Endosome maturation
2011; Springer Nature; Volume: 30; Issue: 17 Linguagem: Inglês
10.1038/emboj.2011.286
ISSN1460-2075
Autores Tópico(s)Extracellular vesicles in disease
ResumoFocus Review31 August 2011free access Endosome maturation Jatta Huotari Jatta Huotari Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Search for more papers by this author Ari Helenius Corresponding Author Ari Helenius Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Search for more papers by this author Jatta Huotari Jatta Huotari Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Search for more papers by this author Ari Helenius Corresponding Author Ari Helenius Institute of Biochemistry, ETH Zurich, Zurich, Switzerland Search for more papers by this author Author Information Jatta Huotari1 and Ari Helenius 1 1Institute of Biochemistry, ETH Zurich, Zurich, Switzerland *Corresponding author. Institute of Biochemistry, ETH Zurich, ETH-Hoenggerberg, HPM E 6.3, Schafmattstrasse 18, Zurich CH-8093, Switzerland. Tel.: +41 44 632 6817; Fax: +41 44 632 1269; E-mail: [email protected] The EMBO Journal (2011)30:3481-3500https://doi.org/10.1038/emboj.2011.286 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Being deeply connected to signalling, cell dynamics, growth, regulation, and defence, endocytic processes are linked to almost all aspects of cell life and disease. In this review, we focus on endosomes in the classical endocytic pathway, and on the programme of changes that lead to the formation and maturation of late endosomes/multivesicular bodies. The maturation programme entails a dramatic transformation of these dynamic organelles disconnecting them functionally and spatially from early endosomes and preparing them for their unidirectional role as a feeder pathway to lysosomes. Introduction Endocytosis is the general term for internalization of fluid, solutes, macromolecules, plasma membrane components, and particles by the invagination of the plasma membrane and the formation of vesicles and vacuoles through membrane fission. In metazoan cells, endocytosed cargo includes a spectrum of nutrients and their carriers, receptor–ligand complexes, fluid, solutes, lipids, membrane proteins, extracellular–matrix components, cell-debris, bacteria, viruses, etc. By sorting, processing, recycling, storing, activating, silencing, and degrading incoming substances and receptors, endosomes are responsible for regulation and fine-tuning of numerous pathways in the cell. Having left endosome research after the early discovery period in the 1980s, and returning to it only recently, one of us (AH) has been impressed by the amount of information that has become available in the meantime on most aspects of endocytosis. Also, it is evident that the central role of endocytosis in cell life and pathogenesis is now much more fully appreciated. The reason for concentrating again on this topic is our interest in host cell entry of animal viruses, the majority of which enter cells by endocytosis and exploit endosomes and the endocytic pathways for penetration into the cytosol (Marsh and Helenius, 1989; Mercer et al, 2010). While virus particles themselves are relative simple and in many cases well characterized, the challenge is to understand the cellular processes used by them, and the responses of the cell to the invasion. Deeper knowledge of endocytosis is urgently required. Incoming viral particles also provide a tool to learn more about the endocytic machinery. In this review, we focus on a relatively narrow topic; the formation and maturation of late endosomes (LEs) in mammalian cells. They are also known as multivesicular bodies, because—although heterogeneous and variable in size and composition—most LEs have a multivesicular morphology, that is, they contain intralumenal vesicles (ILVs). There are many excellent reviews to recommend dealing with endosomes and different aspects of LE maturation. These include the following (Mellman, 1996; Zerial and McBride, 2001; Maxfield and McGraw, 2004; Piper and Katzmann, 2007; Luzio et al, 2007b; van Meel and Klumperman, 2008; Woodman and Futter, 2008; Saftig and Klumperman, 2009; Jovic et al, 2010; Von Bartheld and Altick, 2011). The logistics of the endosome system In a basic, ‘stripped-down’ representation, the classical endocytic pathway has only a few elements (Figure 1). The elements include a recycling circuit for plasma membrane components and their ligands, a degradative system for digestion of macromolecules, and a connecting, unidirectional feeder pathway for transport of fluid and selected membrane components from the recycling circuit to the degradative system. The feeder function is mediated by LEs. LEs also function as a system for mediating transport of lysosomal components from the trans-Golgi network (TGN) to lysosomes. This allows maintenance, diversification, and expansion of the recycling and degradative systems. Finally, the cytosol must be included among