ESCRTing the RABs through conversion Open Access

Endosome maturation is a highly dynamic process in the endosomal system, which sorts and delivers cargoes received from the plasma membrane . During this process, early endosomes transform into late endosomes that fuse with a lysosome to degrade cargoes [1,2,3]. Early endosomes, which are Rab5 positive, have a close-to-neutral pH and exhibit a high amount of cargo and PI(3)P. During their maturation they will change their properties, morphology and localization in the cell. They tend to move during their maturation from the cell periphery to the center [4]. The emerging late endosomes are Rab7 positive and have a more acidic pH. They are characterized by a low amount of cargo on the limiting membrane, high levels of PI(3,5)P₂, and a high number of intraluminal vesicles (ILVs) inside [5,6]. These changes require several processes to take place: Rab conversion [5,7], acidification of the endosomal lumen [8], cargo sorting [9,10], ILV formation [11], cargo recycling [12,13], phosphosinositide phosphate (PIP) conversion [14], membrane tethering [5,15], and endosome motility [16] (Figure 1). Studies performed in our group demonstrated that endosomal acidification depends on Rab conversion, that Rab11-dependent recycling of cargo occurs before and after Rab conversion, and that efficient cargo sorting on the sorting endosome requires factors for endosome recycling and Rab interactions (FERARI) and a kiss-and-run mechanism [18-20]. In addition, the vacuolar tethering complex, homotypic fusion and protein sorting (HOPS) might be involved in the regulation of Rab conversion [21,22]. Furthermore, ESCRT-mediated ILV formation and Rab conversion are co-ordinated through at least four mechanisms: 1. endosomal cargo abundance, 2. the deubiquitinase of the ESCRT machinery USP8 which regulates the endosomal localization and activity of RABEX5, 3. His domain protein tyrosine phosphatase (HD-PTP) (an associated ESCRT factor) that binds to several ESCRT factors and RABAPTIN5, and 4. the binding of MON1/CCZ1 to PI(3)P and the subsequent displacement of RABEX5 [17,23-25] (Figures 1B and 2). For clarity, we will use the Homo sapiens nomenclature throughout this review; please consult Table 1 for alternative names in other species.

In this review, we will first summarize the current knowledge on ESCRT and the factors involved in Rab conversion. Then, we will discuss the role of ESCRT-mediated ILV formation and the MON1/CCZ1-driven Rab conversion during endosome maturation. Finally, we will propose a unified endosome maturation model, incorporating current mechanistic findings of the regulatory role of ESCRTs during the Rab5-to-Rab7 switch, and highlight remaining open questions in the field.

The ESCRT machinery consists of factors belonging to five distinct complexes (ESCRT-0 to ESCRT-III and Vps4) and several additional and associated factors (Figure 3) [27,28]. In Caenorhabditis elegans and H. sapiens over 20 and 30, respectively, different ESCRT factors are encoded in the genome (see Table 1). The five complexes can be functionally divided, in relation to their role during ILV formation on the endosome, into early-acting ESCRTs (0 and I) and late-acting ESCRTs (III and Vps4) (see Figure 3). In general, early ESCRTs are mainly involved in recognizing and corralling of the ubiquitin marked cargo, whereas late ESCRTs are mainly responsible for shaping the ILV through membrane bending and scission.

ESCRT-II is also often simply referred to as early-acting ESCRT because, like ESCRT-0 and ESCRT-I, it is a stable complex, has ubiquitin-binding capabilities and is recruited upstream of ESCRT-III [11,29,30].

ESCRT-mediated ILV formation starts at the early endosome, which receives endocytic cargo from the plasma membrane and where cargo is recognized by early ESCRTs (Figure 3) [10]. This recognition requires that the cargo is ubiquitinated and is mainly performed by ESCRT-0 and ESCRT-I, which have the most ubiquitin-binding sites. Both HRS and STAM (in ESCRT-0) can bind ubiquitin, while TSG101, MVB12 and UBAP1 (in ESCRT-I) also have ubiquitin-binding domains (Figure 3). Both complexes are also believed to associate into larger degradative domains with corralled cargo bound to them [31-38]. Cargoes that are not ubiquitinated avoid the degradative fate and will be recycled back to the plasma membrane or transported to other cellular compartments (see Figure 1) [11,13].

