Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells

The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.     

Rab6-interacting protein 1 links Rab6 and Rab11 function

Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein. R6IP1 also binds to Rab11A in its GTP-bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6-dependent manner but can associate with Rab11-positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.     

A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex

Mammalian cells endocytose a variety of proteins and lipids without utilising clathrin-coated pits. Detailed molecular mechanisms for clathrin-independent endocytosis are unclear. Several markers for this process, including glycosphingolipid-binding bacterial toxin subunits such as cholera toxin B subunit (CTxB), and glycosyl-phosphatidyl-inositol (GPI)-anchored proteins, are found in detergent-resistant membrane fractions (DRMs), or 'lipid rafts'. The Golgi complex constitutes one principal intracellular destination for these markers. Uptake of both CTxB and GPI-anchored proteins may involve caveolae, small invaginations in the plasma membrane (PM). However, the identity of intermediate organelles involved in PM to Golgi trafficking, as well as the function of caveolins, defining protein components of caveolae, are unclear. This paper shows that molecules which partition into DRMs and are endocytosed in a clathrin-independent fashion, accumulate in a discrete population of endosomes en route to the Golgi complex. These endosomes are devoid of markers for classical early and recycling endosomes, but do contain caveolin-1. Caveolin-1-positive endosomes are sites for the sorting of caveolin-1 away from Golgi-bound cargoes, although caveolin-1 itself is unlikely to have a direct function in PM to Golgi transport.     

Rab6 functions in polarized transport in Drosophila photoreceptors

Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected.

The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs.     

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi. In this study, we show that active palmitoylated Ras proteins mainly localize intracellularly at REs and that REs act as a way station along the post-Golgi exocytic pathway to the PM. H-Ras requires two palmitoyl groups for RE targeting. The lack of either or both palmitoyl groups leads to the mislocalization of the mutant proteins to the endoplasmic reticulum, Golgi apparatus, or the PM. Therefore, we demonstrate that palmitoylation directs Ras proteins to the correct intracellular organelles for trafficking and activity.     

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.     

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus.     

GCC185 plays independent roles in Golgi structure maintenance and AP-1-mediated vesicle tethering

GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)-containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.     

SMAP2, a novel ARF GTPase-activating protein, interacts with clathrin and clathrin assembly protein and functions on the AP-1-positive early endosome/trans-Golgi network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.     

TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain-containing Rab guanosine triphosphatase-activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)-positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.     

Golgi-58K can re-localize to late endosomes upon cellular uptake of PS-ASOs and facilitates endosomal release of ASOs

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs can trigger RNase H1 cleavage of cellular target RNAs to modulate gene expression. Internalized PS-ASOs must be released from membraned endosomal organelles, a rate limiting step that is not well understood. Recently we found that M6PR transport between Golgi and late endosomes facilitates productive release of PS-ASOs, raising the possibility that Golgi-mediated transport may play important roles in PS-ASO activity. Here we further evaluated the involvement of Golgi in PS-ASO activity by examining additional Golgi proteins. Reduction of certain Golgi proteins, including Golgi-58K, GCC1 and TGN46, decreased PS-ASO activity, without substantial effects on Golgi integrity. Upon PS-ASO cellular uptake, Golgi-58K was recruited to late endosomes where it colocalized with PS-ASOs. Reduction of Golgi-58K caused slower PS-ASO release from late endosomes, decreased GCC2 late endosome relocalization, and led to slower retrograde transport of M6PR from late endosomes to trans-Golgi. Late endosome relocalization of Golgi-58K requires Hsc70, and is most likely mediated by PS-ASO–protein interactions. Together, these results suggest a novel function of Golgi-58K in mediating Golgi-endosome transport and indicate that the Golgi apparatus plays an important role in endosomal release of PS-ASO, ensuring antisense activity.     

Annexin A1 and A2: roles in retrograde trafficking of Shiga toxin

Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA(2)), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA(2) activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA(2).     

Phosphoinositide-regulated retrograde transport of ricin: crosstalk between hVps34 and sorting nexins

The plant toxin ricin is transported from the plasma membrane via early endosomes and the Golgi apparatus to the endoplasmic reticulum. From this compartment, it enters the cytosol and inhibits protein synthesis. Lipid phosphorylation is an important regulator of vesicular transport, and in the present study we have investigated the role of the phosphatidylinositol (PI) 3-kinase hVps34 in retrograde transport of ricin. Our data demonstrate that transport of ricin from endosomes to the Golgi apparatus in human embryonic kidney cells (HEK 293) is dependent on PI(3)P. By using PI 3-kinase inhibitors, by sequestering the hVps34 product PI(3)P and by expressing mutants of hVps34 or small interfering RNA targeted against its messenger RNA, we show that hVps34 and its product PI(3)P are involved in transport of ricin from endosome to Golgi apparatus. Furthermore, we identify two effector proteins in the hVps34-dependent pathway, namely sorting nexin (SNX) 2 and SNX4. Knockdown of SNX2 or SNX4 inhibits ricin transport to the Golgi apparatus to the same extent as when hVps34 is perturbed. Furthermore, inhibition or knockdown of hVps34 redistributes these proteins. Interestingly, knocking down both SNX2 and SNX4 results in a better inhibition than knocking down only one of them, suggesting that they may act on separate pathways.     

