Early Endosomal Compartments

Early Endosomal Compartments P van der Sluijs, UMC Utrecht, Utrecht, The Netherlands r 2016 Elsevier Inc. All rights reserved.

Endosomes A hallmark of eukaryotic cells is the compartmentization of their cytoplasm which allows for the parallel processing of biochemical reactions with different requirements for optimal execution. All eukaryotic cells have endosomes which constitute a pleiomorphic interconnected membrane system important for many basic cellular processes including nutrient supply, signaling, and plasma membrane protein regulation. In multicellular organisms these features contribute in an essential manner to cellular phenotypes as shape, migration, motility, and division and in doing so are of importance in developmental processes and tissue homeostasis. Endosomes and their role in biology have been firmly established and incorporated in the textbooks. This fact on itself actually bears testimony to the enormous development in imaging technologies that allowed us to rapidly accumulate a wealth of information on an organelle whose existence was unknown in the late 1970s. At that time the lysosome, an acidic degradative organelle was considered to be the destination of all endocytosed molecules and solutes. Observations of Hubbard, Steinman and Cohn, and Goldstein and Brown indicated that the synthesis rate of endocytosed membrane lipid, cell surface proteins, and receptors was insufficient to maintain the surface area of a cell and steady state pools of the proteins they studied (Steinman et al., 1983; Goldstein et al., 1985). Accumulating evidence converged on the notion that salvage pathways had to exist for the return of endocytosed materials back to the cell surface. Early EM studies revealed the existence of prelysosomal vacuoles that were more electronlucent than the typical acid hydrolase containing lysosomes and which could serve as the locale for escape from lysosomal degradation (Helenius et al., 1983). In kinetic experiments on the mechanism of Semliki Forest virus penetration in tissue culture cells Helenius discovered that the low pH-mediated fusion occurred prior to localization of virions in lysosomes, which demonstrated a function for the acidic prelysosomal structures. The subsequent discovery of ATP-driven acidification activity in endosomal membranes cemented the notion that endosomes share the ability with lysosomes to acidify their lumen albeit to a lesser extent (Mellman et al., 1986).

Endosome Heterogeneity Reflects Functional Compartmentalization Microscopy studies showed that some endocytosed molecules like epidermal growth factor and low-density lipoprotein moved centripetally from small peripheral cytoplasmic structures close to the cell surface to more centrally located larger multivesicular organelles and subsequently ‘true’ lysosomes.

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Other tracers of which Transferrin (Tf) and its receptor (TfR) are the archetypical representatives accumulated transiently near the microtubule organizing center before disappearing from the cell without being degraded. Biochemical correlates were derived from internalization experiments followed by cell fractionation where endocytosed tracers initially appeared in a mildly acidic light membrane fraction (‘early endosomes’) and next in more acidic, heavier membranes (‘late endosomes’) with different but overlapping protein composition which stand apart from lysosomes in terms of enrichment profiles for markers and isolation by density gradient centrifugation (Schmid et al., 1988). These and many other microscopy and fractionation studies of increasing sophistication established the paradigm that the term endosome actually encompasses a heterogeneous class of structures both in terms of morphology and function that endow the animal cell with the ability to sort a subset of endocytosed cargo molecules for recycling and re-utilization, while other cargo is dispatched to lysosomes and disposed of by degradation (Geuze et al., 1984; Figure 1). Recent studies have begun to describe the macroscopic properties of the early endosomal system as an ensemble of individual elements that continuously exchange content through fusion and fission. The system analysis approach lends credence to the notion that the net effect of vesicle interactions is the continuous merger into larger ones with increased cargo content (Foret et al., 2012). Interestingly, the quantitative description follows the same mathematical principles as those that were originally derived for the assembly of colloidal particles in larger clusters.

Epithelial Cells Have Spatially Separated, Connected Endosomal Circuits The organization of endocytic organelles and pathways in epithelial cells is more complicated. The polarized phenotype of these cells is a consequence of the interplay between a epithelial polarity establishing program, postional sensors, and a polarized trafficking machinery (Weisz and RodriguezBoulan, 2009; Datta et al., 2011). Maintenance of the chemically distinct apical and basolateral domains is of utmost importance for the barrier function and performance of transport in this cell type. This is most dramatically testified in the rare genetic disease ‘microvillus inclusion disease’ where inactivating mutations in MYO-Vb cause abnormal development of the apical cell surface, mislocalization of apical protein, and severe diarrhea (Müller et al., 2008). Since endocytosis can occur from both apical and basolateral plasma membrane, epithelia essentially duplicated early endosomal compartments. Endocytosed molecules first meet basolateral or apical early endosomes (AEE) that both feed into apical recycling endosomes. From these spatially

Encyclopedia of Cell Biology, Volume 2

doi:10.1016/B978-0-12-394447-4.20015-1

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Plasma mem

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rab7 LE/MVB

Lysosome Figure 1 Overview of generic endosomal compartments. Simplified schematic of the endosomal system and its internal connectivity. Molecules that enter early endosomes (EE) are sorted in two major exit routes that either bring them back to the cell surface directly or via recycling endosomes (RE). The other pathway leads via late endosomes/multivesicular bodies (LE/MVB) to lysosomes or retrieval to the trans Golgi network (TGN). The tubular RE system is sometimes localized in the perinuclear region through interaction with the microtubule organizing center (MTOC). The core endosomal rab GTPases associated with regulation of transport through the conduits are indicated and further described in the main text.

separated early endosomes, material is either returned or transcytosed (for basolaterally internalized molecules) to the apical cell surface, or transported to a common recycling endosome above the nucleus. The common recycling endosome is the sorting station for delivery of cargo to the basolateral plasma membrane or targeting to common lysosomes. Specific sorting signals in cargo molecules are recognized by cytoplasmic machinery for incorporation in apically or basolaterally targeted carriers equipped with the appropriate SNARE proteins for fusion with apical and basolateral domains, respectively (Weisz and Rodriguez-Boulan, 2009; Figure 2).