the essential elements, because it provides a spectrum of transiently associated, peripheral membrane components that support, regulate, and define the pathway (Figure 1). Figure 1.The basic elements of the endocytic machinery. The membrane organelles involve a recycling circuit (the plasma membrane (PM), the EEs, the recycling endosomes, and a variety vesicular carriers), a degradation cycle (lysosomes), and a connecting ‘feeder’ pathway (LEs) from the recycling circuit to the degradative system. The main interacting partner in the Golgi providing lysosomal components is the TGN, which communicates with the PM, EEs and LEs. The recycling circuit has functions independent of the degradative cycle. The degradative cycle is, in turn, a shared ‘facility’ for degradation in the cell and is not only used for substrates delivered via endosomes. The cytosol has a central role by providing peripheral proteins to all the membrane compartments. These proteins define functions such as molecular sorting, membrane fusion and fission, compartment identity, and organelle motility. Download figure Download PowerPoint Of the cargo internalized by ongoing endocytosis in mammalian cells, the majority is recycled back to the plasma membrane via early endosomes (EEs) (Figure 2). In typical mammalian cells, the equivalent of 50–180% of the surface area of the plasma membrane is cycled in and out of the cell every hour (Steinman et al, 1983). The amount of fluid internalized by macrophages corresponds to some 30% of cell volume per hour of which about two-thirds are returned to the extracellular space within about 10–15 min (Steinman et al, 1976, 1983; Besterman and Low, 1983). Figure 2.The endosome/lysosome system. The primary endocytic vesicles deliver their contents and their membrane to EEs in the peripheral cytoplasm. After a period of about 8–15 min during which the EEs accumulate cargo and support recycling to the plasma membrane (directly or via recycling endosomes in the perinuclear region), conversion of the EEs to LE takes place. Thus, as the endosomes are moving towards the perinuclear space along microtubules (MT), the nascent LE are formed inheriting the vacuolar domains of the EE network. They carry a selected subset of endocytosed cargo from the EE, which they combine en route with newly synthesized lysosomal hydrolases and membrane components from the secretory pathway. They undergo homotypic fusion reactions, grow in size, and acquire more ILVs. Their role as feeder system is to deliver this mixture of endocytic and secretory components to lysosomes. To be able to do it, they continue to undergo a maturation process that prepares them for the encounter with lysosomes. The fusion of an endosome with a lysosome generates a transient hybrid organelle, the endolysosome, in which active degradation takes place. What follows is another maturation process; the endolysosome is converted to a classical dense lysosome, which constitutes a storage organelle for lysosomal hydrolases and membrane components. Download figure Download PowerPoint One of the consequences of the active recycling is that transport to lysosomes via LEs is a side pathway limited to a relatively small fraction of internalized fluid and especially membrane components. To enter this side pathway, membrane components undergo stringent selection so that only a specific cohort is transported to lysosomes and degraded. The majority of large particles (such as viruses and ILVs) are also targeted to LEs. The bulk fluid and solutes diverted into this side pathway are not specifically sorted. In addition to ferrying cargo for degradation, LEs transport new lysosomal hydrolases and membrane proteins to lysosomes for the maintenance and amplification of the degradative compartment. Lysosomes depend on the influx of new components, because without incoming endosomal traffic, they loose their intactness, acidity, and perinuclear localization (Bucci et al, 2000). It is evident that the magnitude of the side pathway from EEs to lysosomes is under regulation through some of the cargo. Thus, formation of LEs and inward vesiculation to form ILVs has been shown to increase upon signalling via growth factor receptors. This suggests that the cell adjusts the use of this side pathway according to need (White et al, 2006). In doing so, it probably also adjusts the size of the degradative compartment. How such adjustment is achieved and which factors influence it, is an interesting question that deserves careful study. Unlike the secretory pathway, the endocytic pathway has the advantage that the starting compartment, the extracellular space, is open and accessible. This means that a variety of ligands, fluid, solutes, and particles can be added to cells, and their fate after endocytosis followed in different ways. In mammalian tissue culture cells, the most commonly used cargo markers today are transferrin (Tf) and its receptor (TfR), which faithfully