ESCRT-0 consists of HRS and STAM/STAM2 [39], and it recognizes ubiquitinated degradation cargo through its multiple binding domains and the concomitant detection of PI(3)P by the FYVE domain of HRS [33,40-42]. Interestingly, HRS is ubiquitinated itself by E3 ligases such as NEDD4 [43]. Once recruited, ESCRT-0 starts to concentrate the cargo in degradative subdomains and recruits further ESCRT factors [10] (Figures 2 and 3). One factor recruited to the early endosome at this point is the deubiquitinase USP8 [44]. The early role of USP8 is to deubiquitinate HRS to protect the whole ESCRT-0 from degradation [43,45]. This mechanism is an important regulator of the ESCRT-0 stability [17,24,44,46,47]. Interestingly, HRS does not seem to be the only ubiquitinated early ESCRT factor, as the ubiquitination of STAM, TSG101, and MVB12 has also been demonstrated [37,43,48] (reviewed in [49]).

TSG101 is recruited through ESCRT-0 and is a part of the heterotetrameric ESCRT-I, which consists of TSG101, VPS37, VPS28, and MVB12 or UBAP1 [35,50,51]. ESCRT-I, in general, is able to bind ubiquitin and support the cargo corralling initiated by ESCRT-0, but its specific properties are dependent on the combination of ESCRT-I isoforms incorporated [31,37,52-54]. For example, the use of an MVB12 isoform for the complex assembly adds the potential to bind to PIPs, whereas the incorporation of UBAP1 adds more potential ubiquitin-binding sites to the complex [35,37,55].

In addition to its function in cargo binding, ESCRT-I is involved in the recruitment of the heterotetrameric ESCRT-II [56,57]. This complex consists of EAP30, EAP20, and EAP45 and exhibits a conserved ‘Y’-shaped conformation [57-59]. The GLUE domain of EAP45 interacts with ubiquitin and a variety of PIPs, and the N-terminal basic helix domain of EAP30 has also an affinity to several different PIPs [57,60] (as shown in Figure 3). Moreover, this complex can interact with the ESCRT-III factor CHMP6 through EAP20. This in turn initiates the ESCRT-III filament assembly cascade required for the membrane bending during ILV formation [56,61,62].

The ESCRT-III filament assembly cascade consists of a series of orchestrated filament assemblies, disassemblies, and filament remodeling events facilitated by Vps4 activity [11]. Moreover, studies in yeast showed that USP8 interferes with the assembly/disassembly of ESCRT-III filaments to increase the potential time for cargo deubiquitination and is regulated by ESCRT-III [63-65]. Additionally, the USP8 deubiquitinase seems to be directly involved in filament formation because it actively deubiquitinates ESCRT-III factors to regulate their activities [66,67]. Studies published in recent years defined the dynamics of ESCRT-III filaments [68,69]. In studies performed with purified yeast proteins, it was shown that the formation of vesicles through pinch-off events mediated by ESCRTs requires a stepwise assembly and disassembly of ESCRT-III filaments, which is moderated by Vps4 using ATP [69, 70]. During this cascade of events, conformational changes will cause a reduction in autoinhibition, allowing interactions with the following ESCRT-III subunits [71,72]. The process is probably directional, and biochemical studies in yeast revealed the highly co-ordinated manner in which the core complex of CHMP6, CHMP4, CHMP3, and CHMP2 is assembled [72-74]. These processes support the disconnection of the formed vesicle from the surrounding membrane and recycle the involved ESCRT-III components [75]. It was revealed that the recruitment of ESCRT-III is also dependent on the curvature and tension of the target membrane [76-78] and the functions of ALIX [79-82] and HD-PTP [83,84].

Recently, several studies addressed the regulatory role of ESCRT-mediated ILV formation during endosomal maturation [17,23,24,85,86]. These insights will be discussed in more detail in a later section. For authoritative reviews about this topic, please read [29,72,87].

One of the first [88,89] and best-characterized [90] guanine nucleotide exchange factors (GEFs) is RABEX5 [91]. It belongs to the GEF family of Vps9-domain-containing proteins [92] and is recruited to the early endosome through an interplay of mechanisms where it promotes the recruitment of Rab5A [93]. To enable its own recruitment, RABEX5 contains different domains: zinc finger motif, ubiquitin interacting motif, membrane binding domain, helical bundle domain, Vps9 domain, coiled-coil domain, and proline-rich domain [24,94] (Figure 2A). One of the major recruitment mechanisms for RABEX5 is ubiquitin-dependent and mediated by the zinc finger and ubiquitin-interacting motif (see Figure 2B part I) [95-97]. The ubiquitin interaction is important for the recruitment of the GEF to endosomal compartments [17,26,93,98,99]. The early endosomal targeting domain, consisting of membrane binding and helical bundle domain, is also involved in the recruitment of RABEX5 (Figure 2A and B part I) [100,101]. Moreover, RABEX5 can also be recruited to the early endosome through an indirect mechanism mediated by RABAPTIN5 (also known as RABEP1) [100,102-105]. However, this mechanism is being called into question by recent findings [106] (reviewed in [107]). How all of these recruitment mechanisms interact with each other during RABEX5 recruitment is currently not really understood.

