence, programmed cell death, and pathogen infection [7]. To date, about 40 ATG proteins have been identified in yeast and mammalian cells [21,22]. Among them, the core autophagy machinery is comprised by five functional complexes: the ATG1 complex, the ATG9 complex, the PI3K complex and two conjugation systems including ATG5/ATG12/ATG16 and ATG8. In plants, the majority of the ATG proteins are found to perform conserved roles www.sciencedirect.com

during either basal or stress-induced autophagy as well as in immunity-triggered autophagy [23–25,26,27]. These initial studies have paved the way for our current investigations in plant autophagy, but have also revealed gaps in our knowledge of plant autophagosome-mediated vacuolar degradation. For example, homologs of several key players, including ATG14 that is essential for autophagosome initiation in yeast and mammals, have not as yet been identified in plants. Research on yeast and animal cells suggests that autophagosomes may origin from multiple organelles, including the ER, Golgi and endosomes [28–30]. But currently, the evidence for possible membrane sources for autophagosome formation in plant cells is limited. Recently, by using a GFP fusion reporter (SH3P2-GFP), a conserved model for the sequence of autophagosome formation has been documented that includes an omegasome-like structure or isolation membrane, the encircling compartment eventually leading to the complete double membrane ring-like structure [31,32]. It is very likely that autophagosomes originate from the ER in plant cells, as SH3P2-labelled autophagosomes are often observed with close connection with the ER membrane. SH3P2 may perform similar function to mammalian Bif-1 protein [33,34], in that both have an N-terminus BAR (Bin/ Amphiphysin/Rvs) and C-terminus SH3 (Src Homology 3) domain. In addition, SH3P2 may help to assemble and integrate other non-ATG systems, as it interacts with ATG8 and the ESCRT component FREE1 [13], which we will discuss further in the following section. Thus, SH3P2 probably provides the driving force for the drastic membrane constraints during autophagosome formation, while at the same time recruiting the endocytic machinery via FREE1 for the expansion or maturation of autophagosome. Further support for an ER origin for the phagophore comes from the observation that ATG5 is also dynamically connected to the ER membrane [35]. Threedimensional reconstructions of the growing phagophore by dissecting the connection of ATG5 with either autophagosomal or ER markers reveals that ATG5 decorates the high-curvature edge of the PAS anchored to the ER. It has therefore been suggested that the asymmetric nature of the ER–phagophore interaction would in turn provide the initial polarity required for the establishment of membrane curvature for the growing phagophore. However, how SH3P2 and ATG5 are recruited to the ER remains unknown. Further investigations on the identification of the regulators that are involved in this initiation step are urgently required to fulfill this gap. On the other hand, evidence is accumulating in support of a conserved and unique role for autophagy in regulating specific cargo for vacuolar degradation in plants, which includes ER, mitochondria, peroxisomes, plastids as well Current Opinion in Plant Biology 2015, 28:39–47

42 Cell biology

as pathogens [36,37,38,39,40]. Such cargo molecules seem to be recognized by an autophagic receptor or ATG8-interacting-motif (AIM) containing regulator protein [8,41,42]. Autophagic receptor or AIM-containing proteins usually interact with other proteins at the target sites for ATG8 tethering in order to be degraded in the vacuole, or function as a bridge with other specific autophagy regulator(s). For example, ATG11, despite its canonical role in autophagy, has been shown to participate in selective mitophagy via interactions with ATG1 and ATG8 [38]. Also, a multi-stress regulator TSPO, with a preferential heme binding ability, contains a canonical AIM motif [43]. Recently, it was found that TSPO interacts with PIP2;7, a plasma membrane aquaporin, to negatively regulate its degradation by autophagy, which in turn limits tissue dehydration during exposure to stress conditions [44]. Another ATG8-interacting protein named ATI1, which has been shown to localize to the ER-associated and plastid-associated bodies, is responsible for unnecessary ER and plastid components for degradation in the vacuole via autophagy [45,46]. It has also been reported that ADI3, a suppressor of cell death that binds to the resistance protein Pto and the Pseudomonas syringae effector protein AvrPto, interacts with the ATG8h in controlling programmed cell death [47]. Interestingly, an Arabidopsis exocyst complex has also been predicted to possess consensus autophagyassociated AIM motifs and may be involved in autophagy [48–50]. Although no interaction data have been provided as yet, the exocyst subunit EXO70B1 has been reported to be involved in autophagy-related transport to the vacuole [48]. Thus, these AIM motif containing proteins may serves as a link between the autophagic machinery and specific cargo/ regulators in autophagy, as summarized in Table 1b.

