Towards a molecular understanding of endosomal trafficking by Retromer and Retriever

Endosomes are dynamic intracellular compartments that control the sorting of a constant stream of different transmembrane cargos either for ESCRT‐mediated degradation or for egress and recycling to compartments such as the Golgi and the plasma membrane. The recycling of cargos occurs within tubulovesicular membrane domains and is facilitated by peripheral membrane protein machineries that control both membrane remodelling and selection of specific transmembrane cargos. One of the primary sorting machineries is the Retromer complex, which controls the recycling of a large array of different cargo molecules in cooperation with various sorting nexin (SNX) adaptor proteins. Recently a Retromer‐like complex was also identified that controls plasma membrane recycling of cargos including integrins and lipoprotein receptors. Termed “Retriever,” this complex uses a different SNX family member SNX17 for cargo recognition, and cooperates with the COMMD/CCDC93/CCDC22 (CCC) complex to form a larger assembly called “Commander” to mediate endosomal trafficking. In this review we focus on recent advances that have begun to provide a molecular understanding of these two distantly related transport machineries.     

Retrograde trafficking and plasma membrane recycling pathways of the budding yeast Saccharomyces cerevisiae

The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome‐to‐Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.     

UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.     

Sorting Nexin 17 Regulates ApoER2 Recycling and Reelin Signaling

ApoER2 is a member of the low density-lipoprotein receptor (LDL-R) family. As a receptor for reelin, ApoER2 participates in neuronal migration during development as well as synaptic plasticity and survival in the adult brain. A previous yeast two-hybrid screen showed that ApoER2 is a binding partner of sorting nexin 17 (SNX17) - a cytosolic adaptor protein that regulates the trafficking of several membrane proteins in the endosomal pathway, including LRP1, P-selectin and integrins. However, no further studies have been performed to investigate the role of SNX17 in ApoER2 trafficking and function. In this study, we present evidence based on GST pull-down and inmunoprecipitation assays that the cytoplasmic NPxY endocytosis motif of ApoER2 interacts with the FERM domain of SNX17. SNX17 stimulates ApoER2 recycling in different cell lines including neurons without affecting its endocytic rate and also facilitates the transport of ApoER2 from the early endosomes to the recycling endosomes. The reduction of SNX17 was associated with accumulation of an ApoER2 carboxy-terminal fragment (CTF). In addition, in SNX17 knockdown cells, constitutive ApoER2 degradation was not modified, whereas reelin-induced ApoER2 degradation was increased, implying that SNX17 is a regulator of the receptor''s half-life. Finally, in SNX17 silenced hippocampal and cortical neurons, we underscored a positive role of this endosomal protein in the development of the dendritic tree and reelin signaling. Overall, these results establish the role of SNX17 in ApoER2 trafficking and function and aid in identifying new links between endocytic trafficking and receptor signaling.     

New insights in endosomal dynamics and AMPA receptor trafficking

The trafficking mechanisms that control the density of synaptic AMPA-type glutamate receptors have received significant attention because of their importance for regulating excitatory synaptic transmission and synaptic plasticity in the hippocampus. AMPA receptors are synthesized in the neuronal cell body and reach their postsynaptic targets after a complex journey involving multiple transport steps along different cytoskeleton structures and through various stages of the endocytic pathway. Dendritic spines are important sites for AMPA receptor trafficking and contain the basic components of endosomal recycling. On induction of synaptic plasticity, internalized AMPA receptors undergo endosomal sorting and cycle through early endosomes and recycling endosomes back to the plasma membrane (model for long-term potentiation) or target for degradation to the lysosomes (model for long-term depression). Exciting new studies now provide insight in actin-mediated processes that controls endosomal tubule formation and receptor sorting. This review describes the path of AMPA receptor internalization up to sites of recycling and summarizes recent studies on actin-mediated endosomal receptor sorting.     