Once recruited onto the early endosome, RABEX5 in turn recruits Rab5 and activates it through its Vps9-like GEF domain [93,100,105,108,109] (Figure 2A). The activity of the Vps9 domain is regulated through inhibitory intramolecular interactions involving the ubiquitin-binding domains and the coiled-coil domain [100,105,109]. It has been shown that the inhibitory influence of the coiled-coil domain of RABEX5 can be reduced by interactions with RABAPTIN5 and that the binding of ubiquitin by the two ubiquitin-binding domains increases the GEF activity of RABEX5 [94,100,105,109]. Both mechanisms most probably contribute in vivo to keep the GEF activity of RABEX5 high at the early endosome and to recruit more Rab5 [18,93,106,109]. This leads to the formation of Rab5 nanodomains [110] and the recruitment of numerous effectors, such as the PI(3)P kinase VPS34 [111,112]. It was demonstrated that PI(3)P has a positive effect on the recruitment of Rab5A [113]. This observation fits very well with two other studies, showing that Rab5A GTP actively stimulates PI(3)P production by interacting with the PI (3)P kinase VPS34 complex II [114,115]. The positive feedback loop of Rab5 recruitment and activation, established in this way, must be interrupted in order to successfully switch an endosome from Rab5 to Rab7. Rab conversion mediated by MON1/CCZ1 serves this purpose [5,7].

This central step during endosomal maturation starts with the recruitment of the MON1/CCZ1 complex to the early endosome. It consists of MON1A or B and CCZ1 (short MON1/CCZ1) [25,116,117]. The complex binds to RABEX5 and PI(3)P by coincidence detection for its recruitment [25]. It was shown that the interaction of MON1 with Rab5 is also important for the recruitment of the complex and that the interaction with PIPs is mediated by a basic patch in MON1 that binds to negatively charged PIPs through electrostatic interactions [118-121] (Figure 2B part IV, Figure 1). Moreover, it has been shown that the complex has a third subunit in metazoans, called RMC1. The third subunit is possibly involved in the recruitment of the complex to endosomes [117,122-124]. This idea is further supported by the recently published cryo-electron microscopy structure of MON1-CCZ1-RMC1, which shows that RMC1 has a conserved basic patch which aligns with the lipid-binding site of MON1 and could, thus, potentially support the membrane binding of the trimeric complex [123-125].

After its recruitment and stabilization on the endosomal membrane, the MON1/CCZ1 complex interacts with RABEX5 to displace it from the membrane and interrupt the positive feedback loop that keeps Rab5 activated [25]. Simultaneously, the complex also acts as a GEF for Rab7 and activates Rab7 on the maturing endosome [25,126,127] (Figure 2B part IV, Figure 1).

This GEF function is highly conserved from yeast to man [128-131]. Moreover, the exact mode of action of the GEF was uncovered in the last years through studies with yeast proteins and is based on two effects of MON1/CCZ1 binding to Rab7, which modulate the nucleotide-binding pocket [7,118,129,132,133]. Surprisingly, the third subunit in metazoans seems to have no big influence on the GEF activity of the complex in vitro [118,122]. Thus, the third subunit might serve to stabilized MON1/CCZ1 on endosomal membranes.

An important mechanism for Rab5 to Rab7 conversion is also GTP hydrolysis by Rab5 promoted by GTPase-activating proteins (GAPs) that will lead to Rab5 on membranes to be extracted by the GDP dissociation inhibitor (GDI) [92,134,135]. In a recently published study, it could be shown that MON1/CCZ1 indirectly recruits the novel Rab5 GAP, TBC1D18 (also called RABGAP1L), to endosomes during Rab conversion. Hereby, it supports the dissociation of Rab5 and promotes the Rab switch [1]. In addition, Rab5 is regulated by RabGAP-5, which limits the amount of activated Rab5 and thereby regulates the homotypic fusion and endosomal traffic. This suggests that this GAP is crucial for the function of Rab5 and the subsequent Rab5 to Rab7 conversion [136].

ESCRT-mediated ILV formation and Rab conversion during endosome maturation have to be performed in a way that ensures that the degradation cargo is removed from the limiting membrane and deposited in ILVs before the late endosome finally fuses with a lysosome to degrade the cargo [5]. This basic requirement implies that both processes should be co-ordinated for a successful endosome maturation.

