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Membrane traffic: How do GGAs fit in with the adaptors?
R460
Dispatch
Membrane traffic: How do GGAs fit in with the adaptors? Michael W. Black and Hugh R.B. Pelham
membrane. Recent work with the AP-2 coat has shown that the plasma membrane is identified as a target by the presence of modified phosphatidylinositides [2,3]. AP-2 and other accessory proteins bind to these lipids, recruit clathrin and interact with endocytic signals on membrane proteins, thus sorting them into endocytic vesicles.
The AP-1 adaptor complex has been cast as the major player in clathrin coat formation for vesicular transport from the trans-Golgi to the endocytic pathway. But new results on ‘GGA’ proteins have raised doubts about this paradigm and suggest both a new sorting mechanism and an unexpected complexity in the roles of clathrin. Address: MRC-Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. E-mail: [email protected]
In contrast, it has proven much harder to define the events that allow AP-1 vesicles to form on the Golgi. A key observation for understanding AP-1 recruitment was that the small GTPase ARF-1 is required for the binding of AP-1 to Golgi membranes [4,5]. ARF has a low affinity for membranes when bound to GDP, but when this is exchanged for GTP the membrane-binding conformation of the protein is stabilised. Specific exchange factors associated peripherally with Golgi membranes create a pool of ARF–GTP, which is used to recruit several different coats with roles on the Golgi. In the case of AP-1, a series of biochemical experiments culminated in the demonstration that it could be recruited to artificial liposomes in an ARF-dependent manner [6]. However, an additional unknown factor, found on Golgi membranes and in cytosol, was also required.
Current Biology 2001, 11:R460–R462 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Transport of membrane proteins within cells involves the creation and fusion of transport vesicles. Gathering the correct set of proteins into a vesicle is a function of the protein coat that encases it as it forms, and a variety of different coats have been identified that mediate particular transport steps [1]. The best-characterised coats are those that contain clathrin, a spindly protein that assembles into a wire-netting-like cage over the membrane. Clathrin associates with tetrameric protein complexes termed adaptors (reviewed in [2]): the best characterised of these are the AP-1 and AP-2 complexes. AP-2 is found on the plasma membrane and participates in endocytosis, whereas AP-1 is localised largely on the trans-Golgi network and is implicated in traffic directly from Golgi to endosomes. A third adaptor, AP-3, may also be associated with clathrin and mediates a specialised route for some proteins from Golgi to the endosome/lysosome system.
The so-called ‘GGA’ proteins entered the scene as a result of their ability to bind ARF–GTP [7] and independently by their marked homology to the ‘ear’ domain of γ-adaptin [8,9]. There are three members of this family in animal cells and two in yeast, named GGA proteins for their Golgi localisation, γ-adaptin homology and ARF-binding characteristics. The modular structure of this family was recently examined in detail [10] (Figure 1), and this and other studies have shown that the GGAs are coat proteins that directly link ARF, via their GAT domain [7,8], to clathrin, via their hinge and GAE domains. The GAT domain specifically binds to ARF–GTP, not the GDP-bound cytosolic form, and is sufficient to localise a heterologous protein to the Golgi membrane. Because the GGAs compete for
Vesicle coats are thought to sort membrane proteins by interacting, directly or indirectly, with their cytoplasmic domains. However, if the coats are to sort the membrane proteins, rather than vice versa, a mechanism is required to ensure that a given coat assembles only on the correct Figure 1
0
100 VHS
200
Number of amino acids 300 400 500 600
GAT
Hinge
700
639
Human GGA2
613
Human GGA3
Yeast Gga2p
900
GAE
Human GGA1
Yeast Gga1p
800
723 557 585 Current Biology
Schematic representation of the four-domain structure identified in GGAs from both human and yeast. The current data suggest that the function of each domain is as follows: the VHS domain interacts with cytosolic tails of cargo proteins; the GAT domain binds and stabilises ARF-GTP; the hinge and GAE domains bind to clathrin heavy chains; the GAE domain associates with accessory factors such as γ-synergin. Total numbers of amino acid residues of each protein are noted to the right.
