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Retrograde Transport
Retrograde Transport L Johannes and C Wunder, Institut Curie, Paris, France r 2016 Elsevier Inc. All rights reserved.
Introduction
glycoconjugates having nonreducing terminal galactose (ricin receptors), enters cells via endocytosis and reaches morphologically distinct Golgi membranes. Thus, the first step of the retrograde pathway was defined – trafficking from endosomes to the Golgi apparatus (Figure 1). Scientists were searching for more than 10 years whether endogenous proteins would also use the retrograde pathway, as described by Olsnes for ricin. In 1985, a cellular protein – transferrin receptor – could be identified in leukemia cells by Snider and Rogers (1985), that also traffics from the plasma membrane to the TGN. This initial discovery was followed very soon by several other studies that used similar approaches based on Golgi-specific enzyme modifications. Of these, the characterization of mannose 6-phosphate receptor (MPRs) trafficking by Duncan and Kornfeld (1988) stood out. These authors provided first evidence for a model in which trafficking between endosomes and the TGN is part of the functional cycle of MPRs. Many lysosomal enzymes are posttranslationally modified by mannose 6-phosphate residues. These are recognized by MPRs and shuttled from the TGN to endosomes. The unloaded receptors then return to the TGN for a new transport cycle (Figure 2). Despite the discovery of several endogenous retrograde cargo molecules, toxins were used as discovery tools to study and understand this pathway further. Sandvig et al. (1992) showed for the first time in 1992 that another exogenous protein – the bacterial Shiga toxin (STx) – could reach the ER via the retrograde transport route, similar to ricin. As for ricin, the catalytic A-subunit of Shiga toxin is then translocated to the cytosol to inhibit protein biosynthesis by acting as a N-glycosidase, which cleaves a specific adenine nucleobase
Trafficking of vesicular carriers between the plasma membrane and endomembrane compartments is essential for transport of proteins and other macromolecules to diverse destinations inside and outside of the cell. Membrane trafficking is also required to assure the essential need of cells to maintain membrane homeostasis and to meet specific demands during development, signal perception, and transmission. Two major trafficking routes exist, comprising the endoplasmatic reticulum (ER), the Golgi apparatus, and endosomes: the anterograde and retrograde pathways. Both are highly conserved in the eukaryotic kingdom, including plants and some protozoa, e.g., Toxoplasma gondii. The anterograde pathway delivers proteins and lipids in a vectorial manner from the ER to the plasma membrane. This outward flow of material is counterbalanced by various retrograde trafficking routs. Retrograde cargo proteins and lipids are endocytosed and transported to endosomes, before being delivered to the trans-Golgi network (TGN), and in some instances further to the ER. The discovery of the retrograde trafficking pathway started in 1972 by studying the cellular intoxication process mediated by the plant toxin ricin (Olsnes and Pihl, 1972). Ricin is a highly toxic glycoprotein produced by the castor plant Ricinus communis and inhibits protein biosynthesis by modifying ribosomal ribonucleic acid (RNA) of toxin-exposed cells (Montanaro et al., 1973). Later, Avrameas and colleagues found that even the Golgi apparatus was a trafficking station in the intoxication process of ricin (Gonatas et al., 1975). Hence, the retrograde cargo protein ricin is binding to cell surface
A-subunit B-subunit Shiga toxin A-subunit B-subunit
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Cholera toxin A-subunit B-subunit Ricin
Golgi apparatus
Caveolae Retrograde transport Retrotranslocation
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Figure 1: Toxin entry into cells. Via different uptake routes, cholera toxin, Shiga toxin, and ricin reach early endosomes, from where they then traffic to the ER, via the Golgi apparatus. The catalyic A-subunits of these toxins are then retrotranslocated across the ER membrane to the cytosol where they can meet their molecular targets.
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Wnt Wntless Retromer
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Endoplasmic reticulum
Multivesicular body
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Figure 2 The role of retrograde transport in the cellular functions of Wntless and MPRs. Retromer is of critical importance for the trafficking of Wntless and MPRs from endosomes to the TGN as part of the cellular functions of these receptor molecules. See text for details.
from the 28S rRNA of the ribosomal 60S subunit (reviewed in Johannes and Römer, 2010). These studies have laid the groundwork for the understanding of the trafficking behavior of a whole class of cytotoxic molecules, AB5 toxins, for which no pore forming capacity could be demonstrated and that use the retrograde rout to enter into cells (Figure 1). These toxins are named after their unique subunit composition, containing a single catalytical A-subunit and a homopentameric B-subunit composed of five B-fragments. In some cases, the A-subunit is covalently linked via a disulfide bridge to the B-subunit (Sandvig and van Deurs, 2002). The catalytically inactive B-subunit is binding to cell surface receptors and serves to mediate transport of the AB5 protein complex from the cell surface to the lumen of the ER, via vesicular and tubular carriers and intracellular compartments. The B-subunit retains its binding capacity even in the absence of the A-subunit. However, the complete AB5 holotoxin is required for mediating toxicity. AB5 toxins are a medically important class of bacterial exotoxins, which cause devastating human diseases. Prominent members of this class include the above-mentioned STx, which causes life-threatening diarrhea, dysentery, hemorrhagic colitis, and hemorrhagic uremic syndrome; Escherichia coli heat-labile enterotoxins and cholera toxin (CTx), which cause endemic and epidemic diarrhea and pertussis toxin (PTx), which is the causative agent for whooping cough (Beddoe et al., 2010). Each year, infections with bacteria that produce these toxins affect millions of individuals and cause more than a million deaths. Many cellular functions in development and disease depend on the retrograde transport route, such as wnt morphogen gradient formation (Harterink et al., 2011; Yu et al., 2014; Figure 2), intracellular glucose (Esk et al., 2010), iron and copper ion homeostasis (Tabuchi et al., 2010), and glutamate receptor recycling (Zhang et al., 2012). Besides the poisonous effect of several toxins, the medical importance of the retrograde trafficking route was corroborated by many findings. Its dysfunction causes abnormal elevation of amyloid β-peptide
(Burgos et al., 2010); the nef protein of human immunodeficiency virus (HIV-1) favors immune-evasion by down modulating major histocompatibility complex (MHC) class I (Blagoveshchenskaya et al., 2002) and co-stimulatory molecules (Chaudhry et al., 2008). Recently, a new aspect in modulation of the retrograde pathway was discovered: the intracellular bacterium Legionella pneumophila is able to shut down the retrograde route in order to promote bacterial replication (Finsel et al., 2013). Taken together, understanding the retrograde trafficking pathway is important to develop efficient therapeutic strategies for a wide range of diseases.
Different Retrograde Pathways The above-mentioned ground-breaking papers have proven the existence of a transport connection between plasma membrane/endosomes and TGN/Golgi/ER. The trafficking interface between endosomes and the TGN has become a subject of intense investigation in membrane biology research. The complexity of retrograde transport arises from different pathways between endosomes and the TGN. Endosomes are a complex membrane system of tubulo-vacuolar nature of which early endosomes and late endosomes/lysosomes are the major parts (Huotari and Helenius, 2011). There is an ongoing debate on whether these separate endosomal entities of different protein and lipid compositions are linked by vesicluar carriers, or are transformed from one to the other by a process of maturation. Exactly how very early endosomes arise is not entirely clear, but it seems likely that membranes and volume are mainly derived from primary endocytic vesicles that fuse with each other. Very early endosomes receive cargo proteins through several endocytic pathways, including clathrin-mediated and clathrin-independent endocytic processes – caveolar, clathrin-independent carrier (CLIC) and ARF6-dependent pathways (Mayor and Pagano, 2007). Therefore very early endosomes are heterogeneous in terms of morphology, localization, composition, and function.
