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Clathrin uncoating: Auxilin comes to life
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Clathrin uncoating: Auxilin comes to life Sandra K. Lemmon
The DnaJ protein auxilin has been extensively studied in vitro as a cofactor for uncoating clathrin-coated vesicles by the chaperone Hsc70. Recent studies provide the first evidence that auxilin plays this role in vivo, and work on a new mammalian auxilin suggests the protein may have more complex cellular functions. Address: Department of Molecular Biology and Microbiology, Case Western Reserve, University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4960, USA. E-mail: [email protected] Current Biology 2001, 11:R49–R52 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Clathrin is well known for its roles in vesicle formation at the plasma membrane during endocytosis, and in the sorting of proteins in the trans-Golgi network for delivery to the endosomal system. The formation of clathrincoated vesicles is a complex process involving an array of accessory and regulatory proteins [1]. In animal cells, the AP-1 and AP-2 adaptor proteins capture membrane cargo into clathrin-coated vesicles, while assembly of the clathrin triskelion — with its three heavy and three light chains — into polyhedral lattices is thought to drive membrane deformation during budding (Figure 1). Once
fission of the vesicle has occurred, the clathrin coat must be rapidly shed to enable fusion of the vesicle with its target membrane and recycle coat components for further rounds of vesicle budding. Extensive in vitro studies have indicated that uncoating of clathrin is mediated by the molecular chaperone Hsc70 and a DnaJ cofactor known as auxilin [2–4]. Until recently there were hints, but no proof, that these factors serve this function in vivo ([5,6] for example). Now three new studies [7–9], one published recently in Current Biology [7], have provided the first conclusive evidence that auxilin is required for clathrin recycling in cells. Auxilin 1 is a brain-specific protein that, on the basis of its carboxy-terminal J domain, is included in the DnaJ family of chaperone cofactors (Figure 2a). Other DnaJ proteins have been shown to act cooperatively with molecular chaperones of the heat-shock protein 70 (Hsp70) family, and auxilin 1 interacts via its carboxy-terminal J domain specifically with the Hsp70 family member Hsc70. Auxilin 1 also has an amino-terminal segment related to the tumor suppressor gene product PTEN and the actin-binding protein tensin, and a clathrin-binding domain. A second auxilin, auxilin 2, is expressed more broadly and was identified as cyclin G-associated kinase (GAK), although the significance of this interaction is still not understood
Figure 1 The clathrin-coated vesicle cycle. Adaptor proteins (APs) bind to the membrane, capturing receptor cargo, and clathrin assembles driving invagination of the vesicle. After budding, auxilin recruits Hsc70 to the vesicle for uncoating of clathrin. This step may be regulated by activation of the phosphoinositide phosphatase activity of synaptojanin, which may involve interaction with endophilin’s SH3 domain. Adaptor protein release also seems to be mediated by Hsc70, but requires an additional cytosolic factor(s). Hsc70 may remain associated with triskelions to prime them for reassembly into lattices.
Coat assembly
Bud formation
Fission
Priming
Clathrin release Hsc70 Auxilin Synaptojanin Endophilin
AP release Hsc70 Cytosolic factor(s) Clathrin triskelion
Clathrin lattice
AP complex
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Hsc70
Auxilin
Receptor/ ligand
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Figure 2
(a) Kinase
PTEN/tensin
Clathrin binding
J 1311 GAK/Auxilin 2 910
Auxilin 1
784 C. elegans auxilin TPR
668 Swa2p/Aux1p
(b)
GAK/Auxilin 2 Auxilin 1 C.e. auxilin Swa2p/Aux1p
WIEGKERNIRALLSTLHTVL-WDGESRWTPVGMADLVAPEQVKKHYRRAV WIEGKERNIRALLSTMHTVL-WAGETKWKPVGMADLVTPEQVKKVYRKAV WTQGKERNIRALLGSLHNVL-WEGADRWNQPSMGDLLTPDQIKKHYRKAC WKDGKDDDIRHLLANLSSLLTWCN---WKDVSMQDLVMPKRVKITYMKAV * **. .** ** . .* * * * **. * .* * .*
GAK/Auxilin 2 Auxilin 1 C.e. auxilin Swa2p/Aux1p
LAVHPDKAAGQPYEQ---HAKMIFMELNDAWSEFENQGSRPLF LVVHPDKATGQPYEQ---YAKMIFMELNDAWSEFENQGQKPLY LVVHPDKLTGSPHLS---LAKMAFTELNDAYSKYQNDPAAM AKTHPDKIPESLSLENKMIAENIFSTLSIAWDKFKLQNDIN **** * * * * .