(Ultra)structure and Specialization Early endosomes (EE) consist of a vacuolar part from which one or more tubules emanate (Geuze et al., 1984). The vacuolar portion serves to sort proteins in the degradative pathway and is also known as sorting endosome, which matures through sequential and cooperative recruitment of rab5 and rab7 effector networks into late endosomes (Rink et al., 2005). The acidic endosomal pH causes dissociation of many ligands and receptors whereupon soluble ligands end up

in the vacuolar portion of the endosome (Maxfield and McGraw, 2004). The tubules may detach from the vacuolar part and oftentimes form a network, known as recycling endosomes. These typically contain a transient pool of intracellular receptors that are targeted for recycling to the plasma membrane. In some cells the tubular network has a very characteristic location adjacent to the microtubule organizing center, presumably through interaction with (peri)centriolar proteins (Maxfield and McGraw, 2004). A third exit conduit is used to recycle a subset of endosomal cargo molecules to the trans Golgi network (Bonifacino and Rojas, 2006). Finally, specialized cells have evolved unique endosome exit pathways that are vital to perform their dedicated functions, an example of which is provided by the presynaptic neuron. To prevent depletion of synaptic vesicles during neuronal stimulation, the rapid exocytosis of neurotransmitter is followed by an ultrafast endocytic pathway for retrieval of synaptic vesicles which involves the formation of new synaptic vesicles from early endosomes (Watanabe et al., 2014). A major fraction of internalized cell surface protein is recycled back from early and recycling endosomes to the plasma membrane. Because the surface to volume ratio of a thin tubule is much larger than of a spherical vesicle or the vacular

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Cilium

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Basal Figure 2 Endosomal organization in polarized epithelial cells. Molecules can endocytosed from the apical and basolateral cell surfaces into AEE and basolateral early endosomes (BEE), respectively. A large fraction is recycled directly, or via common recycling endosomes (CRE) or apical recycling endosomes (ARE) to the cells surface from where they originate. Depending on the sorting information present in cargo molecules, some are sorted for transport into the degradative pathway to late endosomes (LE)/lysosomes (L). Other cargo is sorted from the CRE to the cell surface opposite from where they are internalized, a pathway also known as transcytosis. Newly synthesized molecules are sorted form the TGN to the basolateral surface or via the endosomal compartments to various destinations.

part of the endosome, principles of calculus readily explain a passive endosomal sorting activity that directs receptors in tubules and ligand in the vacuolar part. For a long time the geometry-base paradigm was the driving force for the notion that sorting in the recycling pathway occurred by default. Endosomal export has similarities with the process that delivers molecules from the cell surface to early endosomes. Although we generally refer with the single terms endocytosis and recycling to each of them, they actually represent multiple routes that end in, or leave from endosomes, respectively. From a logistic point of view transport pathways can be considered and in fact serve as a means of intracellular communication. Communication within the endosomal system and with the distinct destinations is brought about by vesicular transport pathways of unforeseen and increasing complexity. Vesicle formation is initiated by recruitment of cytoplasmic coat complexes to specific regions of the membrane, selection of cargo molecules, and reshaping the marked membrane

regions into vesicles of defined size between 40 and 80 nm (Bonifacino and Glick, 2004).

Regulation of Endosome Traffic The architectural integrity of the endosomal system depends on sophisticated control systems for coordinating the transport routes for entry and exit of the lipids and proteins needed for maintenance of organelle identity. Two classes of molecules namely small GTPases of the rab and ADP ribosylation factor/ Arf-like (Arf/Arl) families, and phosphoinositides (PIs) are thought to provide compartment specific tags and serve as key regulators of traffic and homeostasis within the endosomal system (Jean and Kiger, 2012). Endosomal membranes can be populated by more than one rab protein and may also display lateral heterogeneity in terms of phosphoinoside species which allows for the coupling of cargo entry and exit from that

Organelles: Structure and Function: Early Endosomal Compartments

membrane region. Although each in its own right can regulate independent processes, the simultaneous presence and concerted activities of a specific rab, Arf/Arl, and phosphoinositide in a membrane region is likely essential to achieve the requisite fidelity in endosomal trafficking pathways.

Rab Proteins The best understood endosome-associated rab proteins are rab4 (early endosomes-recycling endosomes), rab5 (plasma membrane-recycling endosomes), rab7 (late endosomes-lysosomes, rab9 (late endosomes), and rab11 (recycling endosomes). Rab GTPases are molecular switches that cycle between an active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound state. GTPase Activating Proteins (GAPs) accelerate the intrinsic hydrolysis rate, while guanine nucleotide exchange factors (GEFs) catalyze the conversion of the GDP to GTP state. While the GTP-form of rab proteins is bound to the cytosolic leaflet of the endosomal membrane, the GDP form resides in the cytoplasm in a complex with the chaperone rab-GDP dissociation inhibitor (rab-GDI) that shields the hydrophic geranylgeranylgroups in the C-terminal hypervariable region. The rab–rabGDI complex is needed for initial and reversible delivery of rabs to membranes. The specificity of which is provided by signals within the cognate GEF and the rab–rabGDI complex. Elegant studies with mitochondria-targeted cognate GEFs established that rab GEFs serve as the minimal machinery for targeting and activation of rab proteins including endosomal rab5 and rab35 (Barr, 2013). The active form of rabs binds to specific effectors and thereby creates oligomeric protein complexes that form nanodomains within the endosomal membrane. The rab–effector complexes provide spatiotemporal control of an array of processes including those that regulate shape and location of endosomes via interactions with cytoskeleton and motor proteins, phosphoinositide metabolizing enzymes, tethering factors, fusion and fission regulators, and signaling molecules (Wandinger-Ness and Zerial, 2014). Some of the rab effectors pair with several rab proteins whose activity is sequentially organized along the endocytic route. On early endosomes, the rab5 effectors rabenosyn-5, rabaptin, and rabip4's each interact with, and have separate binding domains for rab5 and rab4 while rab4 and rab11 are coupled through such effectors as D-AKAP-2 and GRASP-1 in neurons (Wandinger-Ness and Zerial, 2014). Bivalent effectors of endosomal rab proteins provide an attractive model to spatially link cargo delivery by incoming transport vesicles in the rab5 domain with carriers leaving endosomes from the adjacent but overlapping rab4 domain (Wandinger-Ness and Zerial, 2014). Conceptually this scenario is related to the coupling of exocytosis and re-intake of synaptic vesicles in neurons, or secretory lysosomes at the immunologial synapse in cytoxic lymphocytes. Mechanistic details of the molecular processes supporting this model however need to be established first.

