The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome

The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some but not all ERC vesicles are tethered by contiguous “membrane bridges.” Furthermore, tubular recycling endosomes preferentially traffic CIE cargo and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC.     

Coated endosomal vesicles: sorting and recycling compartment for transferrin in BHK cells.

We have investigated receptor-mediated endocytosis of transferrin (Tf) in baby hamster kidney (BHK) cells, using fluorescence and electron microscopy, and by carrying out colocalization experiments with clathrin antibodies and a fluorescently tagged glycolipid. Early during internalization, Tf was found in small vesicles (100-150 nm in diameter) located at the cell periphery. The ligand remained associated with such vesicles when the latter concentrated towards the cell center, before ending up in the juxtanuclear area. Throughout this vesicular trafficking pathway, clathrin colocalized with Tf. We conclude that Tf is processed intracellularly via small coated endosomal vesicles (CEV) and is not delivered into large tubular endosomes (CURL; compartment for uncoupling receptors and ligands), typical for ligand trafficking to lysosomes. By determining the kinetics of Tf internalization and by comparing the flow of Tf to that of a fluorescent glycolipid, it can also be concluded that CEVs display sorting and recycling properties, implying that small vesicles can be shed from or fuse with CEVs. Acidic pH does not prevent the formation of CEVs, but their intracellular movement, towards the cell center, is impeded.     

ACAP1 promotes endocytic recycling by recognizing recycling sorting signals

Cargo sorting that promotes the transport of cargo proteins from a membrane compartment has been predicted to be unlikely in the endocytic recycling pathways. We now show that ACAP1 binds specifically and directly to recycling cargo proteins. Reducing this interaction for TfR inhibits its recycling. Moreover, ACAP1 binds to two distinct phenylalanine-based sequences in the cytoplasmic domain of TfR that function as recycling sorting signals to promote its transport from the recycling endosome. Taken together, these findings indicate that ACAP1 promotes cargo sorting by recognizing recycling sorting signals.     

Getting active: protein sorting in endocytic recycling

Endocytic recycling returns proteins to the plasma membrane in many physiological contexts. Studies of these events have helped to elucidate fundamental mechanisms that underlie recycling. Recycling was for some time considered to be the exception to a general mechanism of active cargo sorting in multiple intracellular pathways. In recent years, studies have begun to reconcile this seeming disparity and also suggest explanations for why early recycling studies did not detect active sorting. Further articulation of this emerging trend has far-reaching implications for a deeper understanding of many physiological and pathological events that require recycling.     

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Membrane transport: ubiquitylation in endosomal sorting.

In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies. A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor.     

Activity-dependent endocytic sorting of kainate receptors to recycling or degradation pathways

Kainate receptors (KARs) play important roles in the modulation of neurotransmission and plasticity, but the mechanisms that regulate their surface expression and endocytic sorting remain largely unknown. Here, we show that in cultured hippocampal neurons the surface expression of GluR6-containing KARs is dynamically regulated. Furthermore, internalized KARs are sorted into recycling or degradative pathways depending on the endocytotic stimulus. Kainate activation causes a Ca2+- and PKA-independent but PKC-dependent internalization of KARs that are targeted to lysosomes for degradation. In contrast, NMDAR activation evokes a Ca2+-, PKA- and PKC-dependent endocytosis of KARs to early endosomes with subsequent reinsertion back into the plasma membrane. These results demonstrate that GluR6-containing KARs are subject to activity-dependent endocytic sorting, a process that provides a mechanism for both rapid and chronic changes in the number of functional receptors.     

Rab15 effector protein: a novel protein for receptor recycling from the endocytic recycling compartment

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways.     

Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.     

GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment

Glycosylphosphatidylinositol (GPI) anchoring is important for the function of several proteins in the context of their membrane trafficking pathways. We have shown previously that endocytosed GPI-anchored proteins (GPI-APs) are recycled to the plasma membrane three times more slowly than other membrane components. Recently, we found that GPI-APs are delivered to endocytic organelles, devoid of markers of the clathrin-mediated pathway, prior to their delivery to a common recycling endosomal compartment (REC). Here we show that the rate-limiting step in the recycling of GPI-APs is their slow exit from the REC; replacement of the GPI anchor with a transmembrane protein sequence abolishes retention in this compartment. Depletion of endogenous sphingolipid levels using sphingolipid synthesis inhibitors or in a sphingolipid-synthesis mutant cell line specifically enhances the rate of endocytic recycling of GPI-APs to that of other membrane components. We have shown previously that endocytic retention of GPI-APs is also relieved by cholesterol depletion. These findings strongly suggest that functional retention of GPI-APs in the REC occurs via their association with sphingolipid and cholesterol-enriched sorting platforms or 'rafts'.     

Phosphorylation of SNX27 by MAPK11/14 links cellular stress–signaling pathways with endocytic recycling

Endocytosed proteins can be delivered to lysosomes for degradation or recycled to either the trans-Golgi network or the plasma membrane. It remains poorly understood how the recycling versus degradation of cargoes is determined. Here, we show that multiple extracellular stimuli, including starvation, LPS, IL-6, and EGF treatment, can strongly inhibit endocytic recycling of multiple cargoes through the activation of MAPK11/14. The stress-induced kinases in turn directly phosphorylate SNX27, a key regulator of endocytic recycling, at serine 51 (Ser51). Phosphorylation of SNX27 at Ser51 alters the conformation of its cargo-binding pocket and decreases the interaction between SNX27 and cargo proteins, thereby inhibiting endocytic recycling. Our study indicates that endocytic recycling is highly dynamic and can crosstalk with cellular stress–signaling pathways. Suppression of endocytic recycling and enhancement of receptor lysosomal degradation serve as new mechanisms for cells to cope with stress and save energy.     

Rab11-FIP3 is critical for the structural integrity of the endosomal recycling compartment

Rab11-FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient cause loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific because expression of the equivalent Rab11-binding deficient mutant of Rab-coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.     

Structural insights into endosomal sorting complex required for transport (ESCRT-I) recognition of ubiquitinated proteins

The endosomal sorting complex required for transport (ESCRT-I) is a 350-kDa complex of three proteins, Vps23, Vps28, and Vps37. The N-terminal ubiquitin-conjugating enzyme E2 variant (UEV) domain of Vps23 is required for sorting ubiquitinated proteins into the internal vesicles of multivesicular bodies. UEVs are homologous to E2 ubiquitin ligases but lack the conserved cysteine residue required for catalytic activity. The crystal structure of the yeast Vps23 UEV in a complex with ubiquitin (Ub) shows the detailed interactions made with the bound Ub. Compared with the solution structure of the Tsg101 UEV (the human homologue of Vps23) in the absence of Ub, two loops that are conserved among the ESCRT-I UEVs move toward each other to grip the Ub in a pincer-like grasp. The contacts with the UEV encompass two adjacent patches on the surface of the Ub, one containing several hydrophobic residues, including Ile-8(Ub), Ile-44(Ub), and Val-70(Ub), and the second containing a hydrophilic patch including residues Asn-60(Ub), Gln-62(Ub), Glu-64(Ub). The hydrophobic Ub patch interacting with the Vps23 UEV overlaps the surface of Ub interacting with the Vps27 ubiquitin-interacting motif, suggesting a sequential model for ubiquitinated cargo binding by these proteins. In contrast, the hydrophilic patch encompasses residues uniquely interacting with the ESCRT-I UEV. The structure provides a detailed framework for design of mutants that can specifically affect ESCRT-I-dependent sorting of ubiquitinated cargo without affecting Vps27-mediated delivery of cargo to endosomes.     

Rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells

RME-1 is an Eps15-homology (EH)-domain protein that was identified in a genetic screen for endocytosis genes in Caenorhabditis elegans. When expressed in a CHO cell line, the worm RME-1 protein and a mouse homologue are both associated with the endocytic recycling compartment. Here we show that expression of a dominant-negative construct with a point mutation near the EH domain results in redistribution of the endocytic recycling compartment and slowing down of transferrin receptor recycling. The delivery of a TGN38 chimaeric protein to the trans-Golgi network is also slowed down. The function of Rme-1 in endocytic recycling is evolutionarily conserved in metazoans as shown by the protein's properties in C. elegans.     

SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27–Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27–Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.

Coat complexes coordinate cargo recognition with biogenesis of transport carriers during integral membrane protein trafficking. Mechanistic study of the function and evolution of the SNX27:Retromer:ESCPE-1 assembly provides new insight into pathway defects associated with neurodegenerative disease and an interesting comparison with the yeast pentameric Retromer.     

Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1–decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA–mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin–transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca²⁺ switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca²⁺ repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.     

Insulin stimulates the entry of GLUT4 into the endosomal recycling pathway by a quantal mechanism

The insulin-sensitive glucose transporter GLUT4 mediates the uptake of glucose into adipocytes and muscle cells. In this study we have used a novel 96-well plate fluorescence assay to study the kinetics of GLUT4 trafficking in 3T3-L1 adipocytes. We have found evidence for a graded release mechanism whereby GLUT4 is released into the plasma membrane recycling system in a nonkinetic manner as follows: the kinetics of appearance of GLUT4 at the plasma membrane is independent of the insulin concentration; a large proportion of GLUT4 molecules do not participate in plasma membrane recycling in the absence of insulin; and with increasing insulin there is an incremental increase in the total number of GLUT4 molecules participating in the recycling pathway rather than simply an increased rate of recycling. We propose a model whereby GLUT4 is stored in a compartment that is disengaged from the plasma membrane recycling system in the basal state. In response to insulin, GLUT4 is quantally released from this compartment in a pulsatile manner, leaving some sequestered from the recycling pathway even in conditions of excess insulin. Once disengaged from this location we suggest that in the continuous presence of insulin this quanta of GLUT4 continuously recycles to the plasma membrane, possibly via non-endosomal carriers that are formed at the perinuclear region.     

Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles.     

Increased endosomal sorting of ligand to recycling enhances potency of an interleukin-2 analog

An interleukin-2 (IL-2) variant containing adjacent point mutations (L18M/L19S, termed 2D1) displaying binding affinity to the heterotrimeric IL-2 receptor similar to that of wild-type IL-2 (WT) had been previously found to surprisingly exhibit increased bioactivity in a peripheral blood lymphocyte proliferation assay. In order to provide an explanatory mechanism for this unexpected potency enhancement, we hypothesize that altered endocytic trafficking of the 2D1 variant might be responsible by increasing the number of ligand-receptor complexes. We demonstrate here that the internalization kinetics of 2D1 via the high affinity IL-2 receptor are equivalent to those of WT but that a significantly increased fraction of internalized 2D1 is sorted to recycling instead of to lysosomal degradation. We further find a reduced pH sensitivity of binding to IL-2 receptor alpha relative to IL-2 receptor beta compared with WT, which could be responsible for the altered sorting behavior of 2D1 in the acidic endosomal compartment. Accordingly, the 2D1 variant displays a half-life 36 h longer than that of IL-2 in T-lymphocyte culture at concentrations equal to the K(D) of the IL-2 receptor. The extended half-life of intact 2D1 provides enhanced mitogenesis as compared with IL-2. In addition, 2D1 stimulates natural killer cells to a lesser degree than IL-2 at equal concentrations. We conclude that this IL-2 variant provides increased mitogenic stimulation that could not be easily predicted from its cell surface receptor binding affinity while minimizing undesired stimulation of natural killer cells. This concept of altering trafficking dynamics may offer a generalizable approach to generating improvements in the pharmacological efficacy of therapeutic cytokines.