Dynamics of receptor trafficking in tumorigenicity

The transport and sorting of cell surface receptors into membrane-bound intracellular compartments is crucial for cellular homeostasis. Defects in receptor trafficking are associated with several diseases, including cancer. Recent advances in our understanding of mechanisms that control receptor trafficking have highlighted the involvement of membrane trafficking in cell signaling, as well as in biological processes, including cell migration and invasion. In this review, we summarize current knowledge of how cargos, focusing on receptor tyrosine kinases (RTKs) and integrins, are dynamically transported through the endosomal pathway for recycling, and how this promotes spatially restricted signaling microdomains associated with distinct biological responses. We discuss mechanisms through which dysregulation of membrane trafficking contributes to tumorigenesis and potential therapeutic approaches.     

An ESCRT–spastin interaction promotes fission of recycling tubules from the endosome

Mechanisms coordinating endosomal degradation and recycling are poorly understood, as are the cellular roles of microtubule (MT) severing. We show that cells lacking the MT-severing protein spastin had increased tubulation of and defective receptor sorting through endosomal tubular recycling compartments. Spastin required the ability to sever MTs and to interact with ESCRT-III (a complex controlling cargo degradation) proteins to regulate endosomal tubulation. Cells lacking IST1 (increased sodium tolerance 1), an endosomal sorting complex required for transport (ESCRT) component to which spastin binds, also had increased endosomal tubulation. Our results suggest that inclusion of IST1 into the ESCRT complex allows recruitment of spastin to promote fission of recycling tubules from the endosome. Thus, we reveal a novel cellular role for MT severing and identify a mechanism by which endosomal recycling can be coordinated with the degradative machinery. Spastin is mutated in the axonopathy hereditary spastic paraplegia. Zebrafish spinal motor axons depleted of spastin or IST1 also had abnormal endosomal tubulation, so we propose this phenotype is important for axonal degeneration.     

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and endoplasmic reticulum (ER). To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes (EE), recycling endosomes, late endosomes and lysosomes. All cargos pass through EE, but may take different routes to the Golgi. Retromer-dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer-dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-mannose-6-phosphate receptor (CI-M6PR), which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CI-M6PR was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the EE, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer-dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB.     

Endosomal functions in plants

Plant endosomes are highly dynamic organelles that are involved in the constitutive recycling of plasma membrane cargo and the trafficking of polarized plasma membrane proteins such as auxin carriers. In addition, recent studies have shown that surface receptors such as the plant defense-related FLS2 receptor and the brassinosteroid receptor BRI1 appear to signal from endosomes upon ligand binding and internalization. In yeast and mammals, endosomes are also known to recycle vacuolar cargo receptors back to the trans Golgi network and sort membrane proteins for degradation in the vacuole/lysosome. Some of these sorting mechanisms are mediated by the retromer and endosomal sorting complex required for transport (ESCRT) complexes. Plants contain orthologs of all major retromer and ESCRT complex subunits, but they have also evolved variations in endosomal functions connected to plant-specific features such as the diversity of vacuolar transport pathways. This review focuses on recent studies in plants dealing with the regulation of endosomal recycling functions, architecture and formation of multivesicular bodies, ligand-mediated endocytosis and receptor signaling from endosomes as well as novel endosomal markers and the function of endosomes in the transport and processing of soluble vacuolar proteins.     

Out of the ESCPE room: Emerging roles of endosomal SNX-BARs in receptor transport and host–pathogen interaction