Phosphoinositides The endosomal localization of phosphoinositides just like that of rab proteins is also controlled by members of two classes of

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enzymes (Jean and Kiger, 2012). Specific lipid kinases and phosphatases catalyze the formation and consumption of a substrate phosphatidylinositol. Their relative activity or location thereby drives the reversible localization of a given phosphatidylinositol (PI) in a membrane or organelle. The principal endosomal phosphatidylinositol is phosphatidylinositol 3-phosphate (PI(3)P) that to a large extent is produced by the type III kinase Vps34 on endocytic vesicles, where it controls docking and subsequent fusion of early endosomes (Wandinger-Ness and Zerial, 2014). PI(3)P binds to proteins containing a PX (after p40phox and p47phox subunits of NADPH oxidase) or FYVE domain (acronym of Fab1, YOTB, Vac1, EEA1, the founding members of this protein family) and serves as cue for endosomal localization. Some of the FYVE proteins like early endosome antigen (EEA1), rabankyrin, and rabenosyn-5 also interact with active rab5, and the simultaneous binding to PI(3)P and rab5 ensures the specificity and avidity required for their endosomal localization (WandingerNess and Zerial, 2014). PI(3)P generated by Vps34 serves as a docking site for phosphatidylinositol 3-phosphate 5-kinase PIKfyve which converts PI(3)P into PI(3,5)P2, whose effectors are involved in sorting to the multivesicular body (Odorizzi et al., 1998). PI that is funneled through Vps34 is primarily involved in initial stages of multivesicular body formation through the localization of PI(3)P effector hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) to a atypical clathrin-coated region on the vacuolar part of early endosomes (Sachse et al., 2002). The adaptor that links endocytic cargo to the endosomal sorting complex required for transport (ESCRT) coat is Hrs, whose ubiquitin interacting motifs interact with ubiquitinated endosomal cargo molecules tagged for degradation (Umebayashi et al., 2008). Through interactions with signal transducing adaptor molecule (STAM), Hrs assembles into a poly ubiquitin binding platform named ESCRT-0, which constitutes the first stage of ESCRT complex assembly (Bache et al., 2003). A newly identified role of this PI(3)P pool relates to a localization in cytokinetic bridges during mammalian cytokinesis for recruitment of the FYVE-CENT protein (Sagona et al., 2010). Together with the motor kinesin family 13A (KIF13A) and tetratricopeptide repeat domain protein 19 (TTC19), FYVE-CENT is thought to cooperate with the charged multivesicular body protein 4 (CHMP4) subunit of ESCRT-III in abscission. Exocytic pathways leaving early endosomes do not seem to be controlled by Vps34 but instead depend on phosphoinositide 3-kinase C2α (Falasca et al., 2007), a kinase that is far less sensitive to classical inhibitors LY294002 and wortmannin. The question then becomes whether early endosomes contain independent pools of PI(3)P that are involved in different aspects of early endosome function as is the case with PI(4,5)P2 on the plasma membrane. Several recent studies now start to shed light on this question and reveal that the early endosomal system harbors distinct PI(3)P pools. Phosphatidylinositol 3-phosphate kinase (PI3K) C2α not only resides on the plasma membrane but has been found in clathrin-coated vesicles as well and in perinuclear recycling endosomes. The recycling endosome PI(3)P pool generated by PI3K C2α activates rab11 and via a rab11–rabin8–rab8 cascade regulates the function of primary cilia (Franco et al., 2014).

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Coordination of Phosphoinositides and Rab Proteins PI kinases and rab proteins coordinately regulate endosome dynamics via multiple mechanisms. Often these involve the enzymatic activity of the PI kinases as with rab5 and Vps34 in endosome maturation (cf. below). In other instances the PI kinase serves a structural role, not directly related to its enzymatic activity. The function of rab11 in exocytic pathways depends on the interaction with a member of the PI4 kinases namely PI4KIIIβ. The two proteins co-localize on recycling endosomes and the trans Golgi network and the function of rab11 critically relies on PI4KIIIβ (De Graaf et al., 2004). The kinase directly binds rab11 and functionally recruits it to membrane via a novel mechanism that is both dependent and independent of the lipid kinase activity (De Graaf et al., 2004). In fruit fly, rab11 and the downstream effector Nuclear Fallout (ortholog of the human rab11-FIP3 (rab11 family interacting protein 3)) depend on Four Wheel Drive (PI4KIIIβ) for membrane localization, a prerequisite for cytokinesis (Polevoy et al., 2009). Membrane ingression at the end of the asexual erythrocytic stage in the Plasmodium falciparum life cycle similarly depends on rab11 and PI4KIIIβ (McNamara et al., 2013). In the rab11–PI4KIIIβ–FIP3 complex structure (Burke et al., 2014), PI4KIIIβ only makes a single peripheral contact with rab11 switch region I, while molecular interactions with switch region II are absent. As a consequence the rab11 switch regions remain available for simultaneous FIP3 binding and by inference interactions with other effectors. The requirement for PI4KIIlβ enzyme activity likely reflects the role of its product PI (4)P in the recruitment of a DENND4 (differentially expressed in normal and neoplastic cells 4) type rab11 exchange factor (Xiong et al., 2012) to activate rab11 as for the orthologs Ypt32 (yeast protein 32) and Pik1 (yeast 1-phosphatidylinositol 4kinase) in yeast.