The early endosome receives ubiquitinated cargo from the plasma membrane, which accumulates over time and is bound by ESCRT-0 and additional factors. One of these factors is the Rab5GEF RABEX5, which in turn recruits Rab5 and further factors that cause an increase in the PI(3)P concentration in the endosomal membrane. This, together with the high amount of ubiquitinated cargo, enhances the recruitment of ESCRT-0 factors to early endosomes [17,20,137-140].

Once recruited, ESCRT-0 starts to concentrate the cargo on the endosome in specific degradative subdomains [17,32,141-143]. As a consequence of cargo corralling and the recruitment of ESCRT-I to these domains [32,143], the amount of unbound cargo on endosomes is diminished [17] (Figure 2B). This effect is reinforced by the fact that the early endosome stops accepting new cargo from the plasma membrane at a certain point [2,144] and that non-ubiquitinated cargo is recycled either to the plasma membrane or to the trans-Golgi network [10] . Colocalization studies with Rab5 and Rab7 suggest that early ESCRT-mediated cargo corralling is mainly upstream of Rab conversion [17]. Indeed, ESCRT-0 factor HRS preferentially colocalizes with Rab5, and not with Rab7 [17,137,145], and acts mechanistically upstream of MON1/CCZ1 [17]. RABEX5 is dependent on the abundance of ubiquitinated cargo in order to be localized effectively to endosomes [26]. Therefore, cargo corralling by early ESCRTs destabilizes the endosome binding of RABEX5, which in turn supports its displacement by MON1/CCZ1. The presence of RABEX5 and high amounts of PI(3)P on the endosome facilitate MON1/CCZ1 binding, thereby causing Rab7 activation and initiating Rab conversion. Conversely, early ESCRT knockdowns cause the formation of enlarged Rab5-, RABEX5-, ubiquitin- and HRS-positive structures, suggesting that under these conditions, cargo corralling is impaired and RABEX5 remains fairly stably bound to the endosome and cannot be displaced by MON1/CCZ1. In later ESCRT knockdowns, a high colocalization of Rab5 with Rab7 on enlarged ubiquitin-positive structures indicates that the Rab switch could be initiated but not completed because cargo corralling is less efficient and RABEX5 could not be fully displaced [17]. In fact, several publications have already shown that ESCRT deficiencies lead to the formation of enlarged early endosomal structures and hinder endocytic flow [24,146-150]. These structures cannot successfully undergo Rab conversion and, therefore, cannot mature. These conclusions are further supported by the observation of synthetic lethality between mon1(KO) and several ESCRT knockdowns in C. elegans, indicating a connection between ILV formation and Rab conversion [17]. A reduction in the amount of ubiquitin in a mon1(KO) strain reduces the size of early endosomes and causes a partial recruitment of Rab7 to endosomes, even in the absence of its canonical GEF. Together, these data indicate that RABEX5 binding to the endosome is largely driven by its capacity to bind ubiquitin. Consistently, a rabex5 knockdown has a similar effect as the ubiquitin reduction in this strain, and Rab7 is recruited to endosomes [17]. Thus, the amount of ubiquitinated cargo and the function of early ESCRT regulate the timing of Rab conversion. This co-ordination mechanism may explain why EGF treatment, which increases the concentration of EGFR on endosomes, delays Rab conversion in tissue culture cells [151].

USP8 is a deubiquitinase that functions at many stages during endosome maturation. An early role of USP8 is to deubiquitinate HRS, which was previously ubiquitinated by E3 ligases [43,45]. Therefore, without USP8 to protect ESCRT-0 from degradation, early cargo corralling and the very start of endosome maturation would become impossible [17,24,44,46,47]. The localization of USP8 to activated receptors on endosomes is dependent on the large HD-PTP protein [23,84,152]. On the other hand, USP8 also interacts with several late ESCRTs to regulate the assembly/disassembly of ESCRT-III filaments to increase the potential time for cargo deubiquitination [63-65]. USP8 is also directly involved in filament formation by actively deubiquitinating ESCRT-III factors to regulate their activities [66,67]. In addition, USP8 binds directly to and drives the deubiquitination of cargo [84, 153] (Figure 2 and 3).