Dispatch
the same binding site on ARF as that recognised by GTPase activator proteins, they can stabilise this membrane-associated form of ARF. Furthermore, the GAE domain has been observed to interact directly with accessory proteins, such as γ-synergin [11], which also bind to AP-1 complexes. The GGAs thus seem to have all the features needed to create a clathrin coat on Golgi membranes (Figure 2). The GGAs are not, however, present in clathrin-coated vesicles purified from mammalian cells [9]. ARF molecules are also largely absent from these vesicles, presumably because, once their bound GTPs have been hydrolysed, they are released back to the cytosol. The mammalian GGAs, which are monomeric proteins, may not have sufficient affinity for clathrin to remain on vesicles once the ARF is displaced. Loss of GGAs may occur either during vesicle formation in vivo, in which case AP-1 may be able to replace them, or during the process of vesicle purification. Recent studies have shown that the yeast GGAs are more stably associated with clathrin, and can be detected in purified vesicles [12]. Surprisingly, the GGAs do more than recruit a clathrin coat: they also bind the membrane proteins that are the vesicle cargo. In animal cells, major commuters on the Golgi–endosome route are the two mannose-6-phosphate receptors M6PR46 and M6PR300, which sort lysosomal hydrolases. The cytoplasmic tails of these proteins contain a variety of signals, including those for endocytosis and retrieval from endosomes. Detailed studies have established that the Golgito-endosome transport of mannose-6-phosphate receptors requires a signal comprised of an acidic stretch and a dileucine motif [13]. Bonifacino and colleagues [14] have shown that this signal is specifically recognised by the VHS domains of all three GGAs, and can be efficiently discriminated from other, related motifs that have different targeting functions in vivo. Moreover, GGA1 colocalises with mannose-6-phosphate receptors at the Golgi, and GFPtagged versions of GGA1 and M6PR46 can be observed departing together from the Golgi in tubular-vesicular structures in living cells. Finally, expression of a truncated form of GGA1, lacking the clathrin-binding domains, traps mannose-6-phosphate receptors in the trans-Golgi network, as would be expected if the fragment binds the receptors and blocks their recruitment into clathrin-coated vesicles. Independent studies by Nielsen et al. [15] have shown that a different receptor, sortilin, behaves much like the mannose6-phosphate receptors. Like them, sortilin has an acidic dileucine motif in its cytoplasmic tail which is required for sorting from Golgi to endosomes and binds GGA2. In yeast, too, GGAs have been implicated in specific transport from Golgi to late endosomes. Mutations of GGAs not only affect the sorting of vacuolar hydrolases by the sortilin homologue Vps10p [8,9], but also completely abolish the ability to recognise a sorting signal on the late endosomal protein
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Figure 2
Clathrin
GAE
GGA
γ-synergin
GAT ARF1 GTP
VHS
M6PR
Current Biology
A model for the assembly of GGA-containing coats. The GGA proteins (red) are recruited by ARF–GTP and in turn recruit cargo proteins, such as the mannose-6-phosphate receptor (M6PR), as well as clathrin and accessory proteins, such as γ-synergin, which form the vesicle coat.
Pep12p [16], the only cytoplasmic signal identified thus far for Golgi-endosome transport in yeast. While clathrin is also required for this step, AP-1 — the only adaptor that binds clathrin in this organism — is not; indeed, AP-1 has not yet been shown to be essential for any sorting process in yeast. So what is the role of AP-1, long assumed to be responsible for sorting the mannose-6-phosphate receptors in animal cells? It does bind to their tails, but this does not require the di-leucine motif implicated in sorting at the transGolgi network [1,17]. One possibility is that GGAs and AP-1 inhabit the same vesicles, and that AP-1 helps to retain the receptors, yet lacks the affinity needed to recruit them. Alternatively, AP-1 may sort the receptors at a different step. Support for this model has been obtained from observations of mammalian cells that lack a functional AP-1 complex [18], where mannose-6-phosphate receptors accumulate in early endosomes, suggesting that a major role of AP-1 is in early endosome–Golgi recycling. Regardless of its role, the fact remains that AP-1, with the aid of an additional unidentified factor, can be recruited to Golgi membranes by ARF. Could this factor be the GGAs? In principle, AP-1 could bind clathrin that has been recruited to the membrane by GGAs. However, this is unlikely to be the whole story, as the same argument could be applied to AP-2, yet AP-2 is not found on the Golgi. Also, the Golgi localisation of GGAs is completely dependent upon ARF, whereas the unidentified factor