Several functions of the human cell, such as sensing nutrients, cell movement and interaction with the surrounding environment, depend on a myriad of transmembrane proteins and their associated proteins and lipids (collectively termed “cargoes”). To successfully perform their tasks, cargo must be sorted and delivered to the right place, at the right time, and in the right amount. To achieve this, eukaryotic cells have evolved a highly organized sorting platform, the endosomal network. Here, a variety of specialized multiprotein complexes sort cargo into itineraries leading to either their degradation or their recycling to various organelles for further rounds of reuse. A key sorting complex is the Endosomal SNX-BAR Sorting Complex for Promoting Exit (ESCPE-1) that promotes the recycling of an array of cargos to the plasma membrane and/or the trans-Golgi network. ESCPE-1 recognizes a hydrophobic-based sorting motif in numerous cargoes and orchestrates their packaging into tubular carriers that pinch off from the endosome and travel to the target organelle. A wide range of pathogens mimic this sorting motif to hijack ESCPE-1 transport to promote their invasion and survival within infected cells. In other instances, ESCPE-1 exerts restrictive functions against pathogens by limiting their replication and infection. In this review, we discuss ESCPE-1 assembly and functions, with a particular focus on recent advances in the understanding of its role in membrane trafficking, cellular homeostasis and host–pathogen interaction.     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.     

Protein Kinase D-Dependent Trafficking of the Large Herpes simplex Virus Type 1 Capsids from the TGN to Plasma Membrane

The biosynthetic pathway carries cargos from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) via a typical passage through the Golgi. Interestingly, large particles such as procollagen, chylomicrons and some viruses all reach the TGN by atypical routes. Given this dichotomy, we anticipated that such cargos might rely on non-classical machineries downstream of the TGN. Using Herpes simplex virus type 1 (HSV-1) as a model and a synchronized infection protocol that focuses on TGN to plasma membrane transport, the present study revealed the surprising implication of the cellular serine-threonine protein kinase D in HSV-1 egress. These findings, confirmed by a variety of complementary means [pharmacological inhibitors, dominant negative mutant, RNA interference and electron microscopy (EM)], identify one of possibly several cellular factors that modulate the egress of viruses transiting at the TGN. Moreover, the involvement of this kinase, previously known to regulate the transport of small basolateral cargos, highlights the trafficking of both small and exceptionally large entities by a common machinery downstream of the TGN, in sharp contrast to earlier steps of transport. Conceptually, this indicates the TGN is not only a sorting station from which cargos can depart towards different destinations but also a meeting point where conventional and unconventional routes can meet along the biosynthetic pathway. Lastly, given the apical release of HSV-1 in neurons, it opens up the possibility that this kinase might regulate some apical sorting.     

Plant endosomal trafficking pathways

Endosomes regulate both the recycling and degradation of plasma membrane (PM) proteins, thereby modulating many cellular responses triggered at the cell surface. Endosomes also play a role in the biosynthetic pathway by taking proteins to the vacuole and recycling vacuolar cargo receptors. In plants, the trans-Golgi network (TGN) acts as an early/recycling endosome whereas prevacuolar compartments/multivesicular bodies (MVBs) take PM proteins to the vacuole for degradation. Recent studies have demonstrated that some of the molecular complexes that mediate endosomal trafficking, such as the retromer, the ADP-ribosylation factor (ARF) machinery, and the Endosomal Sorting Complexes Required for Transport (ESCRTs) have both conserved and specialized functions in plants. Whereas there is disagreement on the subcellular localization of the plant retromer, its function in recycling vacuolar sorting receptors (VSRs) and modulating the trafficking of PM proteins has been well established. Studies on Arabidopsis ESCRT components highlight the essential role of this complex in cytokinesis, plant development, and vacuolar organization. In addition, post-translational modifications of plant PM proteins, such as phosphorylation and ubiquitination, have been demonstrated to act as sorting signals for endosomal trafficking.     

Ubiquitin in trafficking: The network at work

Targeting of membrane proteins to their proper destination requires specific mechanisms. Protein cargos are included in vesicles that bud off a donor organelle and ultimately fuse with a target organelle, where the cargos are delivered. Endocytosis of transmembrane receptors (e.g., receptor tyrosine kinases, RTKs) follows a common scheme that consists of an internalization reaction and a delivery step, during which cargos are transferred to an endosomal station to be either directed to the lysosome for degradation or recycled back to the cell surface. At each stage along the endocytic route, short motifs within protein cargos and/or post-translational modifications regulate transmembrane receptor sorting. In recent years, studies have shown that ubiquitination acts as a signal for the internalization and sorting of plasma membrane proteins. Here, we present an overview of ubiquitin's role as a ‘signal’ for intracellular trafficking and give examples of the multifaced mechanisms of ubiquitin-regulated RTK endocytosis.     