Intriguingly, USP8 is also involved in other aspects of endosome maturation, namely Rab conversion. It was found to be recruited to Rab5-positive endosomes and USP8 mutants accumulated enlarged early endosomes [24]. Additionally, USP8 binds to the N-terminal Zink Finger-ubiquitin binding motives and the C-terminal coiled-coil of RABEX5 (Figure 2B part III), and this recruitment to early endosomes causes the dissociation of RABEX5. Specifically, the deubiquitination of position K323 on C. elegans RABEX5 leads to its disconnection from endosomes. Last but not the least, MON1/CCZ1 recruitment to endosomes is dependent on USP8 [24]. Taken together, these observations suggest a role of USP8 in the spatiotemporal regulation and co-ordination of ESCRT-driven ILV formation and Rab conversion, even while opening many new questions about the exact mechanisms underlying the different functions of deubiquitination in these processes.

RABAPTIN5 plays a crucial role in keeping RABEX5 active on endosomal membranes. It has been shown to bind to the inhibitory coiled-coil domain of RABEX5, thereby keeping its GEF domain active [105]. Therefore, RABAPTIN5 is an ideal target for RABEX5 regulation. In a separate role from binding USP8, HD-PTP also connects RABAPTIN5 directly to the ESCRT machinery [23]. This connection opens up new avenues to regulate and co-ordinate the two branches of endosome maturation. HD-PTP seems to play an inhibitory role in Rab5 activation, since its depletion enhances Rab5 activity on endosomes. HD-PTP binds initially to the ESCRT-0 subunit STAM2 [84] (Figure 2B part II). Subsequently, HD-PTP interacts with ESCRT-I [83], and in a third step, the Bro1 domain switches from binding ESCRT-0 to binding CHMP4, the first filament forming subunit of ESCRT-III [83]. Finally, direct binding of the Bro1 domain of HD-PTP to RABAPTIN5 was found to be responsible for the down-regulation of RABEX5 activity [23] (Figure 2B part III and IV). This mechanism offers the opportunity for co-ordination inside the ILV formation pathway and regulation of Rab conversion.

The ESCRT-mediated ILV formation and Rab conversion are two important processes during endosome maturation whose co-ordination is based on three different mechanisms. One mechanism is founded on the amount of ubiquitinated degradation cargo on the endosomal surface. This cargo stabilizes RABEX5 on the endosomal membrane and prevents Rab conversion. The ESCRT machinery reduces the amount of cargo during endosome maturation through cargo corralling and ILV generation. This action destabilizes RABEX5 endosome association and allows the displacement by MON1/CCZ1 [17].

Another mechanism is based on the deubiquitinase USP8. This ESCRT factor is recruited to the early endosome through RABEX5 and deubiquitinates the Rab5GEF. This deubiquitination together with the supported recruitment of MON1/CCZ1 causes the dissociation of RABEX5 and allows Rab conversion [24].

In the third mechanism, HD-PTP and RABAPTIN5 are the key players, which influence the association of RABEX5 to the endosome. On the early endosome, HD-PTP binds to ESCRT-0 and ESCRT-I factors and is not available for interactions with RABAPTIN5. During maturation, ESCRT-0 leaves the endosome and ESCRT-III factors get recruited. This causes a binding switch for HD-PTP from ESCRT-0 to ESCRT-III. When early ESCRT-III factors leave the endosome and later factors are recruited, HD-PTP is released and can interact with RABAPTIN5. This interaction interferes most likely with the interaction of RABAPTIN5 and RABEX5 and indirectly promotes Rab conversion [23].

Therefore, it appears that cells have found several ways to co-ordinate ESCRT-mediated ILV formation and Rab conversion. We propose a model taking all three processes into account (Figures 1 and 2). The idea behind this unification was to create an overview of the co-ordination of ESCRT-mediated ILV formation and Rab conversion. The model is also intended as a starting point for further investigations. Given the multiple roles of USP8 and HD-PTP on endosomes, the question arises about how they are regulated. In addition, we do not know whether all these mechanisms have the same relevance in all cell types and organisms.

The authors declare no competing interests.

The work was supported by the Swiss National Science Foundation [310030_197779, 320030_231859 to A.S.] and the University of Basel.

Conceptualization: JAS,DPO, AS; Supervision: AS; Funding aquisition: AS; Visualization: DPO, JAS; Writing-originaldraft: JAS, DPO, AS;

Writing-review & editing: JAS, DPO, AS

We acknowledge the financial support by the Swiss National Science Foundation. We sincerely apologize to all whose valuable work we could not include due to space limitations.

ELR

endocytic lysosome reformation

ESCRT

endosomal sorting complex required for transport

FERARI

factors for endosome recycling and Rab interactions

GAP

GTPase-activating protein

GEF

guanine nucleotide exchange factor

HD-PTP

His domain protein tyrosine phosphatase

HOPS

homotypic fusion and protein sorting

ILV

intraluminal vesicle

MVB

multivesicular body

PIP

phosphosinositide phosphate