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Current Biology Vol 11 No 12
remains on Golgi membranes even in ARF’s absence [6]. It seems more likely that there is a second way to recruit AP-1, possibly involving a direct interaction with ARF [19], allowing the formation of two different classes of clathrincoated vesicles: those with GGAs and those without. This would fit with the observation that, despite overlap between GGA and AP-1 immunofluorescence on Golgi membranes, regions can be seen that appear to have AP-1 but no GGA protein [8]. It is also supported by genetic evidence that yeast AP-1 can function without the GGAs [12]. What could be the purpose of Golgi-derived vesicles containing AP-1 but no GGAs? A possible clue is provided by studies in yeast. Removal of the GGA proteins prevents traffic from Golgi to late endosomes, but not to early endosomes [16]. In contrast, temperature-sensitive clathrin mutants have been observed to mis-sort even those proteins that would normally traffic to early endosomes [20]. This route is a ‘default’ pathway in yeast, as no cytoplasmic signal is required for a membrane protein to enter it — characteristics of the transmembrane domain appear to be the main determinant. Thus, clathrin seems to mediate both a signal-dependent (GGA) pathway to late endosomes and a signal-independent pathway to early endosomes. In agreement with this, the sorting receptor for vacuolar hydrolases (Vps10p) can be found in clathrincoated vesicles even when its cytoplasmic tail is deleted [21]. Extrapolation to animal cells would suggest that there might be a GGA-independent, clathrin-mediated route from the trans-Golgi network to early endosomes. Such a pathway has indeed been invoked for some trans-Golgi network proteins, for example TGN46 [22]. In this case, AP-1 might serve to stabilise clathrin coats but not play a major role in the recruitment of cargo. The creation of vesicles with shared coat components from a single organelle that differ in both cargo and destination seems difficult to envision. However, there is much evidence for the segregation of different lipids and associated proteins into distinct domains within membranes. As the membrane lipid composition can also clearly affect the recruitment of coat proteins [3,6], the lateral segregation of cargoes and some coat components prior to budding could allow such distinct vesicles to form. The discovery and characterisation of GGA proteins has greatly clarified the role of ARF in coat recruitment and protein sorting at the trans-Golgi network, but it has also raised new questions. Are GGAs and AP-1 present in the same vesicles? Can AP-1-containing vesicles be formed without GGAs? If so, what controls their formation and content, or determines their destination? The ability to create vesicles from liposomes in vitro will allow such subtleties to be investigated in detail. Protein sorting is now firmly in the biochemical domain.
References 1. Rohn W, Rouillé Y, Waguri S, Hoflack B: Bi-directional trafficking between the trans-Golgi network and the endosomal/lysosomal system. J Cell Sci 2000, 113:2093-2101. 2. Kirchhausen T: Adaptors for clathrin-mediated traffic. Annu Rev Cell Dev Biol 1999, 15:705-732. 3. Ford M, Pearse B, Higgins M, Vallis Y, Owen D, Gibson A, Hopkins C, Evans P, McMahon H: Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 2001, 291:1051-1055. 4. Stamnes M, Rothman J: The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein. Cell 1993, 73:999-1005. 5. Traub L, Ostrom J, Kornfeld S: Biochemical dissection of AP-1 recruitment onto Golgi membranes. J Cell Biol 1993, 123:561-573. 6. Zhu Y, Drake M, Kornfeld S: ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes. Proc Natl Acad Sci USA 1999, 96: 5013-5018. 7. Boman A, Zhang C, Zhu X, Kahn R: A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi. Mol Biol Cell 2000, 11:1241-1255. 8. Dell’Angelica E, Puertollano R, Mullins C, Aguilar R, Vargas J, Harnell L, Bonifacino J: GGAs: a family of ADP ribosylation factorbinding proteins related to adaptors and associated with the Golgi complex. J Cell Biol 2000, 149:81-93. 9. Hirst J, Lui W, Bright N, Totty N, Seaman M, Robinson M: A family of proteins with g-adaptin and VHS domains facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. J Cell Biol 2000, 149:67-79. 10. Puertollano R, Randazzo P, Presley J, Hartnell L, Bonifacino J: The GGAs promote ARF-dependent recruitment of clathrin to the TGN. Cell 2001, 105:93-102. 