Early Endosome Maturation and Conversion to Late Endosomes Endocytic internalization and early to late endosome conversion and maturation are the best understood trafficking pathways in terms of the interdependence of rab proteins and PI regulators. Rab5 and rab7 both interact with Vps34 and stimulate its enzyme activity. The increased PI(3)P levels in the endosomal rab5 domain serves to recruit proteins with FYVE signature and rab5 binding regions (cf. above) through coincidence detection. PI3Kβ on the cell surface and clathrincoated vesicles is also activated by rab5 binding and generates PI(3,4,5)P3 from PI(4,5)P2 at these locations. Through downstream rab5 interactions with polyphosphate 4-phosphatase and inositol polyphosphate 5-phosphatase, PI(3,4,5) P3 is converted to PI(3,4)P2 and PI(3)P, which in parallel to the PI3K C2α pathway contributes to the formation of a gradient of PI3P from the plasma membrane to early endosomes (Wandinger-Ness and Zerial, 2014). The PI(3)P molecules on early endosomes are turned over via several mechanisms. First, recruitment of PIKfyve converts PI(3)P to PI(3,5)P2. A second pathway involves activation of rab7 which sequesters Rubicon, a negative regulator of UV radiation resistance-associated gene (UVRAG) that binds the

Vps34–p150–beclin1 core complex for fusion on early endosomes (Sun et al., 2010). As a consequence UVRAG activates the homotypic vacuolar protein sorting complex (HOPS) complex, a rab7 effector tethering late endosomes for fusion. The PI conversion pathways are superimposed on a rab cascade where rab5 is replaced by rab7 (Rink et al., 2005). The essentials of the latter include the disruption of the positive cooperativity in the rab5 pathway via which rabex-5–rabaptin5 exchange factor complex contributes to recruitment of additional rab5 effector molecules. Key events in this partially elucidated pathway are docking of the rab7 exchange factor Mon1-Ccz1 (Nordmann et al., 2010) on rab5 and PI(3)P and removal of the rab5 exchange factor. GTP hydrolysis of rab5GTP will cause dissociation of rab5GDP (Poteryaev et al., 2010), recruitment and activation of rab7 and the assembly of a late endosomal domain populated by rab7-GTP and its own effectors such as the HOPS complex, RILP (Rab7-interacting lysosomal protein) and dynein motors (Johansson et al., 2007), and retromer (Rojas et al., 2008). Proteins that do not contain the signals for targeting and degradation in late endosomes/lysosomes are either retrieved to the trans Golgi network or recycled to the plasma membrane. The transport carriers that are used in both exit ways are formed from tubular extensions on early endosomes. In the following sections, these pathways will be discussed in more detail.

Retrieval of Proteins from Endosomes to the Trans Golgi Network Early work by Seaman and Emr (Seaman et al., 1998) led to the identification of retromer as a coat complex required for the return of Vps10, a trans Golgi network (TGN) sorting receptor for the vacuolar enzyme carboxypeptidase Y and counterpart of the mammalian Mannose 6-Phosphate Receptor. The subunits of yeast retromer are organized in two interacting subcomplexes consisting of Vps26–Vps35–Vps29 trimer and a Vps5–Vps17 heterodimer. The first of which is highly conserved, interacts with cargo (Notwehr et al., 2000) and is known as cargo selection complex, while Vps5 and Vps7 are member of the sorting nexin (SNX) family with more sequence diversity. Vps5 and Vps7 contain BAR (Bin–Amphiphysin–Rvs) domains that dimerize into a banana-shaped structure which can sense membrane curvature and generate membrane tubules (Peter et al., 2004). In mammalian cells the cargo selection complex is associated with combinations of SNX1 and SNX2 with either SNX5 or SNX6 that all contain BAR and PX domains (SNX–BAR) proteins (Wassmer et al., 2009). Docking of the cargo selection complex on membranes requires direct binding with rab7 (Rojas et al., 2008) on early/ maturing endosomes, and the affinity of the SNX–BAR subcomplex for PI(3)P in this endosomal membrane domain (Rojas et al., 2008). The Vps29 subunit can recruit TBC1D5, a rab7-GAP whose activity converts rab7 to the inactive GDP form (Seaman et al., 2009) that does not bind retromer. More recent studies in Caenorhabditis elegans and Drosophila melanogaster uncovered a distinct retromer complex consisting of the cargo recognition complex and SNX3, an SNX without BAR domain. This functionally divergent retromer is required

Organelles: Structure and Function: Early Endosomal Compartments

for the retrieval of the wnt receptor wntless from endosomes to the TGN (Harterink et al., 2011) and uses the PX domain of SNX3 for binding to membrane. In accordance with the lack of a BAR domain, the SNX3 retromer concentrates wntless cargo in membrane regions that are distinct from the tubular structures of the archetype SNX-BAR retromer and they represent earlier endocytic domains (Harterink et al., 2011). The existence of an independent second retromer pathway ferrying different cargo from endosomes back to the TGN is a conceptual advance in our understanding of the cellular organization of retrograde transport pathways. Conceivably, the combination of a conserved cargo selection complex with different SNX proteins could generate the requisite diversity for collecting and returning distinct cargo molecules from spatially separated endosomal domains back to the TGN. To complicate matters further, not all traffic from endosomes back to the TGN involves retromer. Cargo molecules including shiga toxin and TGN38 are retrieved from endosomes to the TGN via mechanisms that operate without retromer deployment, and are only beginning to be understood recently. One of them invokes recruitment of the pleckstrin homology (PH) domain protein evectin-2 to recycling endosome domains enriched in phosphatidylserine, which is essential for the return of shiga toxin and TGN38 to the TGN (Uchida et al., 2011). A more detailed description of this and the other salvage routes to the TGN is covered elsewhere in this volume.

