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.     

Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.     

The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A.

TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network, is utilized to study both the structure and function of the trans-Golgi network (TGN). The effects of brefeldin A (BFA) on the TGN were studied in comparison to its documented effects on the Golgi cisternae. During the first 30 min of BFA treatment, the TGN loses its cisternal structure and extends as tubules throughout the cytoplasm. By 60 min, it condenses into a stable structure surrounding the microtubule-organizing center. By electron microscopy, this structure appears as a population of large vesicles, and by immunolabeling, most of these vesicles contain TGN38. TGN38 cycles to the plasma membrane and back, which is shown by addition of TGN38 luminal domain antibodies directly to cell culture media. This results in rapid uptake of antibodies which label the TGN within 30 min, both in its native and BFA-induced conformation. A number of transmembrane proteins have been shown to take this cycling pathway, but TGN38 is unique in that it is the only one predominantly localized to the TGN. To investigate the cycling of TGN38, the endocytic pathway was labeled by internalization of Lucifer Yellow, and in the presence of BFA there was partial colocalization with TGN38. Further studies were carried out in which microtubules were depolymerized, resulting in dispersal of Golgi elements and inhibition of transport from endosomes to lysosomes. TGN38 cycling continues in the absence of microtubules. Taken together, these studies indicate that TGN38 returns from the plasma membrane via the endocytic pathway. We conclude that the TGN is structurally and functionally distinct from the Golgi cisternae, indicating that different molecules control membrane traffic from the Golgi cisternae and from the TGN.     

Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.     

Interaction between p230 and MACF1 is associated with transport of a glycosyl phosphatidyl inositol-anchored protein from the Golgi to the cell periphery

The molecular basis by which proteins are transported along cytoskeletal tracts from the trans-Golgi network (TGN) to the cell periphery remains poorly understood. Previously, using human autoimmune sera, we identified and characterized a TGN protein, p230/Golgin-245, an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, that is anchored to TGN membranes and TGN-derived vesicles by its carboxyl-terminal GRIP domain. To identify molecules that interact with the flexible amino-terminal end of p230, we used this domain as bait to screen a human brain cDNA library in a yeast two-hybrid assay. We found that this domain interacts with the carboxyl-terminal domain of MACF1, a protein that cross-links microtubules to the actin cytoskeleton. The interaction was confirmed by co-immunoprecipitation, an in vitro binding assay, double immunofluorescence images demonstrating overlapped localization in HeLa cells, and co-localization of FLAG-tagged constructs containing the interacting domains of these two proteins with their endogenous partners. Expression in HeLa cells of FLAG-tagged constructs containing the interacting domains of p230 and MACF1 disrupted transport of the glycosyl phosphatidyl inositol-anchored marker protein conjugated with yellow fluorescent protein (YFP-SP-GPI), while trafficking of the transmembrane marker protein, vesicular stomatitis virus glycoprotein conjugated with YFP (VSVG3-GL-YFP), was unaffected. Our results suggest that p230, through its interaction with MACF1, provides the molecular link for transport of GPI-anchored proteins along the microtubule and actin cytoskeleton from the TGN to the cell periphery.     

The regulation of endosome-to-Golgi retrograde transport by tethers and scaffolds

Retrograde transport between endosomes and the trans-Golgi network (TGN) is essential for the recycling of membrane proteins which are involved in a range of biological processes. A variety of machinery components have been identified at the TGN which regulate endosome-to-TGN transport, including small G proteins, SNAREs, tethering factors and scaffold molecules. The challenge is to understand how these regulatory components orchestrate not only the specific docking and fusion of retrograde membrane carriers with the TGN, but also maintain the integrity of this highly dynamic compartment to ensure efficient delivery and export of cargo. Here we review recent advances in defining the form and function of tethers and scaffolds in the regulation of the retrograde transport pathways.     

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.     

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

Protein localization to the TGN was investigated by examining the subcellular distribution of chimeric proteins in which the cytoplasmic and/or transmembrane domains of the TGN protein, TGN38, were substituted for the analogous domains of the plasma membrane protein, Tac. Using immunofluorescence and immunoelectron microscopy, the COOH- terminal cytoplasmic domain of TGN38 was found to be sufficient for localization of the chimeric proteins to the TGN. Deletion analysis identified an 11-amino acid segment containing the critical sequence, YQRL, as being sufficient for TGN localization. TGN localization was abrogated by mutation of the tyrosine or leucine residues in this sequence to alanine, or of the arginine residue to aspartate. In addition to specifying TGN localization, the 11-amino acid segment was active as an internalization signal, although the property of internalization alone was insufficient to confer TGN localization. Overexpression of chimeric proteins containing TGN localization determinants resulted in their detection at the plasma membrane and in intracellular vesicles, and abolished detection of endogenous TGN38. These results suggest that discrete cytoplasmic determinants can mediate protein localization to the TGN, and reveal a novel role for tyrosine-based motifs in this process.     