Rapid cycling of lipid raft markers between the cell surface and Golgi complex

The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 degrees C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.     

A Highlights from MBoC Selection: Cooperation of MICAL-L1, syndapin2, and phosphatidic acid in tubular recycling endosome biogenesis

Endocytic transport necessitates the generation of membrane tubules and their subsequent fission to transport vesicles for sorting of cargo molecules. The endocytic recycling compartment, an array of tubular and vesicular membranes decorated by the Eps15 homology domain protein, EHD1, is responsible for receptor and lipid recycling to the plasma membrane. It has been proposed that EHD dimers bind and bend membranes, thus generating recycling endosome (RE) tubules. However, recent studies show that molecules interacting with CasL-Like1 (MICAL-L1), a second, recently identified RE tubule marker, recruits EHD1 to preexisting tubules. The mechanisms and events supporting the generation of tubular recycling endosomes were unclear. Here, we propose a mechanism for the biogenesis of RE tubules. We demonstrate that MICAL-L1 and the BAR-domain protein syndapin2 bind to phosphatidic acid, which we identify as a novel lipid component of RE. Our studies demonstrate that direct interactions between these two proteins stabilize their association with membranes, allowing for nucleation of tubules by syndapin2. Indeed, the presence of phosphatidic acid in liposomes enhances the ability of syndapin2 to tubulate membranes in vitro. Overall our results highlight a new role for phosphatidic acid in endocytic recycling and provide new insights into the mechanisms by which tubular REs are generated.     

Regulation of Cell Death by Recycling Endosomes and Golgi Membrane Dynamics via a Pathway Involving Src-family kinases,Cdc42 and Rab11a

Actin dynamics and membrane trafficking influence cell commitment to programmed cell death through largely undefined mechanisms. To investigate how actin and recycling endosome (RE) trafficking can engage death signaling, we studied the death program induced by the adenovirus early region 4 open reading frame 4 (E4orf4) protein as a model. We found that in the early stages of E4orf4 expression, Src-family kinases (SFKs), Cdc42, and actin perturbed the organization of the endocytic recycling compartment and promoted the transport of REs to the Golgi apparatus, while inhibiting recycling of protein cargos to the plasma membrane. The resulting changes in Golgi membrane dynamics that relied on actin-regulated Rab11a membrane trafficking triggered scattering of Golgi membranes and contributed to the progression of cell death. A similar mobilization of RE traffic mediated by SFKs, Cdc42 and Rab11a also contributed to Golgi fragmentation and to cell death progression in response to staurosporine, in a caspase-independent manner. Collectively, these novel findings suggest that diversion of RE trafficking to the Golgi complex through a pathway involving SFKs, Cdc42, and Rab11a plays a general role in death signaling by mediating regulated changes in Golgi dynamics.     

Transferrin receptor recycling in the absence of perinuclear recycling endosomes

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.     

ARF1 and ARF4 regulate recycling endosomal morphology and retrograde transport from endosomes to the Golgi apparatus

Small GTPases of the ADP-ribosylation factor (ARF) family, except for ARF6, mainly localize to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We recently showed that class I ARFs (ARF1 and ARF3) localize to recycling endosomes, as well as to the Golgi, and are redundantly required for recycling of endocytosed transferrin. On the other hand, the roles of class II ARFs (ARF4 and ARF5) are not yet fully understood, and the complementary or overlapping functions of class I and class II ARFs have been poorly characterized. In this study, we find that simultaneous depletion of ARF1 and ARF4 induces extensive tubulation of recycling endosomes. Moreover, the depletion of ARF1 and ARF4 inhibits retrograde transport of TGN38 and mannose-6-phosphate receptor from early/recycling endosomes to the trans-Golgi network (TGN) but does not affect the endocytic/recycling pathway of transferrin receptor or inhibit retrograde transport of CD4-furin from late endosomes to the TGN. These observations indicate that the ARF1+ARF4 and ARF1+ARF3 pairs are both required for integrity of recycling endosomes but are involved in distinct transport pathways: the former pair regulates retrograde transport from endosomes to the TGN, whereas the latter is required for the transferrin recycling pathway from endosomes to the plasma membrane.