Interorganellar Communication: Interplay and Processes: Retrograde Transport
The identity and functional attributes of different endosomes is also defined by the association of cytosolic proteins to the cytoplasmic surface of the endosomal membrane (Huotari and Helenius, 2011). Very early endosomes then converge into conventional early endosomes. Their limiting membrane contains a mosaic of tubular and vacuolar domains that differ in composition and function (Zerial and McBride, 2001). These include domains enriched in Rab5, Rab4, Rab11, Arf1/COPI, and retromer (Sonnichsen et al., 2000; Bonifacino and Rojas, 2006). Domains located in the tubular extensions allow molecular sorting and generate transport carriers targeted to distinct organelles, including the plasma membrane, the recycling endosomes, and the TGN for retrograde trafficking (Bonifacino and Rojas, 2006). One of the cytosolic key players in retrograde trafficking is the retromer complex. This complex was shown to be involved in trafficking events from different endosomes to the TGN as well as for recycling to the plasma membrane (Temkin et al., 2011). How specificity is achieved will be discussed later. Pfeffer and colleagues were the first to pinpoint in 1993 an endosomal location from which the MPR recycles to the TGN: late endosomes (Lombardi et al., 1993). In 1998, Johannes and colleagues reported that STx was not found on late endosomal membranes en route to the TGN, which led to the proposal that the toxin bypasses the late endocytic pathway altogether and directly traffics from early endosomes to the TGN (Mallard et al., 1998). Recently, Lieu and Gleeson (2010) suggested that Shiga toxin might in part also traffic from recycling endosomes to the TGN. In summary, retrograde trafficking does occur from different endosomal sites (Figure 3). Routes from late, early, and recycling endosomes seem to operate in parallel. Below, we will discuss these three different scenarios individually. We list key molecular and morphological data that have helped to define them, and point out controversial matters whenever these exist.
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Late Endosomes-to-TGN Trafficking MPRs are the best characterized cargo proteins of the late endosomes-to-TGN retrograde transport process (Rohn et al., 2000). More than 60 lysosomal hydrolases acquire a unique posttranslational modification in the Golgi complex: a mannose 6-phosphate residue, which enables them to bind to MPRs. Two types of MPRs have been identified, the cation-independent (CI-MPR) and the cation-dependent MPR (CD-MPR). Both carry newly synthesized precursor hydrolazes from the Golgi to endosomes. MPRs release their ligands upon encountering the low pH of the late endocytic pathway. The receptors then return from late endosomes to the TGN to start another cycle of biosynthetic enzyme transport.
Cargo Sorting and Budding Lysosomal hydrolaze precursors are transported within the lumen of endocytic carrier vesicles to the lysosome for proteolytic activation. A multitude of sorting signals in the cytoplasmic tail of MPRs has been identified that ensure that MPRs are not lost into lysosomes where they would be degraded. A subset of these sorting signals is recognized by tail interacting protein of 47 kDa (TIP47), and strong arguments have been presented for a role of TIP47 in late endosomes-to-TGN trafficking of MPRs (Diaz et al., 1997), even if some complexity has been noticed that may originate from TIP47's additional functions in lipid droplet formation (Bulankina et al., 2009). TIP47 recognizes a phenylalanine–tryptophan motif in the cytoplasmic tail of CD-MPR (Diaz and Pfeffer, 1998), and a more complex domain of CI-MPR. TIP47 also binds Rab9GTP, upon which its affinity for the CI-MPR is increased threefold (Carroll et al., 2001). Rab9 is itself involved in the retrograde transport of MPRs (Riederer et al., 1994). It has thus been suggested that TIP47 is a coat for MPR sorting into the retrograde pathway.
A-subunit B-subunit Shiga toxin
Hydrolase
Clathrin
MPR Retromer
Early endosome
Recycling endosome
Caveolae
Late endosome
Retro
Golgi apparatus
grade transp or
t
Figure 3 Sites of endosomal retrograde sorting in mammalian cells. Retrograde sorting can operate from different endosomal locations. See text for a detailed discussion of retrogarde trafficking from early, recycling, and late endosomes.
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Docking and Fusion The Rab9 GTPase also binds to the GRIP domain golgin protein GCC185 (Golgi coiled-coil protein) (Reddy et al., 2006) for which tethering functions have been suggested. Tethering is the process by which transport intermediates are captured on target membranes to allow for subsequent N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) complex formation and fusion. GCC185 is required for retrograde transport of MPRs (Reddy et al., 2006; Derby et al., 2007) and interacts with the SNARE protein syntaxin 16 that equally has key functions for retrograde transport from late (Ganley et al., 2008) and early endosomes (see below). For the fusion of late endosomes-derived retrograde transport intermediates, syntaxin 16 is in a SNARE complex with syntaxin 10, Vti1a, and vesicle-associated membrane protein (VAMP3) (Ganley et al., 2008).
Early Endosomes-to-TGN Trafficking Mallard et al. (1998) provided first evidence for a direct transport route from early endosomes to the TGN, using Shiga toxin as a model cargo. This transport interface is used by other exogenous (cholera toxin) and endogenous (TGN38) cargo proteins (Ghosh et al., 1998; Amessou et al., 2007; Lieu et al., 2007). Even MPRs may traffic directly from early endosomes to the TGN, in addition to using the late endosomes-TGN interface. Indeed, MPRs accumulate in early endosomes under many conditions that block retrograde trafficking to the TGN, i.e., depletion for the following proteins: retromer (Seaman et al., 2013), the tSNARE syntaxin 10 (Ganley et al., 2008), the clathrin adapter AP1 (Meyer et al., 2000), the phosphatidylinositol 3-phosphate 5-kinase PIKfyve (Rutherford et al., 2006), and the GRIP domain golgin GCC88 (Lieu et al., 2007). Probably the multitude of sorting signals in the cytosolic tails of MPRs has evolved to permit recycling to the TGN from any stage of the endocytic pathway.