Part 3: crosstalk between the autophagy and the MVB/PVC pathway: shared regulators for vacuolar trafficking In yeast and animal cells, although autophagosomes may directly fuse with the vacuolar membrane, they can also fuse with late endosomal compartments like MVB to become an amphisome for expansion or maturation before their fusion with vacuole/lysosomes, although the interplay between the autophagosome and late endosome is still not well understood [51,52]. In plants, similar fusion events have also been observed (Figure 2). Nevertheless, these fusion events require a certain amount of membrane/lipid as well as membrane remodeling modules to achieve the membrane deformation that is necessary for the dynamical morphological changes. A line of evidence in yeast and mammalian cells has shown that malfunction of some regulators in the conventional MVB– vacuole/lysosome pathway will have severe affects on autophagosome formation as well as autophagic activity [52]. Recently, several MVB/PVC-related regulators have been identified in regulating autophagy in plants (see Table 1b), linking the conventional membrane trafficking system with the autophagic pathway for vacuolar Current Opinion in Plant Biology 2015, 28:39–47

degradation. These include elements of the ESCRT machinery, membrane fusion factors like SNAREs and a small GTPase, as well as components of the retromer complex.

ESCRT machinery Recent evidence from different organisms including plants shows that a dysfunction of ESCRT machinery can affect autophagy, thus supporting the notion that autophagy may require functional ESCRT machinery activity. Most of the atg null mutants typically exhibit early leaf senescence but have no lethal phenotype [7]. A noticeable observation is that depletion of an AMSH3related deubiquitinating enzyme, AMSH1, which interacts with the ESCRT-III subunit VPS2.1, also causes early senescence and hypersensitivity to starvation conditions [53]. Furthermore, accumulations of autophagosomes in the cytoplasm with fewer autophagic bodies in the vacuole were found in their mutants. It is possible that dysfunction of AMSH1 and VPS2.1 may inhibit trafficking of autophagosomes to the vacuole by blocking autophagosome maturation. Another observation made with the mutant of one ESCRT-III component, CHMP1, shows that plastids display severe morphological defects and aberrant division, while degradation of plastid proteins and autophagosome formation are also impaired in chmp1 mutant [37]. These studies all provide strong evidence for a connection between the ESCRT machinery and autophagy. One plausible explanation is that the ESCRT machinery may be required for efficient membrane budding and scission processes for either ILV formation at MVBs or initiation of phagophores and closure of autophagosomes [6]. A further support comes from the identification of FREE1 as an ESCRT component unique to plant, which provides the first evidence for a direct crosstalk between the ESCRT machinery and autophagy [13]. In addition to an essential role in MVB/PVC–vacuole biogenesis and cargo degradation [12,14,54], FREE1 is involved in autophagy via a direct interaction with SH3P2, which binds to ATG8 and has been shown to actively participate in autophagosome formation [31]. It is suggested that FREE1 may function together with the PI3K complex via SH3P2, as pull down assays show that FREE1 precipitates together with SH3P2 and PI3K components. And similar to other ESCRT mutants, FREE1 depletion causes the accumulation of autophagosomes. A striking phenotype of the free1 mutant is the increased association between autophagosomes and MVBs/PVCs (Figure 2e), implying a possible defect during autophagosome and MVB/PVC conversion process.

Rab GTPase and SNARE Another unsolved question in regard to autophagosome– vacuole degradation is the tethering mechanism between the autophagosome membrane and endosome/vacuole. The fusion machinery is evolutionally conserved in plant www.sciencedirect.com

Endocytosis and autophagy crosstalk Zhuang et al. 43

Figure 2

WT (b)

(a)

WT

A

M V A

M

M

WT (d)

(c)

WT

V

V

A

mon1

free1 (f)

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M

M M

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Current Opinion in Plant Biology

Fusion among MVBs/PVCs, autophagosomes and the vacuole in plants. (a) Shown is a representative image of an autophagosome and a MVB captured in the moment of fusion (b), autophagosome and vacuole (c, d) in Arabidopsis thaliana root cells after autophagy induction and the fusion event between the MVB/PVC and autophagosome in free1 mutant (e) and enlarged MVBs/PVCs in mon1 mutant (f) as seen in ultra-thin sections cut from high pressure frozen/freeze-substituted samples. A, autophagsome; M, MVB; V, vacuole. Scale bars = 500 mm.

cells and includes both Rab GTPases and SNARE proteins, which determine the specificity of the fusion process between two membranes [55,56]. Although autophagic bodies, which formed after autophagosome fuses with vacuole, are often observed in plant cells (especially when vacuolar proteolytic activity is inhibited), the www.sciencedirect.com