follow the recycling pathway, and epidermal growth factor (EGF) and its receptor (EGFR), which after ubiquitination of the receptor's cytosolic domain and inclusion in ILVs take the pathway to lysosomes for inactivation and degradation. Fluid uptake is usually followed using fluorescently-labeled dextran and other fluorescent solutes that do not adsorb to the cell surface. The membrane as such can be followed using fluorescent lipid markers (Maier et al, 2002). Viruses and bacterial toxins are also useful tools. In addition to tissue culture cells, the most valuable system for endocytosis studies has been yeast, which seems to have endosomal compartments comparable to animal cells (Lewis et al, 2000; Pelham, 2002). More recently, filamentous fungi such as Aspergillus nidulans, have proven to be excellent systems to study endosomes and their maturation (Penalva, 2010). Caenorhabditis elegans and Drosophila melanogaster also have an impact given the background of strong genetics and the possibility of in situ studies in a multicellular organism (Grant and Sato, 2006; Michelet et al, 2010; Poteryaev et al, 2010). Endocytosis in plant cells is actually quite active (Irani and Russinova, 2009). The most important difference is the apparent lack of an independent EE compartment. The functions of EEs are carried out by an organelle that combines EEs and the TGN (Dettmer et al, 2006; Niemes et al, 2010). Conceptually, the situation in plants and fungi implies that by participating actively in secretory functions, endosomes can be viewed as an extension of the TGN. Early endosomes EEs provide the starting point for LE maturation. Defined initially as the compartment that first receives incoming cargo and fluid (Helenius et al, 1983), EEs are now recognized as the main sorting station in the endocytic pathway. Exactly how EEs arise is not entirely clear, but the membrane and volume is mainly derived from primary endocytic vesicles that fuse with each other. EEs receive endocytic cargo not only through the clathrin-mediated pathway but several other pathways including caveolar-, GEEC-, and ARF6-dependent pathways (Mayor and Pagano, 2007). Typically, an EE accepts incoming vesicles for about 10 min during which time membrane and fluid is rapidly recycled away, while some of the incoming cargo is retained and accumulates over the lifetime of the EE to be included in the LEs (Maxfield and McGraw, 2004). Association of proteins from the cytosol to the cytosolic surface of the EE membrane defines many of its functional attributes. Rab5 is a key component together with its effector VPS34/p150, a phosphatidylinositol 3-kinase (PI(3)K) complex that generates the phosphoinositide (PI) PtdIns(3)P and thus helps to manifest the identity of the organelle (Christoforidis et al, 1999; Zerial and McBride, 2001; Behnia and Munro, 2005). Rab5 follows the endocytic membrane from the beginning through various stages of EE maturation, and is later the main regulator of the conversion to LEs. EEs communicate with the TGN through bidirectional vesicle exchange. The arrival of hydrolases gives them an initial degradative identity further strengthened during maturation of LEs. EEs are heterogenous in terms of morphology, localization, composition, and function (Miaczynska et al, 2004; Lakadamyali et al, 2006; van Meel and Klumperman, 2008). Most of them are relatively small and patrol the peripheral cytoplasm close to the plasma membrane through saltatory movement along microtubules (Nielsen et al, 1999; Hoepfner et al, 2005). The overall distribution of EEs is cell-type dependent. Individual EEs have a complex structure with tubular and vacuolar domains (Figure 3A). Most of the membrane surface area is in the tubules, and much of the volume in the vacuoles. The limiting membrane contains a mosaic of subdomains that differ in composition and function (Zerial and McBride, 2001). They include domains enriched in Rab5, Rab4, Rab11, Arf1/COPI, retromer, and caveolae (Vonderheit and Helenius, 2005; Rojas et al, 2008; Hayer et al, 2010). Many of the domains are located in the tubular extensions where they provide for molecular sorting and generate vesicle carriers targeted to distinct organelles, including the plasma membrane, the recycling endosomes, and the TGN (Bonifacino and Rojas, 2006) (Figure 2). Figure 3.Morphologies of endosomes and lysosomes at the ultrastructural level. (A) Electron micrographs of peripherally located EEs containing HRP-conjugated Tf. They contain vacuolar and tubular domains. Courtesy of Tooze and Hollinshead (1992). Electron micrographs of (B) EE with clathrin lattices and a few ILVs; (C) LE, containing numerous ILVs; (D) endolysosome, with partial electron dense areas; and (E) lysosomes, with electron dense lumen. Images are all from HeLa cells that had been processed for thin section EM. Scale bars in (A): 500 nm and (B–E): 100 nm. Figure 3A is reproduced with kind permission from Rockefeller