Recycling from Early Endosomes to the Plasma Membrane Early studies on transferrin recycling suggested that endosomal recycling occurs by default through geometric sorting into tubules which did not seem to need sorting signals in the cargo (Maxfield and McGraw, 2004). Conceptually this represents an oddity against the common and conserved principles that the cell elsewhere uses to sort proteins in transport carriers for vectorial transport (Bonifacino and Glick, 2004). Over the years, however, a plethora of studies converged on the notion that segregation and deviation from the path to lysosomes depends on signals in the cargo for transport from endosomes to such destinations as plasma membrane (Hsu et al., 2012), TGN (Bonifacino and Rojas, 2006), and melanosomes (Theos et al., 2005). We now will address the recycling routes from endosomes to the plasma membrane. Morphological and biochemical tracer studies using TfR as a marker established the existence of a fast direct recycling route from early endosomes to the plasma membrane and a slow indirect pathway via recycling endosomes (Maxfield and McGraw, 2004). Several small GTPases including arf6, rab4, rab11, and rab35 localize to early endosomes/recycling endosomes and controls distinct stages and modalities of endosome recycling. Arf6 was originally discovered as a regulator TfR recycling pathway (Peters et al., 1995). It does so at multiple levels including the formation of a ACAP1 coat containing clathrin and the GAP protein ACAP1 that recognizes sorting sequence information in the cytoplasmic tail of cargo proteins (Hsu et al., 2012). Arf6 also interacts with the sec10 subunit of the

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octameric exocyst vesicle-tethering complex. Functional studies of this interaction suggest that arf6 specifies delivery of recycling endosome membrane through interaction with the exocyst complex (Prigent et al., 2003). Rab4 and rab11 activities are sequentially organized. Rab4 is a regulator of fast recycling, while rab11 distribution partially overlaps with rab4 and regulates the slow pathway (Wandinger-Ness and Zerial, 2014). Rab35 also regulates a fast recycling pathway (Allaire et al., 2010) together with arf6 (Chesneau et al., 2012). Rab4 acts upstream of rab11 since endocytosed Tf first meets rab4 and subsequently rab11 (Sönnichsen et al., 2000). The function of rab4 is at the level of formation of recycling vesicles from early endosomes as found by in vitro reconstitution assays (Pagano et al., 2004). It does so via recruitment of adaptor complexes of which adaptor protein complex 1 (AP-1) interacts directly (Perrin et al., 2013) and via the effector rabaptin-5α (Deneka et al., 2003) to rab4. The molecular mechanism for this pathway involves a GTPase cascade that is orchestrated by rab4, which results in association of Arl1, type I Arfs and the AP-1, adaptor protein complex 3 (AP-3), and GGA3 (Golgi-localized, γ-adaptin ear-containing, ARF-binding protein) adapter complexes to endosomes (D'Souza et al., 2014). The mechanism of rab11 function in recycling occurs at multiple levels in the pathway. First, through interaction with the sec15 subunit of the octameric exocyst tethering complex (Wu et al., 2005), rab11 can recruit other subunits of the complex to recycling vesicles and thereby regulate fusion at the plasma membrane (Takahashi et al., 2012). Rab11 and its effector rab11-FIP also interact with eps15 homology domain (EHD) ATPase proteins (Naslavsky et al., 2006) that regulate tubule formation and vesiculation (Cai et al., 2013) and endosomal recycling (Pant et al., 2009). Other aspects of Rab11 function in recycling relate to a role in connecting endosomal membrane to the cytoskeleton through motor recruitment. Rab11 interacts in several ways with myosin V motors for motility of recycling vesicles in the peripheral cytoplasm (Welz et al., 2014). Rab11 is essential for recycling endosome tubule morphogenesis and the distribution of the tubules toward the cell periphery through interaction with the microtubule motor KIF13A (Delevoye et al., 2014). Interestingly in melanocytes, KIF13A in complex with the AP-1 adapter is needed for the generation and positioning of peripheral recycling endosomal domains required for sorting of melanogenic enzymes to maturing melanosomes (Delevoye et al., 2009). The relation between recycling endosomes and melanosomes possibly reflects a more common principle that is used in specialized cells with lysosome-related organelles. In several cell types of the immune system including cytotoxic lymphocytes and mast cells, rab 11-positive recycling endosomes supply exocytic machinery to immature lysosome-related organelles that is needed for their maturation and fusion of the latter with the plasma membrane (Menager et al., 2007; Elstak et al., 2011). Investigations into trafficking pathways of specific cargo molecules as LDL-related protein1 (LRP1) (Van Kerkhof et al., 2005), potassium channels (Lunn et al., 2007) and β2-adrenergic receptors (Puthenveedu et al., 2010) unveiled a unforeseen role for SNX17 and SNX27 and retromer in recycling of these ligands. SNX17 and SNX27 lack a BAR domain but

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Plasma membra

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Figure 3 Sorting in endosomal exit pathways. Overview of sorting mechanisms within early endosomes (EE) and the connected tubular endosomal network (TEN). For the sake of simplicity the distinct recycling pathways for Transferrin receptor (TfR), LDL receptor-related protein (LRP) and β2-adrenergic receptor ( β2-AR) are drawn from a continuous network of tubules emanating from the EE and from which recycling carriers bud. Sorting complexes for some of the recycling routes to the plasma membrane and retrieval to the trans Golgi network (TGN) are shown. Cargo destined for degradation such as EGFR is concentrated in the bilayered clathrin coat and subsequently sorted into regions from where intralumenal vesicles are formed and buding into the lumen of multivesicular bodies (MVB).