Global analysis of yeast endosomal transport identifies the vps55/68 sorting complex

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process.     

The proteasome alpha-subunit XAPC7 interacts specifically with Rab7 and late endosomes

Rab7 is a key regulatory protein governing early to late endocytic membrane transport. In this study the proteasome alpha-subunit XAPC7 (also known as PSMA7, RC6-1, and HSPC in mammals) was identified to interact specifically with Rab7 and was recruited to multivesicular late endosomes through this interaction. The protein interaction domains were localized to the C terminus of XAPC7 and the N terminus of Rab7. XAPC7 was not found on early or recycling endosomes, but could be recruited to recycling endosomes by expression of a Rab7-(1-174)Rab11-(160-202) chimera, establishing a central role for Rab7 in the membrane recruitment of XAPC7. Although XAPC7 could be shown to associate with membranes bearing ubiquitinated cargo, overexpression had no impact on steady-state ubiquitinated protein levels. Most notably, overexpression of XAPC7 was found to impair late endocytic transport of two different membrane proteins, including EGFR known to be highly dependent on ubiquitination and proteasome activity for proper endocytic sorting and lysosomal transport. Decreased late endocytic transport caused by XAPC7 overexpression was partially rescued by coexpression of wild-type Rab7, suggesting a negative regulatory role for XAPC7. Nevertheless, Rab7 itself was not subject to XAPC7-dependent proteasomal degradation. Together the data establish the first direct molecular link between the endocytic trafficking and cytosolic degradative machineries.     

Sorting Out the Sorting Functions of Endosomes in Arabidopsis

In animals, sorting of membrane proteins following their internalization from the plasma membrane (PM) by endocytosis occurs through a series of different endosomal compartments. In plants, how and where these sorting events take place is still poorly understood and our current view of the endocytic pathway still largely relies on analogies made from the animal system. However, extensive differences seem to exist between animal and plant endosomal functions, as exemplified by the role of the trans-Golgi network (TGN) as an early endosomal compartment in plants or the functional diversification of conserved sorting complexes. By using the Arabidopsis root tip as a reference model, we and other have begun to shed light on the complexity of the plant endocytic pathways. Notably, we have recently characterized the functions of an endosomal compartment, the SNX1-endosomes, also referred to as the prevacuolar compartment (PVC) or multivesicular bodies (MVB), in the sorting of different cargo proteins, including two related auxin-efflux carriers, PIN1 and PIN2. We have shown that routing decisions take place at this endosomal level, such as the sorting of PIN2 toward the lytic vacuole for degradation or PIN1 toward the PM for recycling.Key Words: Arabidopsis, intracellular trafficking, endocytic recycling, endosomes, MVB, PVC, VPS29, SNX, PIN, cell polarity     

Evidence for a sorting endosome in Arabidopsis root cells

In eukaryotic cells, the endocytic and secretory pathways are key players in several physiological processes. These pathways are largely inter-connected in animal and yeast cells through organelles named sorting endosomes. Sorting endosomes are multi-vesicular compartments that redirect proteins towards various destinations, such as the lysosomes or vacuoles for degradation, the trans-Golgi network for retrograde transport and the plasma membrane for recycling. In contrast, cross-talk between the endocytic and secretory pathways has not been clearly established in plants, especially in terms of cargo protein trafficking. Here we show by co-localization analyses that endosomes labelled with the AtSORTING NEXIN1 (AtSNX1) protein overlap with the pre-vacuolar compartment in Arabidopsis root cells. In addition, alteration of the routing functions of AtSNX1 endosomes by drug treatments leads to mis-routing of endocytic and secretory cargo proteins. Based on these results, we propose that the AtSNX1 endosomal compartment represents a sorting endosome in root cells, and that this specialized organelle is conserved throughout eukaryotes.