SNARE machinery responsible for this process has not been elucidated. Rab7 functions in the maturation of endosomes and autophagosomes as well as their fusion step with the vacuole/lysosome in yeast and animals [56]. Consistently, Current Opinion in Plant Biology 2015, 28:39–47

44 Cell biology

it is known that RABG3b proteins colocalize with ATG8a proteins and RABG3bCA overexpression restores autophagic activity in the atg5-1 mutant [57]. This study implies that autophagosomes may fuse directly with the vacuole, whereas Rab7 functions as a common regulator for either the amphisome–vacuole or autophagosome–vacuole fusion event. However, whether Rab7 malfunction will affect the autophagosome fusing with the MVB/PVC or directly with the vacuole is still unclear, although the mon1 mutant, which disturbs the Rab5-to-Rab7 conversion activity, shows defects in MVB/PVC fusion with the vacuole by forming fragmented vacuoles and enlarged MVBs/PVCs (Figure 2f). In another study, it was shown that dysfunction of the SNARE proteins, VTI12 and VTI11, which affect trafficking into the vacuole, results in a similar phenotype to atg mutants like early leaf senescence [58,59]. Also, overexpression of PVA31, a VAMP (Vesicle associated membrane proteins)-associated protein, leads to early leaf senescence [60]. Therefore, it might be valuable to investigate whether these fusion factors are connected to the ATG system to be involved in autophagosome docking and fusion with endosome/vacuole.

system and the autophagic pathway in regulating autophagy–vacuolar degradation, which has been mainly supported by studies of ESCRT machinery and Rab small GTPases (Figure 3a). In plants, the role of endocytic pathway with an emphasis on the connection with the autophagy is just beginning. Here we have provided a short summary of recent work highlighting the common factors regulating the MVB/PVC–vacuole and autophagosome–vacuole pathways. These include the ESCRT machinery, Rabs and SNARE fusion factors, the ATG machinery and non-ATG machinery. We postulate that the autophagosome–endosome fusion may provide lipid as well as other regulators from other endosome to autophagosome, such as PI3P and endocytic machinery [51,52]. The finding of a direct interaction between FREE1 and SH3P2 provides strong evidence for linking Figure 3

(a)

Arabidopsis thaliana

Saccharomyces cerevisiae

VPS2 AMSH1 FREE1 CHMP1

Vps27 Hse1 Vps23 Vsp28 Vps37 Mvb12 Vps22 Vps25 Vps36 Vps20 Snf7 Vps24 Vps2 Vps4 Vta1

Retromer complex Another piece of evidence for crosstalk between the autophagy and the MVB/PVC pathway comes from a recent study of the retromer machinery. Retromer is an evolutionally machinery for retrograde transport from the endosomes [61]. Plant retromer has been shown to colocalize with MVB/PVC markers, while VPS29 and VPS35A have been shown to play a pivotal role in PVC morphology and the proper localization of Arabidopsis auxin transporter PIN1-GFP, suggesting a complexity of the retromer machinery in cellular trafficking [62,63]. Interestingly, it has recently been found that malfunction of VPS35, a retromer component, impairs autophagy-associated membrane trafficking of immune components, NB-LRR proteins [64]. Noticeably, VPS35 is mainly colocalized with other retromer component and the MVB/ PVC marker RABG3f. However, the question on retromer localization is controversial in plants [63], therefore it is still a question that whether this observation indicates that VPS35 functions predominantly in MVB/PVC alone to regulate autophagy or together with other retromer component(s). A recent study in mammalian cells shows that a Rab GTPase activating protein (GAP) TBC1D5, which interacts with retromer component VPS29, contains two AIM motifs to interact with ATG8 [65]. It is also observed that several retromer components as well as TBC1D5 mediate ATG9 trafficking during autophagy [66,67]. Nevertheless, further studies to investigate interaction partners that relate retromer to autophagy are needed to shed light on these possible connections.

Conclusion and perspectives Much evidence has accumulated in support of the crosstalk between the conventional membrane trafficking Current Opinion in Plant Biology 2015, 28:39–47

ESCRT

Rab5

?

Ypt51 Ypt52 Ypt53

Rab7

RABG3b

Ypt7

Home sapiens HRS STAM1, STAM2 TSG101 VPS28 VPS37A, VPS37B MVB12A, MVBB VPS22(EAP30) VPS25(EAP20) VPS36(EAP45) VPS20(CHMP6) SNF7A, B, C(CHMP4A, B, C) VPS24(CHMP3) VPS2(CHMP2A, B) VPS4(SKD1) VTA1 ALIX Rab5a Rab5b Rab5c Rab7a Rab7b

(b) Vacuole (b)

(a)

(c)

?

