The clathrin heavy chain isoform CHC22 functions in a novel endosomal sorting step

Clathrin heavy chain 22 (CHC22) is an isoform of the well-characterized CHC17 clathrin heavy chain, a coat component of vesicles that mediate endocytosis and organelle biogenesis. CHC22 has a distinct role from CHC17 in trafficking glucose transporter 4 (GLUT4) in skeletal muscle and fat, though its transfection into HEK293 cells suggests functional redundancy. Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells. CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer. In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome–TGN transport as critical for GLUT4 trafficking. Like CHC22, syntaxin 10 is not expressed in mice but is present in humans and other vertebrates, implicating two species-restricted endosomal traffic proteins in GLUT4 transport.     

Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

trans-Golgi network-bound cargo traffic

Cargo following the retrograde trafficking are sorted at endosomes to be targeted the trans-Golgi network (TGN), a central receiving organelle. Though molecular requirements and their interaction networks have been somewhat established, the complete understanding of the intricate nature of their action mechanisms in every step of the retrograde traffic pathway remains unachieved. This review focuses on elucidating known functions of key regulators, including scission factors at the endosome and tethering/fusion mediators at the receiving dock, TGN, as well as a diverse range of cargo.     

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.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes

Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu‐directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF‐B and in the liver in vivo. Copper (10 µm ) caused ATP7B to exit the trans‐Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton‐pump inhibitor bafilomycin‐A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu‐stimulated) case. Overall, loss of acidification‐impaired Cu‐regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane.      

Domains within the GARP subunit Vps54 confer separate functions in complex assembly and early endosome recognition

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking 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.     

Analysis of Articulation Between Clathrin and Retromer in Retrograde Sorting on Early Endosomes

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans -Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes–TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069–7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.     

Lipid Sorting by Ceramide Structure from Plasma Membrane to ER for the Cholera Toxin Receptor Ganglioside GM1

The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane?lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells.?Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid sorting by ceramide structure and provide a molecular explanation for the diversity?and specificity of retrograde trafficking by CT in?host cells.     

Evidence against the acidification hypothesis in cystic fibrosis

The pleiotropic effects of cystic fibrosis (CF) result from themislocalization or inactivity of an apical membrane chloride channel,the cystic fibrosis transmembrane conductance regulator (CFTR). CFTRmay also modulate intracellular chloride conductances and thus affectorganelle pH. To test the role of CFTR in organelle pH regulation, wedeveloped a model system to selectively perturb the pH of a subset ofacidified compartments in polarized cells and determined the effects onvarious protein trafficking steps. We then tested whether these effectswere observed in cells lacking wild-type CFTR and whetherreintroduction of CFTR affected trafficking in these cells. Our modelsystem involves adenovirus-mediated expression of the influenza virusM2 protein, an acid-activated ion channel. M2 expression selectivelyslows traffic through the trans-Golgi network (TGN) andapical endocytic compartments in polarized Madin-Darby canine kidney(MDCK) cells. Expression of M2 or treatment with other pH perturbantsalso slowed protein traffic in the CF cell line CFPAC, suggesting thatthe TGN in this cell line is normally acidified. Expression offunctional CFTR had no effect on traffic and failed to rescue theeffect of M2. Our results argue against a role for CFTR in theregulation of organelle pH and protein trafficking in epithelial cells.

     

Role of PACS-1 in trafficking of human cytomegalovirus glycoprotein B and virus production

The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.     

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

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

Copper-independent lysosomal localisation of the Wilson disease protein ATP7B

In hepatocytes, the Wilson disease protein ATP7B resides on the trans-Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non-hepatic cells. These ATP7B-harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub-domain of the TGN with a lower pH and the TGN-proximal lysosomal compartments. The presence of ATP7B on TGN-lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper-replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN-proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper-independent lysosomal localisation of ATP7B and the importance of TGN-proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway.     

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.     

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.     

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.     

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.     

Arabidopsis TRAPPII is functionally linked to Rab-A,but not Rab-D in polar protein trafficking in trans-Golgi network

The trans-Golgi network (TGN) in plant cells is an independent organelle, displaying rapid association and dissociation with Golgi bodies. In plant cells, the TGN is the site where secretory and endocytic membrane trafficking meet. Cell wall components, signaling molecules and auxin transporters have been found to undergo intracellular trafficking around the TGN. However, how different trafficking pathways are regulated and how different cargoes are sorted in the TGN is poorly defined in plant cells. Using a combined approach of genetic and in vivo imaging, we recently demonstrated that Arabidopsis TRAPPII acts in the TGN and is required for polar targeting of PIN2, but not PIN1, auxin efflux carrier in root tip cells. Here, we report that, TRAPPII in Arabidopsis is required for polar distribution of AUX1, an auxin influx carrier in protophloem cells and epidermal cells of Arabidopsis root tips. In yeast cells, TRAPPII serves as a guanine-nucleotide exchange factor (GEF) for Ypt1 and Ypt31/32 in late Golgi trafficking, while in mammalian cells, TRAPPII acts as a GEF for Rab1 (homolog of yeast Ypt1) in early Golgi trafficking. We show here that TRAPPII in Arabidopsis is functionally linked to Rab-A proteins, homologs of yeast Ypt31/32, but not Rab-D proteins, homologs of yeast Ypt1 and animal Rab1 proteins.