Disturbed vesicular trafficking of membrane proteins in prion disease

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.     

Interactions between the exocytic and endocytic pathways in polarized Madin-Darby canine kidney cells

The compartments involved in polarized exocytosis of membrane proteins are not well defined. In this study we hypothesized that newly synthesized polymeric immunoglobulin receptors are targeted from the trans-Golgi network to endosomes prior to their appearance on the basolateral cell surface of polarized Madin-Darby canine kidney cells. To examine this hypothesis, we have used an assay designed to measure the meeting of newly synthesized receptors with a selective population of apical or basolateral endosomes loaded with horseradish peroxidase. We found that in the course of basolateral exocytosis, the wild-type polymeric immunoglobulin receptor is targeted from the trans-Golgi network to apical and basolateral endosomes. Phosphorylation of a Ser residue in the cytoplasmic tail of the receptor is implicated in this process. The biosynthetic pathway of apically sorted polymeric immunoglobulin receptor mutants similarly traversed apical endosomes, raising the possibility that apical receptors are segregated from basolateral receptors in apical endosomes. The post-endocytic pathway of transcytosing and recycling receptors also passed through apical endosomes. Together, these observations are consistent with the possibility that the biosynthetic and endocytic routes merge into endosomes and justify a model suggesting that endosomal recycling processes govern polarized trafficking of proteins traveling in both pathways.     

Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation

Four isoforms of the Na+/H+ exchanger (NHE6-NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid- to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.     

Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein

We have investigated the intracellular traffic of PrP(c), a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrP(c) is found at the cell surface and in intracellular compartments in SN56 cells. Confocal microscopy and organelle-specific markers suggest that the protein is found in both the Golgi and the recycling endosomal compartment. Perturbation of endocytosis with a dynamin I-K44A dominant-negative mutant altered the steady-state distribution of the GFP-PrP(c), leading to the accumulation of fluorescence in unfissioned endocytic intermediates. These pre-endocytic intermediates did not seem to accumulate GFP-GPI, a minimum GPI-anchored protein, suggesting that PrP(c) trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrP(c) accumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrP(c) but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrP(c) internalizes via a dynamin-dependent endocytic pathway and that the protein is targeted to the recycling endosomal compartment via Rab5-positive early endosomes. These observations indicate that traffic of GFP-PrP(c) is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrP(c) is delivered to classic endosomes after internalization.     

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.     

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.     

Calmodulin modulates hepatic membrane polarity by protein kinase C-sensitive steps in the basolateral endocytic pathway

Membrane polarity is maintained by a complex intermingling of various trafficking pathways, including basolateral and apical endocytosis. The present work was undertaken to better define the role of basolateral endocytic transport in apical membrane homeostasis. When polarized HepG2 hepatoma cells were incubated with calmodulin antagonists, the cells lost their polarity, as reflected by an inhibition of lipid transport of a fluorescent sphingomyelin to the apical membrane and an impediment of its recycling to the basolateral membrane. Instead, an accumulation of the lipid in dilated early endosomal compartments was observed, presumably due to a frustration of vesiculation. Interestingly, lipid transport to the apical pole, lipid recycling to the basolateral membrane and cell polarity were reestablished, while dilated compartments disappeared, when the cells were simultaneously treated with specific inhibitors of protein kinase C (PKC). Consistently, following activation of PKC, extensive dilation/vacuolation of early sorting endosomes was observed, very similar as seen upon treatment with calmodulin antagonists. Thus, the results indicate that membrane trafficking at early steps of the basolateral endocytic pathway in HepG2 cells is regulated by an intricate interplay between calmodulin and PKC. This interference, although not affecting endocytosis as such, compromises cell polarity by impeding membrane trafficking from early endosomes to the apical membrane.     