Cargo Sorting and Budding Clathrin is localized on early endosomes and was shown to be required for the retrograde sorting of Shiga toxin (Saint-Pol et al., 2004). In clathrin-depleted cells Shiga toxin perfectly colocalizes with transferrin receptor at times when the molecule has reached the TGN/Golgi in control conditions, demonstrating that the toxin needs clathrin to leave early endosomes. Different adapters have been identified that function with clathrin in retrograde sorting of Shiga toxin and/ or other retrograde cargo proteins: AP1 (Meyer et al., 2000), epsinR (Saint-Pol et al., 2004), and oculocerebrorenal syndrome of Lowe (inositol polyphosphate 5-phosphatase) (OCRL) (Choudhury et al., 2005). For some cargo proteins with acidic cluster motifs in their cytosolic tail, the phosphofurin acidic cluster sorting protein 1 (PACS1) appears to stabilize binding to AP1, and the CI-MPR accumulates in early endosomes upon overexpression of a dominant negative PACS1 (Scott et al., 2006). The retromer complex was identified first in yeast and then in mammalian cells. It has been shown that retromer is localized on the cytosolic leaflet of early endosomes and
required for retrograde transport. Retromer comprises two subcomplexes, a vps protein heterotrimer (Vps26, Vps29, and Vps35) and a sorting nexin (SNX) dimer. The vps heterotrimer binds cargo proteins (Seaman et al., 2013). However, different cargo proteins are recognized by different vacuolar protein sorting (VPS) proteins of the retromer heterotrimer (A-ALP and Vps10p bind to Vps35p, CI-MPR shows bipartite interactions with VPS35 and VPS29, sortilin to VPS35, divalent metal transporter 1 (DMT1-II) to VPS35, and sorLA to VPS26). The SNX subcomplex senses and/or generates curvature. Latest work from the Cullen laboratory indeed suggests a contribution of the sorting nexins to membrane bending, as purified sorting nexins were shown to deform and tubulate lipid vesicles (van Weering et al., 2012). As such, retromer could function as a coat. However, retromer does not form the same type of electron-dense coats as observed for classical coatomer protein complex-I (COPI), coatomer protein complex-II (COPII), and clathrin. In addition, the molecular mechanism by which retromer sorts cargo seems to be distinct from that of classical membrane coats (Seaman et al., 2013), but somehow similar to other endosomal sorting complexes, like the endosomal sorting complexes required for transport (ESCRT). Retromer seems to regulate trafficking of cargo proteins on non-flat membranes, rather within morphologically distinct tubulovesicular membranes, suggesting a novel mechanism of action. Furthermore, when retromer vps heterotrimer proteins are depleted, retrograde cargocontaining tubules still form that fail to detach from early endosomes (Popoff et al., 2007, 2009). These findings suggest that vps retromer heterotrimer may play a critical role for scission. Whether this function is fulfilled via amphipathic helices/BAR-domains of SNX proteins, or indirectly via recruitment of a scission factor still needs to be determined. One possibility how retromer fulfills a scission function might be via its capacity to recruit the actin nucleation promotion factor Wiskott–Aldrich syndrome protein and scar homolog protein (WASH) and to drive the polymerization of actin on early endosomal membranes (Seaman et al., 2013). A second hypothesis might be an indirect effect of the WASH complex, via the recruitment of dynamin-2 (Derivery et al., 2009). Whether and how dynamin contributes to membrane scission on early endosomes in human cells has not yet been convincingly documented. In yeast, a dynamin-related protein, termed Vps1, promotes fission of SNX-coated retromer tubules on early endosomes (Chi et al., 2014). Important differences exist between the prototypical retromer in yeast and retromer in higher eukaryotes. A key difference is that, in mammalian cells, retromer is not a stable heteropentamer as in yeast, but a much more transient association of the cargo-selective trimer (Vps35, Vps26, and Vps29) and the Snx proteins (Snx1 or Snx2 with Snx5 or Snx6) (Seaman et al., 2013). In addition, retromer exists in different subcomplexes, depending on which SNX protein is involved. In mammalian cells, SNX proteins indeed seem to determine the trafficking route and thereby the specificity of retrograde trafficking. Furthermore, many accessory retromer proteins that were identified from studies in higher eukaryotes are not conserved in yeast. The yeast retromer complex seems to function independent of the WASH complex. Thus, the exact
Interorganellar Communication: Interplay and Processes: Retrograde Transport
scope of functions of retromer accessory proteins is not clear at this stage. Lipids also play critical roles in retrograde sorting. Notably the phosphatidylinositol lipids phosphatidylinositol 3-phosphate (Skånland et al., 2007) and phosphatidylinositol 3,5-bisphosphate (Rutherford et al., 2006) are directly involved in the membrane recruitment of sorting nexins, and may also affect retrograde sorting via Hrs (Popoff et al., 2009). Furthermore, a redistribution of retromer components has been observed in glycosphingolipid-depleted cells (Raa et al., 2009), in agreement with earlier studies that had found a role for raft lipids in retrograde sorting (Falguières et al., 2001). By what mechanism mesoscale raft lipid organization affects the retromer complex, and whether specialized lipid domains are required for retrograde trafficking from early endosomes has yet to be determined.
Docking and Fusion Early endosome-derived vesicles are trafficking further to the TGN, and this transport is microtubule dependent (Hehnly et al., 2006). The Golgi-associated retrograde protein (GARP) complex was identified for tethering of MPR positive vesicles to TGN membranes (Perez-Victoria et al., 2008). The GARP complex is recruited at the level of early endosomes, and then functions at the TGN to promote SNARE complex formation in interaction with syntaxin 6, syntaxin 16, and VAMP4 (PerezVictoria and Bonifacino, 2009), three SNARE proteins that have previously been implicated in early endosomes-to-TGN trafficking (Mallard et al., 2002). Other tethering factors for early endosomal retrograde cargo proteins are GCC88 and GCC185 (Derby et al., 2007; Lieu et al., 2007).
Recycling Endosomes-to-TGN Trafficking In a comparative study it was found that depletion of the tethering factor GCC185 inhibited retrograde transport of Shiga toxin, but not that of the endogenous cargo protein TGN38 (Derby et al., 2007), while the opposite was observed for the depletion of the tethering factor GCC85 (Lieu et al., 2007). Since Shiga toxin, but not TGN38, could be detected in recycling endosomes under different experimental conditions, the authors concluded that TGN38 was transported directly
from early endosomes to the TGN, while Shiga toxin could also transit via recycling endosomes (Lieu and Gleeson, 2010). This conclusion is in apparent contradiction with earlier studies that had described the retrograde trafficking of TGN38 via recycling endosomes (Ghosh et al., 1998), and it is difficult to reconcile with the established function of early/maturing endosomal retromer in the trafficking of Shiga toxin to the TGN. Furthermore, as mentioned above, recycling endosomes remain poorly defined in molecular terms, and whether they have evolved a truly independent retrograde sorting machinery, possibly involving Rab11 (Chaudhry et al., 2008; Wilcke et al., 2000) remains to be established.
Biophysical Mechanisms for Retrograde Trafficking All trafficking events of proteins and lipids between distinct membrane-enclosed organelles are following the same mechanistic principles. The course of action per se for generic trafficking processes is as follows: cargo sorting, membrane bending and bud formation, scission in the neck region of the invaginated bud or tubule, and finally fusion of transport intermediates with acceptor organelles. In the following, we have summarized biophysical mechanisms of each of these steps, based on the list of established retrograde trafficking factors.
Membrane Bending The exact mechanism of membrane bending for retrograde trafficking from early endosomes is not known. However, different SNXs bearing BAR-domains and/or amphipathic helices were analyzed by in vitro studies (van Weering et al., 2012). Therefore, two SNX-driven mechanisms for membrane bending can be envisioned (Figure 4): (1) assymetric insertion of amphipathic helices and/or (2) scaffolding by BAR-domain containing SNXs. Whether SNXs are able to form scaffolds at physiological concentrations such as to drive membrane deformation (Sorre et al., 2012), still needs to be determined. Recently the concerted action of retromer SNXs and the WASH complex on tubule formation was supported by Seaman and colleagues (Freeman et al., 2014). This report indicated the binding of RME-8 to retromer SNXs and the WASH complex
Membrane bending by SNXs SNXs helix insertion
Scaffolding Concentration SNXs with BAR-domain
(a)
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(b)
Figure 4 Membrane bending mechanisms in relation to SNX proteins. (a) Insertion of helices leads to asymmetric expansion of the cytosolic leaflet and to subsequent membrane bending. (b) BAR domain binding can serve to recognize membrane curvature (low BAR-domain protein concentration on membranes), or to drive membrane bending through a scaffolding effect (high concentration).
