Retrograde Transport: Two (or More) Roads Diverged in an Endosomal Tree?

Some proteins and lipids traffic from the plasma membrane to the trans Golgi network (TGN)/Golgi apparatus and the endoplasmic reticulum, via the retrograde transport route. Endosomes are an obligatory through station. Whether early, recycling and late endosomes all hand off material to the TGN have remained a matter of debate. In this review, we give a short historical overview on how retrograde transport was discovered and explored. We then summarize and critically discuss data that have been put forward in favour of the existence of trafficking interfaces between each of the different endocytic localizations and the TGN. We finally point out some conceptual and technological challenges that will have to be met to establish definite conclusions for each of these scenarios.     

Endosome-to-Golgi transport pathways in physiological processes

The trans-Golgi network (TGN) is a major traffic hub of the cell, as it regulates membrane transport in the secretory pathway as well as receiving protein cargo by retrograde transport from endocytic compartments. Retrograde transport between endosomes and the TGN is essential for the recycling of membrane proteins which regulate a range of cellular and development functions. In addition, retrograde transport pathways are exploited by many bacterial toxins to mediate cytotoxicity and by some viral proteins to promote pathogenicity. Recent advances using a range of molecular cell biological strategies have identified multiple retrograde transport pathways each regulated by a distinct set of molecular machinery. Here we review recent advances in this field and highlight the importance of these transport pathways in a range of physiological processes.     

Protein kinase D-mediated anterograde membrane trafficking is required for fibroblast motility

BACKGROUND: Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.RESULTS: To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.CONCLUSIONS: These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration.     

SNAP-tag based proteomics approach for the study of the retrograde route

Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine-tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN-localized SNAP-tagged fusion protein, which allows for their identification. The approach was validated step-by-step using a well explored retrograde cargo protein, the B-subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.     

Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J.Cell Biol.142:923-936).Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.     

Sending out molecules from the TGN

The sorting of secreted cargo proteins and their export from the trans-Golgi network (TGN) remains an enigma in the field of membrane trafficking; although the sorting mechanisms of many transmembrane proteins have been well described. The sorting of secreted proteins at the TGN is crucial for the release of signaling factors, as well as extracellular matrix proteins. These proteins are required for cell–cell communication and integrity of an organism. Missecretion of these factors can cause diseases such as neurological disorders, autoimmune disease, or cancer. The major open question is how soluble proteins that are not associated with the membrane are packed into TGN derived transport carriers to facilitate their transport to the plasma membrane. Recent investigations have identified novel types of protein and lipid machinery that facilitate the packing of these molecules into a TGN derived vesicle. In addition, novel research has uncovered an exciting link between cargo sorting and export in which TGN structure and dynamics, as well as TGN/endoplasmic reticulum contact sites, play a significant role. Here, we have reviewed the progress made in our understanding of these processes.     

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.     

Cargo trafficking between endosomes and the trans-Golgi network

The retrograde membrane transport pathways from endosomes to the trans-Golgi network (TGN) are now recognized as critical intracellular pathways to recycle and shuttle a selective subgroup of membrane proteins, including sorting receptors, membrane-bound enzymes, transporters, as well as providing an avenue for the intracellular transport of various bacterial toxins. Multiple pathways from endosomes to the TGN have now been defined which differ between the cargo transported and the machinery used. Here, we review advances in these pathways and the requirement for TGN organization, and also discuss the development of unbiased analytical approaches to quantitatively track cargo that use these endosome-to-TGN pathways.     

The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.     

Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.     

Cargo trafficking from the trans‐Golgi network towards the endosome

The trans‐Golgi network (TGN) is a major sorting, packing and delivering station of newly synthesised proteins and lipids to their final destination. These cargo molecules follow the secretory pathway, which is a vital part of cellular trafficking machinery in all eukaryotic cells. This secretory pathway is well conserved in all eukaryotes from low‐level eukaryotes, such as yeast, to higher level eukaryotes like mammals. The molecular mechanisms of protein sorting by adaptor proteins, membrane elongation and transport to the final destinations by motor proteins and the cytoskeleton, and membrane pinching‐off by scission proteins must be choreographically managed for efficient cargo delivery, and the understanding of these detailed processes is not yet completed. Functionally, defects in these mechanisms are associated with the pathology of prominent diseases such as acute myeloid leukaemia, Charcot–Marie–Tooth disease, I‐cell disease and Wiskott–Aldrich syndrome. The present review points out the recent advances in our knowledge of the molecular mechanisms involved in the transportation of the cargo from the TGN towards the endosome.     