[10,11]. In addition to the regions of the protein that are similar in sequence to auxilin 1, GAK/auxilin 2 has an amino-terminal serine/threonine kinase domain (Figure 2a). Only the clathrin-binding and DnaJ regions of the two auxilins, however, are required for uncoating activity in vitro [4,6,12]. According to current models, auxilin binds assembled clathrin, recruits ATP-activated Hsc70 via its J domain, and stimulates the chaperone’s ATPase activity [4,13]. Hsc70 then disrupts clathrin–clathrin interactions, leading to release of the clathrin coat. Hsc70 remains bound to disassembled clathrin and may prime triskelions for reassembly [13] (Figure 1), while auxilin is recycled and promotes multiple rounds of uncoating. More recent studies, using dominant-negative mutant forms of Hsc70, support the view that Hsc70 has a role in regulating clathrin disassembly and the clathrin-coated-vesicle cycle in intact cells (S. Newmeyer and S. Schmid, personal communication). But what about auxilin? Two studies [7,9] supporting the inference from the in vitro experiments that auxilin cooperates with Hsc70 in vivo to promote uncoating have been carried out with budding yeast, Saccharomyces cerevisiae. Graham and colleagues [7,14] identified yeast auxilin, which they call Swa2p, in a genetic screen designed to find factors required for ADP-ribosylation factor (ARF)-dependent pathways. ARF is a small GTP-binding protein involved in recruitment of AP-1 and coatomer (COPI) to Golgi membranes [15]. Clathrin-mediated vesicular transport was implicated early on in their screen, as swa5 turned out to be an allele of the clathrin heavy chain gene CHC1 [14].
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Domain structures of auxilins, and sequence alignment of the DnaJ domains. (a) The domain arrangement of human GAK/Auxilin 2 (BAA22623); bovine auxilin 1 (SP Q27974); C. elegans auxilin (AAG02478) and yeast Swa2p/Aux1p (YDR320C) are shown with the kinase, PTEN/tensin, clathrin-binding and DnaJ (J) regions indicated. The TPR domain is only found in yeast auxilin, and all but the worm auxilin contain ‘DPF’ AP-2-binding motifs (black dots). (b) Alignment of carboxy-terminal DnaJ domains from the four sequences indicated above. Identities with three or four matches are blocked. Asterisks and periods underneath indicate identities and conserved residues, respectively. The black line above the alignment notes the conserved sequence HPDK that is important for J domain stimulation of Hsc70 ATPase activity.
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The most notable feature of Swa2p is its carboxy-terminal DnaJ domain, which has greatest sequence similarity — approximately 40% identity — to the J domains of animal auxilins (Figure 2). This suggested that Swa2p might be the yeast clathrin uncoating factor. This same sequence similarity was also recognized in an analysis of the yeast genome sequence by Pichvaee et al. [9], who refer to the gene as AUX1. Swa2p/Aux1p has no other homology to auxilin or GAK, although it does contain a tetratricopeptide repeat (TPR) region (Figure 2), other cases of which have been shown to facilitate protein interactions with Hsc chaperones. The DnaJ domain homology spurred both groups [7,9] on to determine whether Swa2p/Aux1p really is a yeast auxilin. Indeed, Swa2p/Aux1p has an amino-terminal clathrin-binding domain [7,9], and stimulates the ATPase activity of yeast Hsc70 in vitro [7]. Furthermore, Swa2p/Aux1p can recruit mammalian Hsc70 to pre-assembled bovine clathrin baskets, co-assemble with clathrin into coats and stimulate efficient Hsc70-dependent depolymerization of clathrin lattices [9]. These results indicate that Swa2p/Aux1p has the in vitro biochemical properties of auxilin. The two groups [7,9] next tested whether the yeast auxilin-like protein plays a role in uncoating of clathrin in vivo, by generating Swa2p/Aux1p-deficient yeast. They found [7,9] that loss of auxilin results in a phenotype similar to that caused by clathrin deficiency: slowed growth, delayed endocytic delivery to the vacuole of the afactor receptor Ste3p, and inefficient maturation of the vacuolar protein carboxypeptidase Y, which is normally sorted from the trans-Golgi network to the vacuole. Most