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protein FAM21. Interestingly, clathrin could be localized to early endosomes (Popoff et al., 2009; Esk et al., 2010; Freeman et al., 2014), and RME-8 also binds to the clathrin uncoating ATPase Hsc70 (Girard et al., 2005). However, no reports currently exist on interactions between clathrin and components of the retromer machinery. Whether clathrin is directly involved in membrane deformation for other retrograde cargo protein requires further studies but seems likely when considering the function of the clathrin interactors AP1 (Meyer et al., 2000; Natsume et al., 2006), epsinR (Saint-Pol et al., 2004), and OCRL (Choudhury et al., 2005) in retrograde sorting on early endosomes.
Scission The first indication for a molecular player for a scission process of endosomal tubules came from knock down experiments of vps retromer proteins (Popoff et al., 2007, 2009). Endosomal tubules formed that failed to detach from early endosomes, pointing toward a function for retromer or retromer binding proteins in the membrane scission process. Recently, the WASH complex was discovered as a binding partner of retromer and a nucleation point for actin polymerization on early endosomes (Derivery et al., 2009; Gomez and Billadeau, 2009). Pulling forces that are applied to the budding structure by molecular motors or polymerizing actin filaments are expected to reduce the energy barrier involved in neck or tubule fission (Liu et al., 2006). However, even a reduced energy barrier may still require a dedicated scission factor to finalize the reaction. Several lines of evidence indicate that dynamin may provide this function. First, an interaction between WASH and dynamin was identified (Derivery et al., 2009); second, the WASH complex was found at the base of extended tubules (Derivery et al., 2009); third, dynamin inhibition induced extended long tubules on early endosomes (Mesaki et al., 2011). A combined function of actin and dynamin on membrane scission in the retrograde route was recently postulated (Johannes et al., 2014). In summary, at least in human, a model can be proposed for the concerted action of different molecules within the same macromolecular complex. The VPS trimer recruits cargo and SNX proteins, which are deforming the membrane via a BAR domain-dependent scaffolding mechanism and assymetric insertion of amphipathic helices. The RME-8 protein is recruited and to link the SNXs and the WASH subcomplexes for a concerted action in the scission process. WASH is able to initiate actin polymerization and to recruit dynamin.
Biomedical Applications of Retrograde Trafficking Initially, bacterial toxins were used to eliminate cancer cells, or at reduced levels to alter cellular processes that control proliferation, apoptosis, and differentiation. Specificity for cancer cells was reached by coupling cytotoxic toxins to antibodies (immunotoxins) (Becker and Benhar, 2012). However, for most antibody-toxin conjugates clinical evaluation did not go beyond phase I trials for various reasons, mostly linked to toxicity and lack of efficacy. Since several pathogens are using the retrograde pathway for the targeting of cytotoxic proteins into host cells, scientist
are developing tools to use the same pathway for pharmaceutical delivery (Tarrago-Trani and Storrie, 2007). Currently, three different applications of retrograde transport research can be identified (Tartour et al., 2002; Moron et al., 2004; Engedal et al., 2011): the nontoxic B-subunits of AB5 toxins can be used as carriers for cytotoxic drugs in cancer therapy, as carriers for imaging agents, or to deliver antigens to dendritic cells in order to trigger a therapeutic immune response.
Targeted Tumor Therapy Altered glycosylation patterns of tumor cells are a universal feature in carcinogenesis, and may affect cell signaling, adherence, and motility (Hakomori, 1996). Remarkably, human cancers are often characterized by modified glycosphingolipid composition and metabolism, and several tumor-associated antigens have indeed been found to be glycosphingolipids (Hakomori and Zhang, 1997). The retrograde cargo protein STxB recognizes a specific glycosphingolipid, Gb3, which is highly expressed on human cancers (Gariepy, 2001; Johannes and Decaudin, 2005; Engedal et al., 2011). By following the retrograde route, STxB avoids recycling to the plasma membrane or degradation in the late endocytic pathway, which are features that may be of general interest in the development of innovative cancer targeting strategies (reviewed in TarragoTrani and Storrie, 2007). Since intratumoral injection of Shiga toxin inhibits tumor growth (Arab et al., 1999; Ishitoya et al., 2004), STxB might be an appropriate tool for targeted therapy. Following intravenous injection or oral application in mice, STxB has indeed been shown to accumulate in adenocarcinomas of the digestive tract (Janssen et al., 2006). A prodrug composition using the topoisomerase I inhibitor SN38 has been developed that is specifically activated in membranes of the secretory pathway that have been targeted from the outside of cells, via the retrograde route (El Alaoui et al., 2007). Exploitation of this delivery modality may be particularly beneficial in therapeutic strategies requiring prolonged association of the prodrug with tumor cells to ensure efficient prodrug conversion to the active form.
Biomedical Imaging Gb3 overexpressing cancer cells might be visualized by injecting nontoxic STxB molecules coupled to imaging agents (Janssen et al., 2006; Viel et al., 2008). The imaging agents are covalently coupled to the delivery tool and highlight Gb3 expressing cells. The following imaging agents were successfully coupled to STxB and delivered to tumors in mice: florescent STxB (Viel et al., 2008), 18F-labeled STxB followed by Positron emission tomography (PET) imaging (Janssen et al., 2006), and ultrasound contrast agents (Couture et al., 2011).
Immunotherapy Protein toxins have also been used to generate specific cytotoxic T lymphocytes (CTLs) against tumor antigens to which they have been linked chemically or genetically (Smith et al., 2002). The function of the toxin-antigen conjugates is in this case to facilitate cytosolic entry of antigens, which are then degraded by
Interorganellar Communication: Interplay and Processes: Retrograde Transport
proteasomes and displayed on MHC class I molecules to prime a CTL response. This approach to generate therapeutic vaccines has been used with adenylate cyclase of Bordetella pertussis (Fayolle et al., 1996), Pseudomonas exotoxin A (PEx A) (Donnelly et al., 1993), E. coli bacterial heat-labile enterotoxin (Hearn et al., 2004), and STxB (Haicheur et al., 2000). For example, antigens that are chemically coupled to STxB are selectively targeted to dendritic cells expressing the Gb3 receptor, and it has been shown that the MHC class I-restricted presentation of the vectorized antigens is proteasome- and transporter-associated with antigen processing (TAP) dependent (Haicheur et al., 2000). This strongly suggests that the exogenous antigens are indeed delivered to the cytosolic compartment. In mice, the delivery of exogenous antigens to dendritic cells induces a potent cytotoxic T-lymphocyte response and protects the animals against experimentally induced tumor growth (Vingert et al., 2006; Pere et al., 2011; Badoual et al., 2013; Sandoval et al., 2013). One could envisage the design of new synthetic molecules that interact with target cells similarly to the toxins discussed above, and mimic the toxin trafficking pathway to deliver molecules specifically to the Golgi or ER in order to correct enzymatic defects, or elicit toxicity for the treatment of cancer. Moreover, given its avoidance of the lysosome, perhaps this route could be of better use for gene delivery and enable prolonged survival of gene therapy vectors after targeted cell entry.