11. Takatsu H, Yoshino K, Nakayama K: Adaptor γ-ear homology domain conserved in γ-adaptin and GGA proteins that interact with γ-synergin. Biochem Biophys Res Comm 2000, 271:719-725. 12. Costaguta G, Stefan C, Bensen E, Emr S, Payne G: Yeast GGA coat proteins function with clathrin in Golgi to endosome transport. Mol Biol Cell 2001, in press. 13. Chen H, Yuan J, Lobel P: Systematic mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor cytoplasmic domain. An acidic cluster containing a key aspartate is important for function in lysosomal enzyme sorting. J Biol Chem 1997, 14:7003-7012. 14. Puertollano R, Aguilar R, Gorshkova I, Crouch R, Bonifacino J: Sorting of mannose 6-phosphate receptors mediated by the GGAs. Science 2001, 292:1712-1716. 15. Nielsen M, Madsen P, Christensen E, Nykjaer A, Gliemann J, Kasper D, Pohlmann R, Petersen C: The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein. EMBO J 2001, 20:2180-2190. 16. Black M, Pelham H: A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins. J Cell Biol 2000, 151:587-600. 17. Honing S, Sosa M, Hille-Rehfeld A, Figura KV: The 46 kDa mannose 6-phosphate receptor contains multiple binding sites for clathrin adaptors. J Biol Chem 1997, 272:19884-19890. 18. Meyer C, Zizioli D, Lausmann S, Eskelinen E, Hamann J, Saftig P, Figura K, Schu P: µ1A-adaptin-deficient mice: lethality, loss of AP-1 binding and rerouting of mannose 6-phosphate receptors. EMBO J 2000, 19:2193-2203. 19. Austin C, Hinners I, Tooze S: Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules. J Biol Chem 2000, 275:21862-21869. 20. Redding K, Seeger M, Payne G, Fuller R: The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p. Mol Biol Cell 1996, 7:1667-1677. 21. Deloche O, Yeung B, Payne G, Schekman R: Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrincoated vesicles. Mol Biol Cell 2001, 12:475-485. 22. Banting G, Maile R, Roquemore E: The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrinmediated endocytosis. J Cell Sci 1998, 111:3451-3458.
Dispatch
Membrane traffic: How do GGAs fit in with the adaptors? Michael W. Black and Hugh R.B. Pelham
membrane. Recent work with the AP-2 coat has shown that the plasma membrane is identified as a target by the presence of modified phosphatidylinositides [2,3]. AP-2 and other accessory proteins bind to these lipids, recruit clathrin and interact with endocytic signals on membrane proteins, thus sorting them into endocytic vesicles.
The AP-1 adaptor complex has been cast as the major player in clathrin coat formation for vesicular transport from the trans-Golgi to the endocytic pathway. But new results on ‘GGA’ proteins have raised doubts about this paradigm and suggest both a new sorting mechanism and an unexpected complexity in the roles of clathrin. Address: MRC-Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. E-mail: [email protected]
In contrast, it has proven much harder to define the events that allow AP-1 vesicles to form on the Golgi. A key observation for understanding AP-1 recruitment was that the small GTPase ARF-1 is required for the binding of AP-1 to Golgi membranes [4,5]. ARF has a low affinity for membranes when bound to GDP, but when this is exchanged for GTP the membrane-binding conformation of the protein is stabilised. Specific exchange factors associated peripherally with Golgi membranes create a pool of ARF–GTP, which is used to recruit several different coats with roles on the Golgi. In the case of AP-1, a series of biochemical experiments culminated in the demonstration that it could be recruited to artificial liposomes in an ARF-dependent manner [6]. However, an additional unknown factor, found on Golgi membranes and in cytosol, was also required.
Current Biology 2001, 11:R460–R462 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Transport of membrane proteins within cells involves the creation and fusion of transport vesicles. Gathering the correct set of proteins into a vesicle is a function of the protein coat that encases it as it forms, and a variety of different coats have been identified that mediate particular transport steps [1]. The best-characterised coats are those that contain clathrin, a spindly protein that assembles into a wire-netting-like cage over the membrane. Clathrin associates with tetrameric protein complexes termed adaptors (reviewed in [2]): the best characterised of these are the AP-1 and AP-2 complexes. AP-2 is found on the plasma membrane and participates in endocytosis, whereas AP-1 is localised largely on the trans-Golgi network and is implicated in traffic directly from Golgi to endosomes. A third adaptor, AP-3, may also be associated with clathrin and mediates a specialised route for some proteins from Golgi to the endosome/lysosome system.