MVB/PVC FREE1

Autophagosome SH3P2

Rab7 Current Opinion in Plant Biology

Crosstalk between MVBs/PVCs, autophagosomes and the vacuole. (a). Comparison of shared ESCRT machinery and Rab GTPases in MVB–vacuole and autophagosome–vacuole pathways among Arabidopsis thaliana, Saccharomyces cerevisiae and Homo sapiens. (b) MVBs/PVCs and autophagosomes may deliver cargo into the vacuole via direct membrane fusion (a, b), both of which may be regulated by Rab7. On the other hand, autophagosome may fuse with the endosome for expansion/maturation, which is bridged by the ESCRT component FREE1 and the autophagy-related regulator SH3P2. The final fusion process between the amphisome and the vacuole might be mediated by Rab7 (c, question mark). www.sciencedirect.com

Endocytosis and autophagy crosstalk Zhuang et al. 45

the ESCRT machinery and autophagy pathway in plants. Interestingly, in mammalian cell, a recent study has also been reported that another ESCRT associated protein Alix interacts with ATG12/ATG3 [68], further supporting the direct interconnection between ESCRT and autophagy machineries. However, there are still many unsolved questions for our understanding of the underlying mechanism. Whether autophagosomes homotypically fuse with one another or engage in a maturation process by fusion with the MVB/PVC before being delivered to the vacuole remains to be seen (Figure 3). New approaches and tools like genetic screening are needed to identify specific regulators to connect endocytic and autophagic machinery. A recent study by suppressor screening of the lon2 mutant, a peroxisomal protease, demonstrates that autophagy is required for proper degradation of peroxisomes in a LON2 dependent-manner [69]. A forward genetic screen for mutants that suppress the seedling lethal phenotype of FREE1-RNAi transgenic plants has identified two putative sof mutants, which provide useful resources for the identification of possible new components associated with FREE1 [70]. Therefore, it is a future challenge to investigate the plant ATG machinery at the molecular level for interconnections with other membrane trafficking systems.

9.

Haas TJ, Sliwinski MK, Martinez DE, Preuss M, Ebine K, Ueda T, Nielsen E, Odorizzi G, Otegui MS: The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 2007, 19:1295-1312.

10. Cai Y, Zhuang X, Gao C, Wang X, Jiang L: The Arabidopsis endosomal sorting complex required for transport III regulates internal vesicle formation of the prevacuolar compartment and is required for plant development. Plant Physiol 2014, 165:1328-1343. 11. Reyes FC, Buono RA, Roschzttardtz H, Di Rubbo S, Yeun LH, Russinova E, Otegui MS: A novel endosomal sorting complex required for transport (ESCRT) component in Arabidopsis thaliana controls cell expansion and development. J Biol Chem 2014, 289:4980-4988. 12. Gao C, Luo M, Zhao Q, Yang R, Cui Y, Zeng Y, Xia J, Jiang L: A  unique plant ESCRT component FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol 2014, 24:2556-2563. This is the first study reporting plant unique ESCRT component FREE1 mediated internal vesicle formation of the MVB/PVC in plants. 13. Gao C, Zhuang X, Cui Y, Fu X, He Y, Zhao Q, Zeng Y, Shen J,  Luo M, Jiang L: Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc Natl Acad Sci U S A 2015, 112:1886-1891. This study demonstrated that the plant unique FREE1 serve as dual roles in both vacuolar protein transport and autophagic degradation pathways in plants.

Acknowledgements

14. Kolb C, Nagel MK, Kalinowska K, Hagmann J, Ichikawa M,  Anzenberger F, Alkofer A, Sato MH, Braun P, Isono E: FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol 2015, 167:1361-1373. The authors reported that FYVE1 played an important role in vacuole biogensis and intercelluar trafficking in plant.

This work was supported by grants from the Research Grants Council of Hong Kong (CUHK466610, 466011, 465112, 466613, CUHK2/CRF/11G, C4011-14R, and AoE/M-05/12), NSFC/RGC (N_CUHK406/12), the National Natural Science Foundation of China (31270226 and 31470294), the Chinese Academy of Sciences-Croucher Funding Scheme for Joint Laboratories, Shenzhen Basic Research Project (JCYJ20120619150052041), and Shenzhen Peacock Project (KQTD201101).

15. Isono E, Katsiarimpa A, Muller IK, Anzenberger F, Stierhof YD,  Geldner N, Chory J, Schwechheimer C: The deubiquitinating enzyme AMSH3 is required for intracellular trafficking and vacuole biogenesis in Arabidopsis thaliana. Plant Cell 2010, 22:1826-1837. This study showed the AMSH3 played an essential role for degradation of ubiquitinated membrane proteins in plant cells.

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