instead contain post synaptic density (PDZ) and FERM (4.1 protein, ezrin, radixin, moesin) domains. The PDZ domain of SNX27 interacts with signals in the cytoplasmic tail of the β2adrenerigic receptor (Lauffer et al., 2010) and is needed together with the cargo selection complex for rapid retrieval of the receptor to the plasma membrane (Temkin et al., 2011). Retromer-mediated recycling of β2-adrenergic occurs from rab4 -associated endosomal tubules that are decorated with F-actin and distinct from the tubules primarily involved in TfR recycling (Puthenveedu et al., 2010). The actin patches are generated by the multisubunit Wiskott–Aldrich syndrome protein and SCAR homolog (WASH) complex, a nucleation promoting factor that binds Vps35 (Gomez and Billadeau, 2009) and SNX27 (Temkin et al., 2011). WASH activates actin-related proteins 2/3 (Arp2/3) complex and drives actin branching on the endosomal tubules (Derivery et al., 2009). WASH-regulated actin dynamics form a prerequisite for sorting β2-adrenergic receptor to the cell surface (Puthenveedu et al., 2010). The extensive interactions between WASH and retromer suggest an attractive model in which actin and retromer-dependent remodeling of the endosomal surface cooperate for efficient recycling.

With this new role of the cargo selection complex in endosomal recycling, the functions of retromer have now diversified beyond those of the SNX-BAR and SNX3 – retromer complexes at the endosome TGN interface. In accord with a central role of SNX27-retromer in endosome recycling, many plasma membrane proteins have been identified that rely on SNX27 for recycling and bypassing of the degradative pathway (Steinberg et al., 2013; Figure 3).

Conclusion and Perspectives Nature's choice to allocate cellular functions to discrete compartments importantly shaped our thinking of the strategies to decipher the workings of a cell. By taking apart the cell part and studying individual elements, we made fast and great progress in understanding the ramifications of the endo-lysosomal system within the core fabric of life. We charted and partially delineated the complexity of pathways leading to and from this highly dynamic organelle. We also discovered many molecules that safeguard the uninterrupted flow of information within the endosomal system and from the upstream

Organelles: Structure and Function: Early Endosomal Compartments

and downstream neighboring organelles of the trafficking routes. Given the massive flux of proteins and lipids that enter and leave the endosomal system we need to address now how compartmental integrity is maintained. In other words, how are input and output of the distinct pathways integrated, what is the nature and hierarchy of the control systems that coordinate and achieve homeostasis. These questions also ask for applying control and system theory to provide quantitative information needed for describing and predicting the behavior of the system in terms of response to, for instance, nutritional alterations. Perhaps related to this problem is the need to explore whether and how the separate protein complexes that regulate endosomal recycling cooperate to sustain a specific pathway. We may ask which of the rab proteins co-organizes the SNX27retromer pathway. Not only will this bring us closer to an understanding of the organizational principles of exit routes, we may also learn whether the rab7-SNX-BAR retromer coincidence detection reflects an example of a general principle. In recent years it has become evident that endosomes directly and functionally interact with the endoplasmic reticulum, the largest cellular compartment and store for ions and lipids. Signaling of the epidermal growth factor (EGF) receptor involves contacts between endosomes and endoplasmic reticulum membrane containing the protein tyrosine phosphatase PTP1B (Eden et al., 2010). Importantly endoplasmic reticulum contacts were recently shown to determine the sites, timing, and efficiency of endosome fission events (Rowland et al., 2014). Conceivably, such contacts may contribute to, or control sorting processes within the endosomal system. Elucidating the molecular principles underlying the contact sites may therefore be of great interest to understand the dynamics of the endosomal system as well as of the endoplasmic reticulum.

See also: Organelles: Structure and Function: The Endoplasmic Reticulum

References Allaire, P.D., Marat, A.L., Dall'a Armi, C., et al., 2010. The Connecdenn DENN domain: A GEF for rab35 mediating cargo-specific exit from early endosomes. Molecular Cell 37, 370–382. Bache, K.G., Brech, A., Mehlum, A., Stenmark, H., 2003. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. Journal of Cell Biology 162, 435–442. Barr, F.A., 2013. Rab GTPases and membrane identity: Causal or inconsequential. Journal of Cell Biology 202, 191–199. Bonifacino, J.S., Glick, B.S., 2004. The mechanisms of vesicle budding and fusion. Cell 116, 153–166. Bonifacino, J.S., Rojas, R., 2006. Retrogade transport from endosomes to the trans Golgi network. Nature Reviews Molecular Cell Biology 7, 568–579. Burke, J.E., Inglis, A.J., Peisic, O., et al., 2014. Structures of PI4KIIIbeta complexes show simultaneous recruitment of rab11 and its effectors. Science 344, 1035–1038. Cai, B., Giridharan, S.S., Zhang, J., et al., 2013. Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes. Journal of Biological Chemistry 288, 30172–30180. Chesneau, L., Dambournet, D., Machicoane, M., et al., 2012. An ARF6/rab35 GTPase cascade for endocytic recycling and successful cytokinesis. Current Biology 22, 147–153.