The so-called ‘GGA’ proteins entered the scene as a result of their ability to bind ARF–GTP [7] and independently by their marked homology to the ‘ear’ domain of γ-adaptin [8,9]. There are three members of this family in animal cells and two in yeast, named GGA proteins for their Golgi localisation, γ-adaptin homology and ARF-binding characteristics. The modular structure of this family was recently examined in detail [10] (Figure 1), and this and other studies have shown that the GGAs are coat proteins that directly link ARF, via their GAT domain [7,8], to clathrin, via their hinge and GAE domains. The GAT domain specifically binds to ARF–GTP, not the GDP-bound cytosolic form, and is sufficient to localise a heterologous protein to the Golgi membrane. Because the GGAs compete for
Vesicle coats are thought to sort membrane proteins by interacting, directly or indirectly, with their cytoplasmic domains. However, if the coats are to sort the membrane proteins, rather than vice versa, a mechanism is required to ensure that a given coat assembles only on the correct Figure 1
0
100 VHS
200
Number of amino acids 300 400 500 600
GAT
Hinge
700
639
Human GGA2
613
Human GGA3
Yeast Gga2p
900
GAE
Human GGA1
Yeast Gga1p
800
723 557 585 Current Biology
Schematic representation of the four-domain structure identified in GGAs from both human and yeast. The current data suggest that the function of each domain is as follows: the VHS domain interacts with cytosolic tails of cargo proteins; the GAT domain binds and stabilises ARF-GTP; the hinge and GAE domains bind to clathrin heavy chains; the GAE domain associates with accessory factors such as γ-synergin. Total numbers of amino acid residues of each protein are noted to the right.
Dispatch
the same binding site on ARF as that recognised by GTPase activator proteins, they can stabilise this membrane-associated form of ARF. Furthermore, the GAE domain has been observed to interact directly with accessory proteins, such as γ-synergin [11], which also bind to AP-1 complexes. The GGAs thus seem to have all the features needed to create a clathrin coat on Golgi membranes (Figure 2). The GGAs are not, however, present in clathrin-coated vesicles purified from mammalian cells [9]. ARF molecules are also largely absent from these vesicles, presumably because, once their bound GTPs have been hydrolysed, they are released back to the cytosol. The mammalian GGAs, which are monomeric proteins, may not have sufficient affinity for clathrin to remain on vesicles once the ARF is displaced. Loss of GGAs may occur either during vesicle formation in vivo, in which case AP-1 may be able to replace them, or during the process of vesicle purification. Recent studies have shown that the yeast GGAs are more stably associated with clathrin, and can be detected in purified vesicles [12]. Surprisingly, the GGAs do more than recruit a clathrin coat: they also bind the membrane proteins that are the vesicle cargo. In animal cells, major commuters on the Golgi–endosome route are the two mannose-6-phosphate receptors M6PR46 and M6PR300, which sort lysosomal hydrolases. The cytoplasmic tails of these proteins contain a variety of signals, including those for endocytosis and retrieval from endosomes. Detailed studies have established that the Golgito-endosome transport of mannose-6-phosphate receptors requires a signal comprised of an acidic stretch and a dileucine motif [13]. Bonifacino and colleagues [14] have shown that this signal is specifically recognised by the VHS domains of all three GGAs, and can be efficiently discriminated from other, related motifs that have different targeting functions in vivo. Moreover, GGA1 colocalises with mannose-6-phosphate receptors at the Golgi, and GFPtagged versions of GGA1 and M6PR46 can be observed departing together from the Golgi in tubular-vesicular structures in living cells. Finally, expression of a truncated form of GGA1, lacking the clathrin-binding domains, traps mannose-6-phosphate receptors in the trans-Golgi network, as would be expected if the fragment binds the receptors and blocks their recruitment into clathrin-coated vesicles. Independent studies by Nielsen et al. [15] have shown that a different receptor, sortilin, behaves much like the mannose6-phosphate receptors. Like them, sortilin has an acidic dileucine motif in its cytoplasmic tail which is required for sorting from Golgi to endosomes and binds GGA2. In yeast, too, GGAs have been implicated in specific transport from Golgi to late endosomes. Mutations of GGAs not only affect the sorting of vacuolar hydrolases by the sortilin homologue Vps10p [8,9], but also completely abolish the ability to recognise a sorting signal on the late endosomal protein
R461
Figure 2
Clathrin
GAE
GGA
γ-synergin
GAT ARF1 GTP
VHS
M6PR
Current Biology
A model for the assembly of GGA-containing coats. The GGA proteins (red) are recruited by ARF–GTP and in turn recruit cargo proteins, such as the mannose-6-phosphate receptor (M6PR), as well as clathrin and accessory proteins, such as γ-synergin, which form the vesicle coat.