See also: Vertical Integration: Applications: Neurogenesis in the Adult Brain
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Introduction
glycoconjugates having nonreducing terminal galactose (ricin receptors), enters cells via endocytosis and reaches morphologically distinct Golgi membranes. Thus, the first step of the retrograde pathway was defined – trafficking from endosomes to the Golgi apparatus (Figure 1). Scientists were searching for more than 10 years whether endogenous proteins would also use the retrograde pathway, as described by Olsnes for ricin. In 1985, a cellular protein – transferrin receptor – could be identified in leukemia cells by Snider and Rogers (1985), that also traffics from the plasma membrane to the TGN. This initial discovery was followed very soon by several other studies that used similar approaches based on Golgi-specific enzyme modifications. Of these, the characterization of mannose 6-phosphate receptor (MPRs) trafficking by Duncan and Kornfeld (1988) stood out. These authors provided first evidence for a model in which trafficking between endosomes and the TGN is part of the functional cycle of MPRs. Many lysosomal enzymes are posttranslationally modified by mannose 6-phosphate residues. These are recognized by MPRs and shuttled from the TGN to endosomes. The unloaded receptors then return to the TGN for a new transport cycle (Figure 2). Despite the discovery of several endogenous retrograde cargo molecules, toxins were used as discovery tools to study and understand this pathway further. Sandvig et al. (1992) showed for the first time in 1992 that another exogenous protein – the bacterial Shiga toxin (STx) – could reach the ER via the retrograde transport route, similar to ricin. As for ricin, the catalytic A-subunit of Shiga toxin is then translocated to the cytosol to inhibit protein biosynthesis by acting as a N-glycosidase, which cleaves a specific adenine nucleobase
Trafficking of vesicular carriers between the plasma membrane and endomembrane compartments is essential for transport of proteins and other macromolecules to diverse destinations inside and outside of the cell. Membrane trafficking is also required to assure the essential need of cells to maintain membrane homeostasis and to meet specific demands during development, signal perception, and transmission. Two major trafficking routes exist, comprising the endoplasmatic reticulum (ER), the Golgi apparatus, and endosomes: the anterograde and retrograde pathways. Both are highly conserved in the eukaryotic kingdom, including plants and some protozoa, e.g., Toxoplasma gondii. The anterograde pathway delivers proteins and lipids in a vectorial manner from the ER to the plasma membrane. This outward flow of material is counterbalanced by various retrograde trafficking routs. Retrograde cargo proteins and lipids are endocytosed and transported to endosomes, before being delivered to the trans-Golgi network (TGN), and in some instances further to the ER. The discovery of the retrograde trafficking pathway started in 1972 by studying the cellular intoxication process mediated by the plant toxin ricin (Olsnes and Pihl, 1972). Ricin is a highly toxic glycoprotein produced by the castor plant Ricinus communis and inhibits protein biosynthesis by modifying ribosomal ribonucleic acid (RNA) of toxin-exposed cells (Montanaro et al., 1973). Later, Avrameas and colleagues found that even the Golgi apparatus was a trafficking station in the intoxication process of ricin (Gonatas et al., 1975). Hence, the retrograde cargo protein ricin is binding to cell surface
A-subunit B-subunit Shiga toxin A-subunit B-subunit
Endosome
Cholera toxin A-subunit B-subunit Ricin
Golgi apparatus
Caveolae Retrograde transport Retrotranslocation
Endoplasmic reticulum Nucleus
Figure 1: Toxin entry into cells. Via different uptake routes, cholera toxin, Shiga toxin, and ricin reach early endosomes, from where they then traffic to the ER, via the Golgi apparatus. The catalyic A-subunits of these toxins are then retrotranslocated across the ER membrane to the cytosol where they can meet their molecular targets.
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Wnt Wntless Retromer
Endosome
Hydrolase MPR
Golgi apparatus Retrograde transport
Endoplasmic reticulum
Multivesicular body
Lysosome Nucleus
Figure 2 The role of retrograde transport in the cellular functions of Wntless and MPRs. Retromer is of critical importance for the trafficking of Wntless and MPRs from endosomes to the TGN as part of the cellular functions of these receptor molecules. See text for details.
from the 28S rRNA of the ribosomal 60S subunit (reviewed in Johannes and Römer, 2010). These studies have laid the groundwork for the understanding of the trafficking behavior of a whole class of cytotoxic molecules, AB5 toxins, for which no pore forming capacity could be demonstrated and that use the retrograde rout to enter into cells (Figure 1). These toxins are named after their unique subunit composition, containing a single catalytical A-subunit and a homopentameric B-subunit composed of five B-fragments. In some cases, the A-subunit is covalently linked via a disulfide bridge to the B-subunit (Sandvig and van Deurs, 2002). The catalytically inactive B-subunit is binding to cell surface receptors and serves to mediate transport of the AB5 protein complex from the cell surface to the lumen of the ER, via vesicular and tubular carriers and intracellular compartments. The B-subunit retains its binding capacity even in the absence of the A-subunit. However, the complete AB5 holotoxin is required for mediating toxicity. AB5 toxins are a medically important class of bacterial exotoxins, which cause devastating human diseases. Prominent members of this class include the above-mentioned STx, which causes life-threatening diarrhea, dysentery, hemorrhagic colitis, and hemorrhagic uremic syndrome; Escherichia coli heat-labile enterotoxins and cholera toxin (CTx), which cause endemic and epidemic diarrhea and pertussis toxin (PTx), which is the causative agent for whooping cough (Beddoe et al., 2010). Each year, infections with bacteria that produce these toxins affect millions of individuals and cause more than a million deaths. Many cellular functions in development and disease depend on the retrograde transport route, such as wnt morphogen gradient formation (Harterink et al., 2011; Yu et al., 2014; Figure 2), intracellular glucose (Esk et al., 2010), iron and copper ion homeostasis (Tabuchi et al., 2010), and glutamate receptor recycling (Zhang et al., 2012). Besides the poisonous effect of several toxins, the medical importance of the retrograde trafficking route was corroborated by many findings. Its dysfunction causes abnormal elevation of amyloid β-peptide
(Burgos et al., 2010); the nef protein of human immunodeficiency virus (HIV-1) favors immune-evasion by down modulating major histocompatibility complex (MHC) class I (Blagoveshchenskaya et al., 2002) and co-stimulatory molecules (Chaudhry et al., 2008). Recently, a new aspect in modulation of the retrograde pathway was discovered: the intracellular bacterium Legionella pneumophila is able to shut down the retrograde route in order to promote bacterial replication (Finsel et al., 2013). Taken together, understanding the retrograde trafficking pathway is important to develop efficient therapeutic strategies for a wide range of diseases.
Different Retrograde Pathways The above-mentioned ground-breaking papers have proven the existence of a transport connection between plasma membrane/endosomes and TGN/Golgi/ER. The trafficking interface between endosomes and the TGN has become a subject of intense investigation in membrane biology research. The complexity of retrograde transport arises from different pathways between endosomes and the TGN. Endosomes are a complex membrane system of tubulo-vacuolar nature of which early endosomes and late endosomes/lysosomes are the major parts (Huotari and Helenius, 2011). There is an ongoing debate on whether these separate endosomal entities of different protein and lipid compositions are linked by vesicluar carriers, or are transformed from one to the other by a process of maturation. Exactly how very early endosomes arise is not entirely clear, but it seems likely that membranes and volume are mainly derived from primary endocytic vesicles that fuse with each other. Very early endosomes receive cargo proteins through several endocytic pathways, including clathrin-mediated and clathrin-independent endocytic processes – caveolar, clathrin-independent carrier (CLIC) and ARF6-dependent pathways (Mayor and Pagano, 2007). Therefore very early endosomes are heterogeneous in terms of morphology, localization, composition, and function.