University Press; © 2009 Rockefeller University Press. Originally published in J Cell Biol 118: 813–830. doi: 10.1083/jcb.118.4.813. Download figure Download PowerPoint The formation of ILVs begins already in EEs. For this the cytosolic surface of the EE membrane has characteristic ‘plaques’ containing clathrin and components of the endosomal sorting complex required for transport (ESCRT), machinery responsible for sorting of ubiquitinated membrane proteins into ILVs (Raiborg et al, 2002; Sachse et al, 2002) (Figure 3B). The lumen of the vacuolar EE domains often contains several ILVs; in HepG2 cells there are 1–8 ILVs (van Meel and Klumperman, 2008). EEs are weakly acidic (pH 6.8–5.9) (Maxfield and Yamashiro, 1987), and contain a relatively low Ca2+ concentration (Gerasimenko et al, 1998). The traffic between endosomes and the TGN is a continuously ongoing process that has been extensively studied. It is responsible for the delivery of lysosomal and removal of endosomal components during endosome maturation. It occurs at the level of EEs, maturing LEs, and possibly for some time after the fusion of LEs with lysosomes. At the endosome level, the sorting and vesicle formation for transport to the TGN depends on factors such as Rab7, Rab9, and the retromer complex (Bonifacino and Hurley, 2008; Pfeffer, 2009). The retromer is a multimeric complex composed of sorting nexins and other proteins recruited to the cytosolic surface of EEs and maturing LEs. LEs and lysosomes Mature LEs are typically round or oval and have a diameter of 250–1000 nm (Figure 3C). They have a low bouyant density, and a high negative surface charge (Bayer et al, 1998; Falguieres et al, 2008). The limiting membrane contains lysosomal membrane proteins such as LAMP1 and the lumen contains a complement of acid hydrolases. The lumen also has numerous ILVs (often up to ⩾30) with a diameter of about 50–100 nm. The pH ranges between pH 6.0–4.9 (Maxfield and Yamashiro, 1987). LEs formed in the peripheral cytoplasm move to the perinuclear area of the cell where they fuse with each other to form larger bodies and to undergo transient (‘kiss-and-run’) fusions and eventually full fusions with lysosomes and pre-existing hybrid organelles between endosomes and lysosomes (Luzio et al, 2007b) (Figure 2). By fusing with lysosomes, the LEs follow what is essentially a unidirectional, dead-end pathway. Whereas most of the components of LEs are degraded in the lysosomal environment, others are longer lived and contribute, as already mentioned, to the maintenance and generation of lysosomes. It has been suggested that a few components such as mannose-6-phosphate receptors, tetraspanins, and SNAREs may escape degradation through vesicle trafficking after LE fusion with lysosomes, but this pathway is not well characterized. To determine whether a multivesicular organelle is a LE, or a fusion product between LEs and lysosomes is not always straightforward because the hybrid organelles contain components from both fusion partners (Figure 3D and E). We will call these hybrid organelles endolysosomes, to distinguish them from classical dense, primary lysosomes and from lysosomes generated through fusion with phagosomes, macropinosomes, and autophagosomes. It is in the endolysosomes that most of the hydrolysis of endocytosed cargo takes place. The lysosomal compartment as a whole consists of a collection of vacuoles of heterogeneous composition, morphology, location, and density. The heterogeneity is due to the diversity of cargo and the existence of several feeder pathways of which the classical endosome pathway is a major one. Heterogeneity is also generated by the varying degree of cargo degradation within individual vacuoles, and by fusion events between the various vacuoles. The classical lysosomes of high buoyant density and high hydrolase content correspond to the end point of the degradation process. They serve in part as storage vacuoles for lysosomal components ready to be redeployed. These components include the hydrolases, the limiting membrane protected by LAMPs, and other substances resistant to degradation. They also contain slowly degraded lumenal lipids often present as multilamellar membrane whirls. Taken together, the EEs, LEs, endolysosomes, and lysosomes provide a dynamic and adaptable continuum (Von Bartheld and Altick, 2011). The pathway is elusive because the organelles are scattered, and undergoing continuous maturation, transformation, fusion, and fission. Specific protein and lipid components are only partially useful as molecular markers because the majority is either transiently associated with the organelles or follow the organelles through several steps of transformation. In the pathway, events occur, moreover, non-synchronously. A cohort of cargo molecules simultaneously internalized from the plasma membrane may, for example, arrive at a certain acidic pH value in a time window spread over hours (Kielian et al, 