CNTD1’s crossover act

Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins? In this paper, we show that disruption of SM homeostasis at the trans-Golgi network (TGN) by treatment of HeLa cells with d-ceramide-C6, which was converted together with phosphatidylcholine to short-chain SM and diacylglycerol by SM synthase, led to the segregation of Golgi-resident proteins from each other. We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other. Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.     

The role of secretory and endocytic pathways in the maintenance of cell polarity

Epithelial cells line virtually every organ cavity in the body and are important for vectorial transport through epithelial monolayers such as nutrient uptake or waste product excretion. Central to these tasks is the establishment of epithelial cell polarity. During organ development, epithelial cells set up two biochemically distinct plasma membrane domains, the apical and the basolateral domain. Targeting of correct constituents to each of these regions is essential for maintaining epithelial cell polarity. Newly synthesized transmembrane proteins destined for the basolateral or apical membrane domain are sorted into separate transport carriers either at the TGN (trans-Golgi network) or in perinuclear REs (recycling endosomes). After initial delivery, transmembrane proteins, such as nutrient receptors, frequently undergo multiple rounds of endocytosis followed by re-sorting in REs. Recent work in epithelial cells highlights the REs as a potent sorting station with different subdomains representing individual targeting zones that facilitate the correct surface delivery of transmembrane proteins.     

Requirement of the human GARP complex for mannose 6-phosphate-receptor-dependent sorting of cathepsin D to lysosomes

The biosynthetic sorting of acid hydrolases to lysosomes relies on transmembrane, mannose 6-phosphate receptors (MPRs) that cycle between the TGN and endosomes. Herein we report that maintenance of this cycling requires the function of the mammalian Golgi-associated retrograde protein (GARP) complex. Depletion of any of the three GARP subunits, Vps52, Vps53, or Vps54, by RNAi impairs sorting of the precursor of the acid hydrolase, cathepsin D, to lysosomes and leads to its secretion into the culture medium. As a consequence, lysosomes become swollen, likely due to a buildup of undegraded materials. Missorting of cathepsin D in GARP-depleted cells results from accumulation of recycling MPRs in a population of light, small vesicles downstream of endosomes. These vesicles might correspond to intermediates in retrograde transport from endosomes to the TGN. Depletion of GARP subunits also blocks the retrograde transport of the TGN protein, TGN46, and the B subunit of Shiga toxin. These observations indicate that the mammalian GARP complex plays a general role in the delivery of retrograde cargo into the TGN. We also report that a Vps54 mutant protein in the Wobbler mouse strain is active in retrograde transport, thus explaining the viability of these mutant mice.     

The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network.

Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins. Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport. Mutant cells lacking the Age2 and Gcs1 proteins cease proliferation, accumulate membranous structures resembling Berkeley bodies, and are unable to properly process and localize the vacuolar hydrolase carboxypeptidase (CPY) and the vacuolar membrane protein alkaline phosphatase (ALP), which are transported from the TGN to the vacuole by distinct transport routes. Immunofluorescence studies localizing the proteins ALP, Kex2 (a TGN resident protein), and Vps10 (the CPY receptor for transport from the TGN to the vacuole) suggest that inadequate function of this ArfGAP pair leads to a fragmentation of TGN, with effects on secretion and endosomal transport. Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.     

Targeting of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule.

Previous studies suggested that varicella-zoster virus (VZV) envelope glycoproteins (gps) are selectively transported to the trans-Golgi network (TGN) and that the cytosolic domain of gpI (gE) targets it to the TGN. To identify targeting signals in the gpI cytosolic domain, intracellular protein trafficking was studied in transfected cells expressing chimeric proteins in which a full-length or mutated gpI cytosolic domain was fused to the gpI transmembrane domain and interleukin-2 receptor (tac) ectodomain. Expressed protein was visualized with antibodies to tac. A targeting sequence (AYRV) and a second, acidic amino acid-rich region of the gpI cytosolic domain (putative signal patch) were each sufficient to cause expressed protein to colocalize with TGN markers. This targeting was lost when the tyrosine of the AYRV sequence was replaced with glycine or lysine, when arginine was replaced with glutamic acid, or when valine was substituted with lysine. In contrast, tyrosine could be replaced by phenylalanine and valine could be substituted with leucine. Mutation of alanine to aspartic acid or deletion of alanine abolished TGN targeting. Exposure of transfected cells to antibodies to the tac ectodomain revealed that the TCN targeting of expressed tac-gpI chimeric proteins occurred as a result of selective retrieval from the plasmalemma. These data suggest that the AYRV sequence and a second signaling patch in the cytosolic domain of gpI are responsible for its targeting to the TGN. The observations also support the hypothesis that the TGN plays a critical role in the envelopment of VZV.     

Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning

The trans-Golgi Network (TGN) sorts molecular “addresses” and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca²⁺ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.     

Caveolae and sorting in the trans-Golgi network of epithelial cells.

VIP21 is a 21 kDa membrane protein present in TGN-derived transport vesicles isolated from the epithelial MDCK cell line. The membrane topology and subcellular localization of VIP21 were studied using antibodies against the N- and C-terminal domains. The protein was found to have a structure with little or no exposure to the exoplasmic side of the membrane. VIP21 was localized to the TGN, consistent with its presence in TGN-derived transport vesicles. Unexpectedly, it was also very abundant in the non-clathrin-coated plasma membrane invaginations called caveolae. We have previously proposed that VIP21 is associated with glycosphingolipid-enriched membrane domains in the TGN which may be involved in the sorting of proteins into vesicles directed to the apical plasma membrane. Caveolae are specialized lipid structures with similarities to the glycolipid microdomains in the TGN. The presence of VIP21 in both locations suggests that the mechanisms governing inclusion of proteins into caveolar plasma membrane domains are related to the processes of protein and lipid sorting at the TGN. This connection is confirmed by the recent finding that the amino acid sequence of VIP21 is almost identical to that of caveolin, a protein previously localized to caveolae.     

Intracellular trafficking of LRP9 is dependent on two acidic cluster/dileucine motifs

LDL receptor-related protein 9 (LRP9) is a distant member of the low-density lipoprotein receptor (LDLR) superfamily. To date, there are no reports on the cellular distribution of LRP9 or the signals responsible for its localization. Here, we investigated the intracellular localization and trafficking of LRP9. Using confocal microscopy, we demonstrated that LRP9 was not present at the plasma membrane but co-localized with various markers of the trans-Golgi network (TGN) and endosomes. This co-localization was dependent on the presence of two acidic cluster/dileucine (DXXLL) motifs in the cytoplasmic tail of LRP9, which interact with GGA proteins, clathrin adaptors involved in transport between the TGN and endosomes. LRP9 is the first example of a transmembrane protein with an internal GGA-binding sequence in addition to the usual C-terminal motif. An inactivating mutation (LL --> AA) in both DXXLL motifs, which completely inhibited the interaction of LRP9 with GGA proteins, led to an intracellular redistribution of LRP9 from the TGN to early endosomes and the cell surface, indicating that the two DXXLL motifs are essential sorting determinants of LRP9. In conclusion, our results suggest that LRP9 cycles between the TGN, endosomes and the plasma membrane through a GGA dependent-trafficking mechanism.     

Sorting of plant vacuolar proteins is initiated in the ER

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor–ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer‐mediated recycling of the plant VSR BP80 starts at the trans‐Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co‐expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII‐mediated transport route. Retention of soluble cargo despite ongoing COPII‐mediated bulk flow can only be explained by an interaction with membrane‐bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER‐anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR–ligand interaction. It also implies that the retromer‐mediated recycling route for the VSRs leads from the TGN back to the ER.     

African swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane

Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.     

Cell-free transport from the trans-golgi network to late endosome requires factors involved in formation and consumption of clathrin-coated vesicles

Transport between the trans-Golgi network (TGN) and late endosome represents a conserved, clathrin-dependent sorting event that separates lysosomal from secretory cargo molecules and is also required for localization of integral membrane proteins to the TGN. Previously, we reported a cell-free reaction that reconstitutes transport from the yeast TGN to the late endosome/prevacuolar compartment (PVC) and requires the PVC t-SNARE Pep12p. Here, we report that factors required both for formation of clathrin-coated vesicles at the TGN (the Chc1p clathrin heavy chain and the Vps1p dynamin homolog) and for vesicle fusion at the PVC (the Vps21p rab protein and Vps45p SM (Sec1/Munc18) protein) are required for cell-free transport. The marker for TGN-PVC transport, Kex2p, is initially present in a clathrin-containing membrane compartment that is competent for delivery of Kex2p to the PVC. A Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC. Antibodies against the Kex2p and Vps10p C-tails selectively block transport of Kex2p and the Kex2-Vps10p chimera. The requirements for factors involved in vesicle formation and fusion, the identification of the donor compartment as a clathrin-containing membrane, and the need for accessibility of C-tail sequences argue that the TGN-PVC transport reaction involves selective incorporation of TGN cargo molecules into clathrin-coated vesicle intermediates. Further biochemical dissection of this reaction should help elucidate the molecular requirements and hierarchy of events in TGN-to-PVC sorting and transport.