Interorganellar Communication: Interplay and Processes: Retrograde Transport
The identity and functional attributes of different endosomes is also defined by the association of cytosolic proteins to the cytoplasmic surface of the endosomal membrane (Huotari and Helenius, 2011). Very early endosomes then converge into conventional early endosomes. Their limiting membrane contains a mosaic of tubular and vacuolar domains that differ in composition and function (Zerial and McBride, 2001). These include domains enriched in Rab5, Rab4, Rab11, Arf1/COPI, and retromer (Sonnichsen et al., 2000; Bonifacino and Rojas, 2006). Domains located in the tubular extensions allow molecular sorting and generate transport carriers targeted to distinct organelles, including the plasma membrane, the recycling endosomes, and the TGN for retrograde trafficking (Bonifacino and Rojas, 2006). One of the cytosolic key players in retrograde trafficking is the retromer complex. This complex was shown to be involved in trafficking events from different endosomes to the TGN as well as for recycling to the plasma membrane (Temkin et al., 2011). How specificity is achieved will be discussed later. Pfeffer and colleagues were the first to pinpoint in 1993 an endosomal location from which the MPR recycles to the TGN: late endosomes (Lombardi et al., 1993). In 1998, Johannes and colleagues reported that STx was not found on late endosomal membranes en route to the TGN, which led to the proposal that the toxin bypasses the late endocytic pathway altogether and directly traffics from early endosomes to the TGN (Mallard et al., 1998). Recently, Lieu and Gleeson (2010) suggested that Shiga toxin might in part also traffic from recycling endosomes to the TGN. In summary, retrograde trafficking does occur from different endosomal sites (Figure 3). Routes from late, early, and recycling endosomes seem to operate in parallel. Below, we will discuss these three different scenarios individually. We list key molecular and morphological data that have helped to define them, and point out controversial matters whenever these exist.
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Late Endosomes-to-TGN Trafficking MPRs are the best characterized cargo proteins of the late endosomes-to-TGN retrograde transport process (Rohn et al., 2000). More than 60 lysosomal hydrolases acquire a unique posttranslational modification in the Golgi complex: a mannose 6-phosphate residue, which enables them to bind to MPRs. Two types of MPRs have been identified, the cation-independent (CI-MPR) and the cation-dependent MPR (CD-MPR). Both carry newly synthesized precursor hydrolazes from the Golgi to endosomes. MPRs release their ligands upon encountering the low pH of the late endocytic pathway. The receptors then return from late endosomes to the TGN to start another cycle of biosynthetic enzyme transport.
Cargo Sorting and Budding Lysosomal hydrolaze precursors are transported within the lumen of endocytic carrier vesicles to the lysosome for proteolytic activation. A multitude of sorting signals in the cytoplasmic tail of MPRs has been identified that ensure that MPRs are not lost into lysosomes where they would be degraded. A subset of these sorting signals is recognized by tail interacting protein of 47 kDa (TIP47), and strong arguments have been presented for a role of TIP47 in late endosomes-to-TGN trafficking of MPRs (Diaz et al., 1997), even if some complexity has been noticed that may originate from TIP47's additional functions in lipid droplet formation (Bulankina et al., 2009). TIP47 recognizes a phenylalanine–tryptophan motif in the cytoplasmic tail of CD-MPR (Diaz and Pfeffer, 1998), and a more complex domain of CI-MPR. TIP47 also binds Rab9GTP, upon which its affinity for the CI-MPR is increased threefold (Carroll et al., 2001). Rab9 is itself involved in the retrograde transport of MPRs (Riederer et al., 1994). It has thus been suggested that TIP47 is a coat for MPR sorting into the retrograde pathway.
A-subunit B-subunit Shiga toxin
Hydrolase
Clathrin
MPR Retromer
Early endosome
Recycling endosome
Caveolae
Late endosome
Retro
Golgi apparatus
grade transp or
t
Figure 3 Sites of endosomal retrograde sorting in mammalian cells. Retrograde sorting can operate from different endosomal locations. See text for a detailed discussion of retrogarde trafficking from early, recycling, and late endosomes.
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Docking and Fusion The Rab9 GTPase also binds to the GRIP domain golgin protein GCC185 (Golgi coiled-coil protein) (Reddy et al., 2006) for which tethering functions have been suggested. Tethering is the process by which transport intermediates are captured on target membranes to allow for subsequent N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) complex formation and fusion. GCC185 is required for retrograde transport of MPRs (Reddy et al., 2006; Derby et al., 2007) and interacts with the SNARE protein syntaxin 16 that equally has key functions for retrograde transport from late (Ganley et al., 2008) and early endosomes (see below). For the fusion of late endosomes-derived retrograde transport intermediates, syntaxin 16 is in a SNARE complex with syntaxin 10, Vti1a, and vesicle-associated membrane protein (VAMP3) (Ganley et al., 2008).
Early Endosomes-to-TGN Trafficking Mallard et al. (1998) provided first evidence for a direct transport route from early endosomes to the TGN, using Shiga toxin as a model cargo. This transport interface is used by other exogenous (cholera toxin) and endogenous (TGN38) cargo proteins (Ghosh et al., 1998; Amessou et al., 2007; Lieu et al., 2007). Even MPRs may traffic directly from early endosomes to the TGN, in addition to using the late endosomes-TGN interface. Indeed, MPRs accumulate in early endosomes under many conditions that block retrograde trafficking to the TGN, i.e., depletion for the following proteins: retromer (Seaman et al., 2013), the tSNARE syntaxin 10 (Ganley et al., 2008), the clathrin adapter AP1 (Meyer et al., 2000), the phosphatidylinositol 3-phosphate 5-kinase PIKfyve (Rutherford et al., 2006), and the GRIP domain golgin GCC88 (Lieu et al., 2007). Probably the multitude of sorting signals in the cytosolic tails of MPRs has evolved to permit recycling to the TGN from any stage of the endocytic pathway.