199

D'Souza, R.S., Semus, R., Billings, E.A., et al., 2014. Rab4 orchestrates a small GTPase cascade for recruitment of adaptor proteins to early endosomes. Current Biology 24, 1187–1198. Datta, A., Bryant, D.M., Mostoiv, K.E., 2011. Molecular regulation of lumen morphogenesis. Current Biology 21, R126–R136. De Graaf, P., Zwart, W.T., Van Dijken, R.A.J., et al., 2004. Phosphatidylinositol 4kinasebeta is critical for functional association of rab11 with the Golgi complex. Molecular Biology of the Cell 15, 2038–2047. Delevoye, C., Hurbain, I., Tenza, D., et al., 2009. AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis. Journal of Cell Biology 187, 247–264. Delevoye, C., Miserey-Lenkei, S., Montagnac, G., et al., 2014. Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A. Cell Reports 6, 445–454. Deneka, M., Neeft, M., Popa, I., et al., 2003. Rabaptin-5a/rabaptin-4 serves as a linker between rab4 and γ1-adaptin in membrane recycling from endosomes. EMBO Journal 22, 2645–2657. Derivery, E., Sousa, C., Gautier, J.J., et al., 2009. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex. Developmental Cell 17, 712–723. Eden, E.R., White, I.J., Tsapara, A., Futter, C.E., 2010. Membrane contacts between endosomes and ER provide sites for PTP1B-epidermal growth factor receptor interaction. Nature Cell Biology 12, 267–272. Elstak, E.D., Neeft, M., Nehme, N.T., et al., 2011. Munc13-4 rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane. Blood 118, 1570–1578. Falasca, M., Hughes, W.E., Dominguez, V., et al., 2007. The role of phosphoinositide 3-kinase C2alpha in insuin signaling. Journal of Biological Chemistry 282, 28226–28236. Foret, L., Dawson, J.E., Villaseor, R., et al., 2012. A general theoretical framework to infer endosomal network dynamics from quantitative images. Current Biology 22, 1381–1390. Franco, I., Gulluni, F., Campa, C.C., et al., 2014. PI3 class II alpha controls spatially restricted endosomal PtdIns3P and rab11 activation to promote primary cilium function. Developmental Cell 28, 647–658. Geuze, H.J., Slot, J.W., Strous, G.J., et al., 1984. Intracellular receptor sorting during endocytosis: Comparative immuno electron microscopy of multiple receptors in rat liver. Cell 37, 195–204. Goldstein, J.L., Brown, M.S., Anderson, R.G., Russell, D.W., Schneider, W.J., 1985. Receptor-mediated endocytosis: Concepts emerging from the LDL receptor system. Annual Review of Cell and Developmental Biology 1, 1–39. Gomez, T.S., Billadeau, D.D., 2009. A FAM21 containing WASH complex regulates retromer dependent sorting. Developmental Cell 17, 699–711. Harterink, M., Port, F., Lorenowicz, M.J., et al., 2011. A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biology 13, 914–923. Helenius, A., Mellman, I., Wall, D., Hubbard, A.L., 1983. Endosomes. Trends in Biochemical Sciences 8, 45–50. Hsu, V.W., Bai, M., Li, J., 2012. Getting active: Protein sorting in endocytic recycling. Nature Reviews Molecular Cell Biology 13, 323–328. Jean, S., Kiger, A.A., 2012. Coordination between rab GTPase and phosphoinositide regulation and functions. Nature Reviews Molecular Cell Biology 13, 463–470. Johansson, M., Rocha, N., Zwart, W.T., et al., 2007. Activation of endosomal dynein motors by stepwise assembly of rab7-RILP-p150Glued, ORP1L and the receptor betaIIIspectrin. Journal of Cell Biology 176, 459–471. Lauffer, B.E.L., Melero, C., Temkin, P., et al., 2010. SNX27 mediates PDZ-directed sorting from endosomes to the plasma membrane. Journal of Cell Biology 190, 565–574. Lunn, M.L., Nassirpour, R., Arrabit, C., et al., 2007. A unique sorting nexin regulates trafficking of potassium channels via a PDZ domain interaction. Nature Neuroscience 10, 1249–1259. Maxfield, F.R., McGraw, T.E.M., 2004. Endocytic recycling. Nature Reviews Molecular Cell Biology 5, 121–132. McNamara, C.W., Lee, M.C., Lim, C.S., et al., 2013. Targeting plasmodium PI(4)K to eliminate malaria. Nature 504, 248–253. Mellman, I., Fuchs, R., Helenius, A., 1986. Acidification of the endocytic and exocytic pathways. Annual Review of Biochemistry 55, 712–722. Menager, M.M., Menasche, G., Romao, M., et al., 2007. Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4. Nature Immunology 8, 257–267. Müller, T., Hess, M.W., Schiefermeier, N., et al., 2008. MYO VB mutations cause mocrovillus inclusion disease and disrupt epithelial cell polarity. Nature Genetics 40, 1163–1165.