Direct Pathway from Early/Recycling Endosomes to the Golgi Apparatus Revealed through the Study of Shiga Toxin B-fragment Transport

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.     

A Highlights from MBoC Selection: FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.     

Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport

Regulation of membrane transport is controlled by small G proteins, which include members of the Rab and Arf families. Whereas the role of the classic Arf family members are well characterized, many of the Arf-like proteins (Arls) remain poorly defined. Here we show that Arl5a and Arl5b are localised to the trans-Golgi in mammalian cells, and furthermore have identified a role for Arl5b in the regulation of retrograde membrane transport from endosomes to the trans-Golgi network (TGN). The constitutively active Arl5b (Q70L)-GFP mutant was localised efficiently to the Golgi in HeLa cells whereas the dominant-negative Arl5b (T30N)-GFP mutant was dispersed throughout the cytoplasm and resulted in perturbation of the Golgi apparatus. Stable HeLa cells expressing GFP-tagged Arl5b (Q70L) showed an increased rate of endosome-to-Golgi transport of the membrane cargo TGN38 compared with control HeLa cells. Depletion of Arl5b by RNAi resulted in an alteration in the intracellular distribution of mannose-6-phosphate receptor, and significantly reduced the endosome-to-TGN transport of the membrane cargo TGN38 and of Shiga toxin, but had no affect on the anterograde transport of the cargo E-cadherin. Collectively these results suggest that Arl5b is a TGN-localised small G protein that plays a key role in regulating transport along the endosome-TGN pathway.     

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).     

Redundant roles of BIG2 and BIG1, guanine-nucleotide exchange factors for ADP-ribosylation factors in membrane traffic between the trans-Golgi network and endosomes

BIG2 and BIG1 are closely related guanine-nucleotide exchange factors (GEFs) for ADP-ribosylation factors (ARFs) and are involved in the regulation of membrane traffic through activating ARFs and recruiting coat protein complexes, such as the COPI complex and the AP-1 clathrin adaptor complex. Although both ARF-GEFs are associated mainly with the trans-Golgi network (TGN) and BIG2 is also associated with recycling endosomes, it is unclear whether BIG2 and BIG1 share some roles in membrane traffic. We here show that knockdown of both BIG2 and BIG1 by RNAi causes mislocalization of a subset of proteins associated with the TGN and recycling endosomes and blocks retrograde transport of furin from late endosomes to the TGN. Similar mislocalization and protein transport block, including furin, were observed in cells depleted of AP-1. Taken together with previous reports, these observations indicate that BIG2 and BIG1 play redundant roles in trafficking between the TGN and endosomes that involves the AP-1 complex.     

Possible implication of Golgi-nucleating function for the centrosome

The Golgi apparatus breaks down at mitosis, resulting in the dispersal of Golgi-resident proteins. In NRK cells, however, subsets of both TGN38 and golgin-97, but not ManII and GM130, remained associated with the centrosome throughout the cell cycle. This centrosome association of TGN38 and golgin-97 was not disrupted by treatment with brefeldin A, additional inducers of retrograde trafficking and inhibitors of either kinases or protein phosphatases. Anchoring of the Golgi apparatus within the juxtanuclear region depends on microtubules; the association of TGN38 and golgin-97 subsets with the centrosome, however, was insensitive to nocodazole treatment. Drugs such as PDMP, which block Golgi dispersal both by nocodazole, despite microtubule depolymerization, and by inducers of retrograde trafficking, strengthened the microtubule-nucleating activity of the centrosome. These observations cumulatively suggest the centrosome is implicated in nucleation of the Golgi apparatus through interactions with Golgi-resident proteins, such as TGN38 and golgin-97.     

Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.     

Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal‐Like Compartments and the Golgi Complex

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post‐Golgi and it has been speculated that they pass through endosomal‐like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well‐established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant‐like secretory pathway .     

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.