Cargo Sorting and Budding Clathrin is localized on early endosomes and was shown to be required for the retrograde sorting of Shiga toxin (Saint-Pol et al., 2004). In clathrin-depleted cells Shiga toxin perfectly colocalizes with transferrin receptor at times when the molecule has reached the TGN/Golgi in control conditions, demonstrating that the toxin needs clathrin to leave early endosomes. Different adapters have been identified that function with clathrin in retrograde sorting of Shiga toxin and/ or other retrograde cargo proteins: AP1 (Meyer et al., 2000), epsinR (Saint-Pol et al., 2004), and oculocerebrorenal syndrome of Lowe (inositol polyphosphate 5-phosphatase) (OCRL) (Choudhury et al., 2005). For some cargo proteins with acidic cluster motifs in their cytosolic tail, the phosphofurin acidic cluster sorting protein 1 (PACS1) appears to stabilize binding to AP1, and the CI-MPR accumulates in early endosomes upon overexpression of a dominant negative PACS1 (Scott et al., 2006). The retromer complex was identified first in yeast and then in mammalian cells. It has been shown that retromer is localized on the cytosolic leaflet of early endosomes and
required for retrograde transport. Retromer comprises two subcomplexes, a vps protein heterotrimer (Vps26, Vps29, and Vps35) and a sorting nexin (SNX) dimer. The vps heterotrimer binds cargo proteins (Seaman et al., 2013). However, different cargo proteins are recognized by different vacuolar protein sorting (VPS) proteins of the retromer heterotrimer (A-ALP and Vps10p bind to Vps35p, CI-MPR shows bipartite interactions with VPS35 and VPS29, sortilin to VPS35, divalent metal transporter 1 (DMT1-II) to VPS35, and sorLA to VPS26). The SNX subcomplex senses and/or generates curvature. Latest work from the Cullen laboratory indeed suggests a contribution of the sorting nexins to membrane bending, as purified sorting nexins were shown to deform and tubulate lipid vesicles (van Weering et al., 2012). As such, retromer could function as a coat. However, retromer does not form the same type of electron-dense coats as observed for classical coatomer protein complex-I (COPI), coatomer protein complex-II (COPII), and clathrin. In addition, the molecular mechanism by which retromer sorts cargo seems to be distinct from that of classical membrane coats (Seaman et al., 2013), but somehow similar to other endosomal sorting complexes, like the endosomal sorting complexes required for transport (ESCRT). Retromer seems to regulate trafficking of cargo proteins on non-flat membranes, rather within morphologically distinct tubulovesicular membranes, suggesting a novel mechanism of action. Furthermore, when retromer vps heterotrimer proteins are depleted, retrograde cargocontaining tubules still form that fail to detach from early endosomes (Popoff et al., 2007, 2009). These findings suggest that vps retromer heterotrimer may play a critical role for scission. Whether this function is fulfilled via amphipathic helices/BAR-domains of SNX proteins, or indirectly via recruitment of a scission factor still needs to be determined. One possibility how retromer fulfills a scission function might be via its capacity to recruit the actin nucleation promotion factor Wiskott–Aldrich syndrome protein and scar homolog protein (WASH) and to drive the polymerization of actin on early endosomal membranes (Seaman et al., 2013). A second hypothesis might be an indirect effect of the WASH complex, via the recruitment of dynamin-2 (Derivery et al., 2009). Whether and how dynamin contributes to membrane scission on early endosomes in human cells has not yet been convincingly documented. In yeast, a dynamin-related protein, termed Vps1, promotes fission of SNX-coated retromer tubules on early endosomes (Chi et al., 2014). Important differences exist between the prototypical retromer in yeast and retromer in higher eukaryotes. A key difference is that, in mammalian cells, retromer is not a stable heteropentamer as in yeast, but a much more transient association of the cargo-selective trimer (Vps35, Vps26, and Vps29) and the Snx proteins (Snx1 or Snx2 with Snx5 or Snx6) (Seaman et al., 2013). In addition, retromer exists in different subcomplexes, depending on which SNX protein is involved. In mammalian cells, SNX proteins indeed seem to determine the trafficking route and thereby the specificity of retrograde trafficking. Furthermore, many accessory retromer proteins that were identified from studies in higher eukaryotes are not conserved in yeast. The yeast retromer complex seems to function independent of the WASH complex. Thus, the exact
Interorganellar Communication: Interplay and Processes: Retrograde Transport
scope of functions of retromer accessory proteins is not clear at this stage. Lipids also play critical roles in retrograde sorting. Notably the phosphatidylinositol lipids phosphatidylinositol 3-phosphate (Skånland et al., 2007) and phosphatidylinositol 3,5-bisphosphate (Rutherford et al., 2006) are directly involved in the membrane recruitment of sorting nexins, and may also affect retrograde sorting via Hrs (Popoff et al., 2009). Furthermore, a redistribution of retromer components has been observed in glycosphingolipid-depleted cells (Raa et al., 2009), in agreement with earlier studies that had found a role for raft lipids in retrograde sorting (Falguières et al., 2001). By what mechanism mesoscale raft lipid organization affects the retromer complex, and whether specialized lipid domains are required for retrograde trafficking from early endosomes has yet to be determined.
Docking and Fusion Early endosome-derived vesicles are trafficking further to the TGN, and this transport is microtubule dependent (Hehnly et al., 2006). The Golgi-associated retrograde protein (GARP) complex was identified for tethering of MPR positive vesicles to TGN membranes (Perez-Victoria et al., 2008). The GARP complex is recruited at the level of early endosomes, and then functions at the TGN to promote SNARE complex formation in interaction with syntaxin 6, syntaxin 16, and VAMP4 (PerezVictoria and Bonifacino, 2009), three SNARE proteins that have previously been implicated in early endosomes-to-TGN trafficking (Mallard et al., 2002). Other tethering factors for early endosomal retrograde cargo proteins are GCC88 and GCC185 (Derby et al., 2007; Lieu et al., 2007).
Recycling Endosomes-to-TGN Trafficking In a comparative study it was found that depletion of the tethering factor GCC185 inhibited retrograde transport of Shiga toxin, but not that of the endogenous cargo protein TGN38 (Derby et al., 2007), while the opposite was observed for the depletion of the tethering factor GCC85 (Lieu et al., 2007). Since Shiga toxin, but not TGN38, could be detected in recycling endosomes under different experimental conditions, the authors concluded that TGN38 was transported directly
from early endosomes to the TGN, while Shiga toxin could also transit via recycling endosomes (Lieu and Gleeson, 2010). This conclusion is in apparent contradiction with earlier studies that had described the retrograde trafficking of TGN38 via recycling endosomes (Ghosh et al., 1998), and it is difficult to reconcile with the established function of early/maturing endosomal retromer in the trafficking of Shiga toxin to the TGN. Furthermore, as mentioned above, recycling endosomes remain poorly defined in molecular terms, and whether they have evolved a truly independent retrograde sorting machinery, possibly involving Rab11 (Chaudhry et al., 2008; Wilcke et al., 2000) remains to be established.
Biophysical Mechanisms for Retrograde Trafficking All trafficking events of proteins and lipids between distinct membrane-enclosed organelles are following the same mechanistic principles. The course of action per se for generic trafficking processes is as follows: cargo sorting, membrane bending and bud formation, scission in the neck region of the invaginated bud or tubule, and finally fusion of transport intermediates with acceptor organelles. In the following, we have summarized biophysical mechanisms of each of these steps, based on the list of established retrograde trafficking factors.
Membrane Bending The exact mechanism of membrane bending for retrograde trafficking from early endosomes is not known. However, different SNXs bearing BAR-domains and/or amphipathic helices were analyzed by in vitro studies (van Weering et al., 2012). Therefore, two SNX-driven mechanisms for membrane bending can be envisioned (Figure 4): (1) assymetric insertion of amphipathic helices and/or (2) scaffolding by BAR-domain containing SNXs. Whether SNXs are able to form scaffolds at physiological concentrations such as to drive membrane deformation (Sorre et al., 2012), still needs to be determined. Recently the concerted action of retromer SNXs and the WASH complex on tubule formation was supported by Seaman and colleagues (Freeman et al., 2014). This report indicated the binding of RME-8 to retromer SNXs and the WASH complex
Membrane bending by SNXs SNXs helix insertion
Scaffolding Concentration SNXs with BAR-domain
(a)
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(b)
Figure 4 Membrane bending mechanisms in relation to SNX proteins. (a) Insertion of helices leads to asymmetric expansion of the cytosolic leaflet and to subsequent membrane bending. (b) BAR domain binding can serve to recognize membrane curvature (low BAR-domain protein concentration on membranes), or to drive membrane bending through a scaffolding effect (high concentration).