200

Organelles: Structure and Function: Early Endosomal Compartments

Naslavsky, N., Rahajeng, J., Sharma, M., Jovic, M., Caplan, S., 2006. Interactions between EHD proteins and rab11-FIP2: A role for EHD3 in erly endosomal transport. Molecular Biology of the Cell 17, 163–177. Nordmann, M., Cabrera, M., Perz, A., et al., 2010. The Mon1-CCz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Current Biology 20, 1654–1659. Notwehr, S.F., Ha, S.A., Bruinsma, P., 2000. Sorting of yeast membrane proteins into a endosome to Golgi pathway involves direct interaction of their cytosolic domains. Journal of Cell Biology 151, 297–310. Odorizzi, G., Babst, M., Emr, S.D., 1998. Fab1p PtdIns(3)P5-kinase function essential for protein sorting in the multivesicular body. Cell 95, 847–858. Pagano, A., Crottet, P., Prescianotto-Baschong, C., Spiess, M., 2004. In vitro formation of recycling vesicles from endosomes requires AP-1/clathrin and is regulated by rab4 and the connector rabaptin-5. Molecular Biology of the Cell 15, 4990–5000. Pant, S., Sharma, M., Patel, K., et al., 2009. AMPH-1/amphiphysin/Bin1 functions with RME-1/EHD1 in endocytic recycling. Nature Cell Biology 11, 1399–1410. Perrin, L., Lacas-Gervais, S., Gilleron, J., et al., 2013. Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1. Journal of Cell Science 126, 4950–4962. Peter, B.J., Kent, H.M., Mills, I.G., et al., 2004. BAR domains as sensors of membrane curvature: The amphiphysin BAR structure. Science 303, 495–499. Peters, P., Hsu, V., Ooi, C.E., et al., 1995. Overexpression of wild type and mutant ARF1 and ARF6: Distinct perturbations of nonoverlapping membrane compartments. Journal of Cell Biology 128, 1003–1017. Polevoy, G., Wei, H.C., Wong, R., et al., 2009. Dual roles for the drosophila PI4kinase four wheel drive in localizing rab11 during cytokinesis. Journal of Cell Biology 187, 847–858. Poteryaev, D., Datta, S., Ackema, K., Zerial, M., Spang, A., 2010. Identification of the switch in erarly to late endosome transition. Cell 141, 497–508. Prigent, M., Dubois, T., Raposo, G., et al., 2003. ARF6 controls post-endocytic recycling through its downstream exocyst complex effector. Journal of Cell Biology 163, 1111–1121. Puthenveedu, M.A., Lauffer, B., Temkin, P.V.R., et al., 2010. Sequence dependent sorting of recycling proteins by actin-stabilized endosomal microdomains. Cell 143, 761–773. Rink, J., Ghigo, E., Kalaidzidis, Y., Zerial, M., 2005. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749. Rojas, R., Van Vlijmen, T., Mardones, G., et al., 2008. Regulation of retromer recruitment to endosomes by sequential action of rab5 and rab7. Journal of Cell Biology 183, 513–526. Rowland, A.A., Chitwood, P.J., Phillips, M.J., Voeltz, G.K., 2014. ER contact sites define the position and timing of endosome fission. Cell 159, 1027–1041. Sachse, M., Urbe, S., Oorschot, V., Strous, G.J., Klumperman, J., 2002. Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting towards lysosomes. Molecular Biology of the Cell 13, 1313–1328. Sagona, A.P., Nezis, I.P., Pedersen, N.M., et al., 2010. PtdIns(3 P) controls cytokinesis through KIF13A mediated recruitment of FYVE-CENT to the midbody. Nature Cell Biology 12, 362–371. Schmid, S.L., Fuchs, R., Male, P., Mellman, I., 1988. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell 52, 73–83. Seaman, M.N., Harbour, M.E., Tattersall, D., Read, E., Bright, N., 2009. Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small

GTPase Rab7 and inhibited by the Rab-GAP TBC1D5. Journal of Cell Science 122, 2371–2382. Seaman, M.N.J., McCaffery, J.M., Emr, S.D., 1998. A membrane coat complex essential for endosome to Golgi retrograde transport in yeast. Journal of Cell Biology 142, 655–682. Sönnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J., Zerial, M., 2000. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of rab4, rab5, and rab11. Journal of Cell Biology 149, 901–913. Steinberg, F., Gallon, M., Winfield, M., et al., 2013. A global analysis of SNX27retromer assembly and cargo specificity reveals a function in glucose ad metal ion transport. Nature Cell Biology 15, 461–471. Steinman, R.M., Mellman, I., Muller, W.A., Cohn, Z.A., 1983. Endocytosis and the recycling of plasma membrane. Journal of Cell Biology 96, 1–27. Sun, Q., Westphal, W., Wong, K.N., Tan, I., Zhong, Q., 2010. Rubicon controls endosome maturation as a Rab7 effector. Proceedings of the National Academy of Sciences 107, 19338–19343. Takahashi, S., Kubo, K., Waguri, S., et al., 2012. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. Journal of Cell Science 125, 4049–4057. Temkin, P., Lauffer, B., Jager, S., et al., 2011. SNX27 mediates retromer tuubule entry ad endosome to plasma membrane trafficking of signalling receptors. Nature Cell Biology 13, 715–721. Theos, A.C., Tenza, D., Martina, J.A., et al., 2005. Functions of adaptor protein AP-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes. Molecular Biology of the Cell 16, 5356–5372. Uchida, Y., Hasegawa, J., Chinnapen, D., et al., 2011. Intracelluar phosphatidylserine is essential for retrograde membrane traffic through endosomes. Proceedings of the National Academy of Sciences of the United States of America 108, 15846–15851. Umebayashi, K., Stenmark, H., Yoshimori, T., 2008. Ubc4/5 and c-abl continue to ubiquitinate EGF receptor after internalization to facilitate polyubiquitination and degradation. Molecular Biology of the Cell 19, 3454–3462. Van Kerkhof, B., Lee, J., McCormick, L., et al., 2005. Sorting nexin 17 facilitates LRP recycling in the early endosome. EMBO Journal 24, 2851–2861. Wandinger-Ness, A., Zerial, M., 2014. Rab proteins and the compartmentalization of the endosomal system. Cold Spring Harbor Perspectives in Biology 6, a022509. Wassmer, T., Attar, N., Harterink, M., et al., 2009. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport with carrier recognition by the trans Golgi network. Developmental Cell 17, 110–122. Watanabe, S., Trimbuch, T., Camacho-Perez, M., et al., 2014. Clathrin regenerates synaptic vesicles from endosomes. Nature 515, 228–233. Weisz, O.A., Rodriguez-Boulan, E., 2009. Apical trafficking in epithelial cells: Signals, clusters and motors. Journal of Cell Science 122, 4253–4266. Welz, T., Wellbourne-Wood, J., Kerkhoff, E., 2014. Orchestration of cell surface proteins by rab11. Trends in Cell Biology 24, 407–415. Wu, S., Mehta, S.Q., Pichaud, F., Bellen, H.J., Quiocho, F.A., 2005. Sec15 interacts with rab11 via a novel domain and affects rab11 localization in vivo. Nature Structural & Molecular Biology 12, 879–885. Xiong, T., Bayat, V., Jaiswal, M., et al., 2012. CRAG is a GEF for rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells. PLoS Biology 10, E1001438.