1986). The ambiguity, heterogeneity, and lack of synchrony in the endocytic system may, in part, explain a certain lack of common, universally agreed concepts and models for endosome function and maturation. Formation of LEs LEs are derived from the vacuolar domains of EEs. By itself this constitutes molecular sorting because in addition to a large fraction of the fluid, the vacuolar domains of EEs have a different composition than the tubules. In the lumen, they contain ligands dissociated from their receptors as well as proteins and solutes internalized as components of the bulk fluid. The ILVs, and other large particles such as incoming viruses, are also present in the vacuolar elements of EEs. The membrane probably contains most of the cholesterol and sphingolipid-rich lipid rafts, membrane protein aggregates, V-ATPases, clathrin, ESCRT complexes, and a selected spectrum of membrane proteins destined for degradation (Ukkonen et al, 1986; Mukherjee et al, 1999; Mukherjee and Maxfield, 2004). The formation of a new LE is preceded by the generation of a Rab7 domain (Rink et al, 2005; Vonderheit and Helenius, 2005). This leads to the transient formation of a hybrid Rab5/Rab7 endosome. As discussed below, Rab7 is recruited to the EE by Rab5-GTP. How the tubules and the rest of the EE domains are lost during the formation of an LE is not entirely clear, but there are two possibilities. Evidence obtained by analysing large, spherical, juxtanuclear Rab5-positive endosomes supports a mechanism in which Rab5 after recruiting Rab7 is converted to the GDP-bound form and dissociates together with its effectors. A process like this was observed by live microscopy (Rink et al, 2005). Rab5 was lost within a few minutes, and replaced with Rab7. Another mechanism envisages a fission event that separates parts of the hybrid endosome containing the nascent Rab7 domain from the rest. Gruenberg and Stenmark (2004) call the newly formed LEs endosomal carrier vesicles (ECVs), and suggest that these serve as transporters of cargo to a stable LE compartment before delivery to lysosomes. While following viruses after endocytosis in small peripheral endosomes by live microscopy, we found support for such splitting of the endosomes. We observed microtubule-dependent events in which the virus and the Rab7 domain separated from the rest of the endosome containing Rab5, Rab4, and Arf1 (Vonderheit and Helenius, 2005). Consistent with this, it has been shown that dynein-mediated pulling forces are critical for the separation of endosomal elements containing the recycling ligand Tf from the lysosome-targeted ligand EGF (Driskell et al, 2007). Similar observations were recently made by a group that proposed a role for dynamin in fission of LEs from EEs (Mesaki et al, 2011). It is possible that different mechanisms exist for the peripheral and perinuclear populations of EEs, with gradual maturation occurring in perinuclear endosomes and a fission-based mechanism in peripherally located, relatively small and motile endosomes. Maturation of LEs Regardless of the mechanism of initial formation, the newly formed LEs continue to undergo a multitude of changes (Table I). As a result, by the time they fuse with lysosomes some 10–40 min later, they have completed a remarkable transformation leaving them with few similarities to EEs. The maturation process involves exchange of membrane components, movement to the perinuclear area, a shift in choice of fusion partners, formation of additional ILVs, a drop in lumenal pH, acquisition of lysosomal components, and a change in morphology (Table I). The programme is closely coordinated and regulated by factors recruited to the surface of the limiting membrane from the cytosol. Table 1. The endosome maturation programme Rab switch. Rab5 is exchanged for Rab7. This switch reprograms the association of effector proteins from the cytosol and redefines many of the properties of the endosomes. Other Rabs, such as Rab4, Rab11, are Rab22 also lost, while Rab9 is added. Formation of ILVs. Ubiquitinated cargo recruits machinery from the cytosol (ESCRT and other factors) that induce inward-budding of the limiting membrane, and thus the formation of ILVs containing membrane proteins and lipids targeted for lysosomal degradation. Capacitation of microphagocytosis-like mechanisms for the inclusion of cytoplasmic proteins and RNAs, of ILV backfusion with the limiting membrane, and exosome release. Acidification. The lumenal pH drops from values above pH 6 to pH 6.0–4.9. PI conversion. PtdIns(3)P is converted to PtdIns(3,5)P(2), and some of the PtdIns(3)P is sorted into the ILVs. Change in size and morphology. The tubular extensions present on EEs are lost and the endosomes acquire a round or oval shape and grow in size. Loss of recycling with the plasma membrane. Recycling receptors are lost from the organelle and recycling of membrane and fluid to the cell surface stops. Gain of lysosomal hydrolases