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protein FAM21. Interestingly, clathrin could be localized to early endosomes (Popoff et al., 2009; Esk et al., 2010; Freeman et al., 2014), and RME-8 also binds to the clathrin uncoating ATPase Hsc70 (Girard et al., 2005). However, no reports currently exist on interactions between clathrin and components of the retromer machinery. Whether clathrin is directly involved in membrane deformation for other retrograde cargo protein requires further studies but seems likely when considering the function of the clathrin interactors AP1 (Meyer et al., 2000; Natsume et al., 2006), epsinR (Saint-Pol et al., 2004), and OCRL (Choudhury et al., 2005) in retrograde sorting on early endosomes.
Scission The first indication for a molecular player for a scission process of endosomal tubules came from knock down experiments of vps retromer proteins (Popoff et al., 2007, 2009). Endosomal tubules formed that failed to detach from early endosomes, pointing toward a function for retromer or retromer binding proteins in the membrane scission process. Recently, the WASH complex was discovered as a binding partner of retromer and a nucleation point for actin polymerization on early endosomes (Derivery et al., 2009; Gomez and Billadeau, 2009). Pulling forces that are applied to the budding structure by molecular motors or polymerizing actin filaments are expected to reduce the energy barrier involved in neck or tubule fission (Liu et al., 2006). However, even a reduced energy barrier may still require a dedicated scission factor to finalize the reaction. Several lines of evidence indicate that dynamin may provide this function. First, an interaction between WASH and dynamin was identified (Derivery et al., 2009); second, the WASH complex was found at the base of extended tubules (Derivery et al., 2009); third, dynamin inhibition induced extended long tubules on early endosomes (Mesaki et al., 2011). A combined function of actin and dynamin on membrane scission in the retrograde route was recently postulated (Johannes et al., 2014). In summary, at least in human, a model can be proposed for the concerted action of different molecules within the same macromolecular complex. The VPS trimer recruits cargo and SNX proteins, which are deforming the membrane via a BAR domain-dependent scaffolding mechanism and assymetric insertion of amphipathic helices. The RME-8 protein is recruited and to link the SNXs and the WASH subcomplexes for a concerted action in the scission process. WASH is able to initiate actin polymerization and to recruit dynamin.
Biomedical Applications of Retrograde Trafficking Initially, bacterial toxins were used to eliminate cancer cells, or at reduced levels to alter cellular processes that control proliferation, apoptosis, and differentiation. Specificity for cancer cells was reached by coupling cytotoxic toxins to antibodies (immunotoxins) (Becker and Benhar, 2012). However, for most antibody-toxin conjugates clinical evaluation did not go beyond phase I trials for various reasons, mostly linked to toxicity and lack of efficacy. Since several pathogens are using the retrograde pathway for the targeting of cytotoxic proteins into host cells, scientist
are developing tools to use the same pathway for pharmaceutical delivery (Tarrago-Trani and Storrie, 2007). Currently, three different applications of retrograde transport research can be identified (Tartour et al., 2002; Moron et al., 2004; Engedal et al., 2011): the nontoxic B-subunits of AB5 toxins can be used as carriers for cytotoxic drugs in cancer therapy, as carriers for imaging agents, or to deliver antigens to dendritic cells in order to trigger a therapeutic immune response.
Targeted Tumor Therapy Altered glycosylation patterns of tumor cells are a universal feature in carcinogenesis, and may affect cell signaling, adherence, and motility (Hakomori, 1996). Remarkably, human cancers are often characterized by modified glycosphingolipid composition and metabolism, and several tumor-associated antigens have indeed been found to be glycosphingolipids (Hakomori and Zhang, 1997). The retrograde cargo protein STxB recognizes a specific glycosphingolipid, Gb3, which is highly expressed on human cancers (Gariepy, 2001; Johannes and Decaudin, 2005; Engedal et al., 2011). By following the retrograde route, STxB avoids recycling to the plasma membrane or degradation in the late endocytic pathway, which are features that may be of general interest in the development of innovative cancer targeting strategies (reviewed in TarragoTrani and Storrie, 2007). Since intratumoral injection of Shiga toxin inhibits tumor growth (Arab et al., 1999; Ishitoya et al., 2004), STxB might be an appropriate tool for targeted therapy. Following intravenous injection or oral application in mice, STxB has indeed been shown to accumulate in adenocarcinomas of the digestive tract (Janssen et al., 2006). A prodrug composition using the topoisomerase I inhibitor SN38 has been developed that is specifically activated in membranes of the secretory pathway that have been targeted from the outside of cells, via the retrograde route (El Alaoui et al., 2007). Exploitation of this delivery modality may be particularly beneficial in therapeutic strategies requiring prolonged association of the prodrug with tumor cells to ensure efficient prodrug conversion to the active form.
Biomedical Imaging Gb3 overexpressing cancer cells might be visualized by injecting nontoxic STxB molecules coupled to imaging agents (Janssen et al., 2006; Viel et al., 2008). The imaging agents are covalently coupled to the delivery tool and highlight Gb3 expressing cells. The following imaging agents were successfully coupled to STxB and delivered to tumors in mice: florescent STxB (Viel et al., 2008), 18F-labeled STxB followed by Positron emission tomography (PET) imaging (Janssen et al., 2006), and ultrasound contrast agents (Couture et al., 2011).
Immunotherapy Protein toxins have also been used to generate specific cytotoxic T lymphocytes (CTLs) against tumor antigens to which they have been linked chemically or genetically (Smith et al., 2002). The function of the toxin-antigen conjugates is in this case to facilitate cytosolic entry of antigens, which are then degraded by
Interorganellar Communication: Interplay and Processes: Retrograde Transport
proteasomes and displayed on MHC class I molecules to prime a CTL response. This approach to generate therapeutic vaccines has been used with adenylate cyclase of Bordetella pertussis (Fayolle et al., 1996), Pseudomonas exotoxin A (PEx A) (Donnelly et al., 1993), E. coli bacterial heat-labile enterotoxin (Hearn et al., 2004), and STxB (Haicheur et al., 2000). For example, antigens that are chemically coupled to STxB are selectively targeted to dendritic cells expressing the Gb3 receptor, and it has been shown that the MHC class I-restricted presentation of the vectorized antigens is proteasome- and transporter-associated with antigen processing (TAP) dependent (Haicheur et al., 2000). This strongly suggests that the exogenous antigens are indeed delivered to the cytosolic compartment. In mice, the delivery of exogenous antigens to dendritic cells induces a potent cytotoxic T-lymphocyte response and protects the animals against experimentally induced tumor growth (Vingert et al., 2006; Pere et al., 2011; Badoual et al., 2013; Sandoval et al., 2013). One could envisage the design of new synthetic molecules that interact with target cells similarly to the toxins discussed above, and mimic the toxin trafficking pathway to deliver molecules specifically to the Golgi or ER in order to correct enzymatic defects, or elicit toxicity for the treatment of cancer. Moreover, given its avoidance of the lysosome, perhaps this route could be of better use for gene delivery and enable prolonged survival of gene therapy vectors after targeted cell entry.
See also: Vertical Integration: Applications: Neurogenesis in the Adult Brain
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