Pep12p [16], the only cytoplasmic signal identified thus far for Golgi-endosome transport in yeast. While clathrin is also required for this step, AP-1 — the only adaptor that binds clathrin in this organism — is not; indeed, AP-1 has not yet been shown to be essential for any sorting process in yeast. So what is the role of AP-1, long assumed to be responsible for sorting the mannose-6-phosphate receptors in animal cells? It does bind to their tails, but this does not require the di-leucine motif implicated in sorting at the transGolgi network [1,17]. One possibility is that GGAs and AP-1 inhabit the same vesicles, and that AP-1 helps to retain the receptors, yet lacks the affinity needed to recruit them. Alternatively, AP-1 may sort the receptors at a different step. Support for this model has been obtained from observations of mammalian cells that lack a functional AP-1 complex [18], where mannose-6-phosphate receptors accumulate in early endosomes, suggesting that a major role of AP-1 is in early endosome–Golgi recycling. Regardless of its role, the fact remains that AP-1, with the aid of an additional unidentified factor, can be recruited to Golgi membranes by ARF. Could this factor be the GGAs? In principle, AP-1 could bind clathrin that has been recruited to the membrane by GGAs. However, this is unlikely to be the whole story, as the same argument could be applied to AP-2, yet AP-2 is not found on the Golgi. Also, the Golgi localisation of GGAs is completely dependent upon ARF, whereas the unidentified factor
R462
Current Biology Vol 11 No 12
remains on Golgi membranes even in ARF’s absence [6]. It seems more likely that there is a second way to recruit AP-1, possibly involving a direct interaction with ARF [19], allowing the formation of two different classes of clathrincoated vesicles: those with GGAs and those without. This would fit with the observation that, despite overlap between GGA and AP-1 immunofluorescence on Golgi membranes, regions can be seen that appear to have AP-1 but no GGA protein [8]. It is also supported by genetic evidence that yeast AP-1 can function without the GGAs [12]. What could be the purpose of Golgi-derived vesicles containing AP-1 but no GGAs? A possible clue is provided by studies in yeast. Removal of the GGA proteins prevents traffic from Golgi to late endosomes, but not to early endosomes [16]. In contrast, temperature-sensitive clathrin mutants have been observed to mis-sort even those proteins that would normally traffic to early endosomes [20]. This route is a ‘default’ pathway in yeast, as no cytoplasmic signal is required for a membrane protein to enter it — characteristics of the transmembrane domain appear to be the main determinant. Thus, clathrin seems to mediate both a signal-dependent (GGA) pathway to late endosomes and a signal-independent pathway to early endosomes. In agreement with this, the sorting receptor for vacuolar hydrolases (Vps10p) can be found in clathrincoated vesicles even when its cytoplasmic tail is deleted [21]. Extrapolation to animal cells would suggest that there might be a GGA-independent, clathrin-mediated route from the trans-Golgi network to early endosomes. Such a pathway has indeed been invoked for some trans-Golgi network proteins, for example TGN46 [22]. In this case, AP-1 might serve to stabilise clathrin coats but not play a major role in the recruitment of cargo. The creation of vesicles with shared coat components from a single organelle that differ in both cargo and destination seems difficult to envision. However, there is much evidence for the segregation of different lipids and associated proteins into distinct domains within membranes. As the membrane lipid composition can also clearly affect the recruitment of coat proteins [3,6], the lateral segregation of cargoes and some coat components prior to budding could allow such distinct vesicles to form. The discovery and characterisation of GGA proteins has greatly clarified the role of ARF in coat recruitment and protein sorting at the trans-Golgi network, but it has also raised new questions. Are GGAs and AP-1 present in the same vesicles? Can AP-1-containing vesicles be formed without GGAs? If so, what controls their formation and content, or determines their destination? The ability to create vesicles from liposomes in vitro will allow such subtleties to be investigated in detail. Protein sorting is now firmly in the biochemical domain.
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