Endocytic host cell machinery plays a dominant role in intracellular trafficking of incoming human immunodeficiency virus type 1 in human placental trophoblasts

Vertical transmission of human immunodeficiency virus type 1 (HIV-1) is the primary cause of infection by this retrovirus in infants. In this study, we report for the first time that there is a correlation between endocytic uptake of HIV-1 and virus gene expression in polarized trophoblasts. To shed light on the relationship between endocytosis and the fate of HIV-1 in polarized trophoblasts, the step-by-step movements of HIV-1 within the endocytic compartments were tracked by confocal imaging. Incoming virions were initially located in early endosomes. As time progressed, virions accumulated in late endosomes. HIV-1 was also found in apical recycling endosomes and at the basolateral pole. Experiments performed with indicator cells revealed that HIV-1 is recycled and transcytosed. These data indicate that the intracellular trafficking of HIV-1 upon entry into polarized human trophoblasts is a complex process which requires the active participation of the endocytic host cell machinery.     

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

Tracking down the elusive early endosome

Despite significant progress in understanding protein trafficking and compartmentation in plants, the identification and protein compartmentalization for organelles that belong to both the secretory and endocytic pathways have been difficult because protein trafficking has generally been studied separately in these two pathways. However, recent data indicate that the trans-Golgi network serves as an early endosome merging the secretory and endocytic pathways in plant cells. Here, we discuss the proteins identified as markers for post-Golgi compartments in these two pathways and propose that the trans-Golgi network is a pivotal organelle with multiple sorting domains for post-Golgi protein trafficking in plant cells.     

Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells

Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.     

Rab8 regulates basolateral secretory, but not recycling, traffic at the recycling endosome

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the mu1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.     

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.     

Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator

Endocytic motifs in the carboxyl terminus of cystic fibrosis transmembrane conductance regulator (CFTR) direct internalization from the plasma membrane by clathrin-mediated endocytosis. However, the fate of such internalized CFTR has remained unknown. Internalized membrane proteins can be either targeted for degradation or recycled back to the plasma membrane. Using cell surface biotinylation and antibody uptake studies, we show that CFTR undergoes constitutive endocytosis and recycling back to the plasma membrane. Expression of dominant negative Rme-1 (a protein that regulates exit from the endosomal recycling compartment) in CFTR-expressing cells results in the expansion of recycling compartments. Transferrin, a marker for the endosomal recycling compartment, and CFTR accumulate in these enlarged recycling endosomes. Such accumulation leads to a loss of cell surface CFTR because it is prevented from being recycled back to the cell surface. In contrast, traffic of the low-density lipoprotein (LDL) is unaffected by the expression of dominant negative Rme-1. In addition, chimeras containing the extracellular domain of the transferrin receptor and the carboxyl terminal tail of CFTR also enter Rme-1-regulated recycling compartments and accumulate in these compartments containing dominant negative Rme-1, suggesting that in addition to endocytic signals, the carboxyl terminal tail of CFTR also contains intracellular traffic information.     

Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments

Polarized epithelial cells sort newly synthesized and recycling plasma membrane proteins into distinct trafficking pathways directed to either the apical or basolateral membrane domains. While the trans‐Golgi network is a well‐established site of protein sorting, increasing evidence indicates a key role for endosomes in the initial trafficking of newly synthesized proteins. Both basolateral and apical proteins have been shown to traverse endosomes en route to the plasma membrane. In particular, apical proteins traffic through either subapical early or recycling endosomes. Here we use the SNAP tag system to analyze the trafficking of the apical protein gp135, also known as podocalyxin. We show that newly synthesized gp135 traverses the apical recycling endosome, but not the apical early endosomes (AEEs). In contrast, post‐endocytic gp135 is delivered to the AEE before recycling back to the apical membrane. The pathways pursued by the newly synthesized and recycling gp135 populations do not detectably intersect, demonstrating that the biosynthetic and post‐endocytic pools of this protein are subjected to distinct sorting processes.      

Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.     

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.     

Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medical recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.     

RhoB-dependent modulation of postendocytic traffic in polarized Madin-Darby canine kidney cells

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein-tagged wild-type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin-Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant-negative RhoBN19 or dominant-active RhoBV14 altered postendocytic traffic of ligand-receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral-to-apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.     

Efficiency of Immunotoxin Cytotoxicity Is Modulated by the Intracellular Itinerary

Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F_v)-PE38), are proposed to traffic to the trans-Golgi network (TGN) and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO) cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity – presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.