and membrane proteins. These lysosomal components are transported mainly from the TGN. Some of them are active already in the maturing endosomes. Switch in fusion specificity. The endosomes can no longer fuse with EEs. Instead, they acquire the necessary tethering complexes and SNAREs to fuse with each other, with lysosomes, and with autophagosomes. The conversion of CORVET to HOPS complex on membranes. The HOPS/CORVET complexes are involved in a number of processes, including membrane tethering, the Rab5/7 switch, and mediating SNARE assembly. A switch in cytoplasmic motility. The endosomes associate with a new set of microtubule-dependent motors that allow them to move into the perinuclear region of the cell. Changes in lumenal ionic environment. In addition to the drop in pH, there is an increase in Cl−, and changes in Ca2+, Na+, K+ concentrations. Change in temperature sensitivity. Unlike earlier steps in endocytosis, LE formation or their fusion with lysosomes is blocked at temperatures below 19–20°C. Decrease in buoyant density and increase in negative surface charge. These properties are used to isolate LEs. Before considering the conversion in more detail, it is important to ask why LEs are subject to such a dramatic transformation. What is the reason for this complex maturation programme? The general answer is that the programme is in place to close down recycling and other functions of EEs and to allow the union of LEs with the degradative compartment. Similar maturation using some of the same factors occurs in phagosomes, autophagosomes, and probably macropinosomes before they fuse with LEs and lysosomes (Eskelinen, 2008; Kinchen and Ravichandran, 2008; Kerr and Teasdale, 2009). Since lysosomes constitute a point-of-no-return for most macromolecules and lipids, the cargo contents of LEs must be narrowed down to molecules and particles that need to be degraded, and to cargo that the LEs and lysosomes require for their functionality. Some of the membrane-bound cargo, moreover, needs to be presented in a form easily digested by hydrolases, which may explain in part the formation and the composition of ILVs. The limiting membrane of the LEs must, on the other hand, be rendered resistant to the hydrolases by inclusion of lysosomal glycoproteins such as LAMPs. Moreover, interactions with the cytoskeleton have to be altered so that the LEs can move to the region of the cytoplasm where the lysosomes are localized. LEs also need new tethering factors and SNAREs to be able to fuse with each other, with lysosomes, and perhaps with macropinosomes and autophagosomes. In the following sections, we will focus on some of the events and factors involved in endosome maturation. These include the Rab GTPase switch, phosphatydylinositide conversion, Arf1/COPI association, ILV biogenesis, acidification, and LE motility. Finally, we will discuss some of the roles that LEs have beyond the degradative function. The Rab switch Rab GTPases, especially Rab5 and Rab7 (Vps21p and Ypt7p in yeast, respectively), provide the most important organelle identity markers and master regulators in the endocytic pathway. Rab5 itself, its guanine-nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), GDP dissociation inhibitors (GDIs), and GDI displacement factors (GDFs) as well as its effectors determine the functions of EEs (Chavrier et al, 1990; Ullrich et al, 1994; Rink et al, 2005; Jovic et al, 2010). Rab7 and its corresponding factors have a similar role in LEs and lysosomes (Chavrier et al, 1990; Meresse et al, 1995; Tjelle et al, 1996). Endosome maturation involves a conversion from Rab5 to Rab7 (Rink et al, 2005; Vonderheit and Helenius, 2005; Poteryaev et al, 2010). The conversion can be blocked by expressing a constitutively active mutant of Rab5 (Q79L), and by depletion of VPS39, a subunit of the HOPS complex (Rink et al, 2005). This results in the formation of hybrid endosomal compartments with markers for both EEs and LEs. These abnormal hybrid compartments seem to arise from homotypic fusions of EEs and heterotypic fusions with LEs or lysosomes, and are found to accumulate ILVs (Rosenfeld et al, 2001; Hirota et al, 2007; Wegner et al, 2010). Expression of the Rab5(Q79L) mutant results in sorting defects of both recycling and degradative cargo. Lysosome biogenesis is also affected as the amount of lysosomes in dense Percoll gradient fractions is greatly reduced (Rosenfeld et al, 2001). Initially, Rabex-5, a GEF for Rab5, is recruited to EEs where it activates Rab5 (Horiuchi et al, 1997; Barr and Lambright, 2010). Interestingly, in addition to its GEF activity, Rabex-5 also possesses ubiquitin (Ub) E3 ligase activity and can bind to ubiquitinated proteins, which is indeed required for its association with EE membranes (Mattera and Bonifacino, 2008). In the cytosol Rabex-5 itself is monoubiquitinated and it is thought that a cycle of Ub binding an
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