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importantly, swa2-∆/aux1-∆ mutants secrete a precursor form of the mating pheromone α-factor, the final processing of which is dependent upon a group of proteases localized in the trans-Golgi network [7]. This phenotype is a hallmark of a clathrin defect, which causes the escape of trans-Golgi network membrane proteins to the cell surface in yeast. Auxilin-deficient cells were found [7,9] also to exhibit a major loss of triskelia from the cytosol, and to have significantly more membrane-associated or assembled clathrin than normal cells. The absence of auxilin was associated with a significant shift of clathrin, the Golgi clathrin adaptor protein AP-1, and the cargo proteins procarboxypeptidase Y and Ste3p into clathrin-coated vesicle fractions [9]. Finally, 50–100 nm vesicles coated with clathrin were found to accumulate in auxilin-depleted, but not in normal cells [9], further supporting the conclusion that auxilin has a role in the uncoating of clathrin in vivo. Evidence that auxilin has a similar function in a metazoan organism has been obtained by Greener et al. [8], who identified an auxilin homologue, CeAUX, of the nematode worm Caenorhabditis elegans (Figure 2). This group used RNA-mediated interference (RNAi) to reduce the auxilin level in vivo. They found that internalization of a yolk protein into growing oocytes was severely perturbed in the auxilin-deficient worms, and progeny arrested development during early larval stages. Many somatic cell types producing a form of clathrin heavy chain tagged with the green fluorescent protein (GFP) displayed increased punctate fluorescence, perhaps reflecting increased association of clathrin with vesicles or membranes; alternatively, the auxilin defect might have caused a compensatory increase in the level of clathrin expression. Indeed the production of GFP–clathrin from a transgene appeared to increase the ability of auxilin-deficient larvae to survive past early stages, suggesting that overproduction of clathrin might compensate for the inability to recycle clathrin efficiently. The conclusion that auxilin deficiency affects clathrin recycling was further supported by analysis using the fluorescence recovery after photobleaching (FRAP) technique: fluorescent GFP–clathrin puncta in cells from auxilin-deficient animals showed little recovery after photobleaching, compared to wild-type worms. Now that in vitro observations of clathrin uncoating by auxilin/Hsc70 has been validated by in vivo evidence, a number of questions remain. First, how is uncoating regulated? What prevents premature lattice release prior to fission of the coated vesicle from the membrane? The clathrin light chain might be involved, as suggested in earlier studies, even though it is not required for uncoating in vitro [3]. Perhaps vesicle acidification upon pinching off from the membrane serves as a switch for uncoating. Another possibility is that phosphorylation
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regulates the uncoating process in vivo. Kinases are known to be associated with clathrin-coated vesicles ([16], for example), and both yeast and mammalian auxilins are heavily phosphorylated [7,16]. The kinase domain in GAK/auxilin 2 might play some regulatory role in coat disassembly. Phosphorylation of the large subunits of the adaptor proteins AP-1 and AP-2 decreases their affinity for clathrin [17], and the consequent reduction in the interactions between the adaptor proteins and clathrin ‘legs’ could shift the equilibrium in favour of lattice disassembly. It seems unlikely, however, that the kinase domain of GAK has this regulatory role, as it exhibits no kinase activity towards the AP-1 and AP-2 large chains in vitro [6]. Synaptojanin 1, a polyphosphoinositide phosphatase that acts on the phosphoinositide lipids PI(4,5)P2 and PI(3,4,5)P3, has been implicated as a potential regulator of clathrin-coated vesicle uncoating in vivo. Synaptojanin is highly enriched in synapses and binds to a number of proteins involved in endocytosis [1]. Recent studies [18,19] have shown that synaptojanin 1 knockout mice and C. elegans unc-26 synaptojanin mutants have increased numbers of clathrin-coated vesicles in nerve endings, consistent with a defect in uncoating after synaptic vesicle endocytosis. Endophilin, a protein with a Src homology 3 (SH3) domain that binds synaptojanin, may also be important for clathrin uncoating, as inhibition of endophilin–synaptojanin interaction in vivo also leads to accumulation of clathrin-coated vesicles in the synapse [20]. Mutation of Inp53p, one of three yeast synaptojanins, was found to exacerbate the trans-Golgi network defects caused by a temperature-sensitive mutation of clathrin heavy chain [21], though it is not yet known whether this leads to accumulation of clathrin-coated vesicles. Finally, the mammalian auxilin domain related to PTEN is intriguing as, like synaptojanin, PTEN is a phosphoinositide phosphatase [22]. Auxilin lacks amino acids required for phosphatase activity, but binding of phosphoinositides might directly regulate auxilin function. Another important issue is the mechanism by which the heterotetrameric adaptor proteins, AP-1 and AP-2, dissociate from clathrin-coated vesicles once the outer triskelion lattice has disassembled. The adaptor protein shell would still present a barrier for subsequent vesicle fusion, and adaptor proteins would need to be recycled for further rounds of clathrin-coated vesicle formation (Figure 1). Mammalian and yeast auxilins contain ‘DPF’ motifs (Figure 2), which are known to bind to the appendage domain of the AP-2 α chain and, as predicted, GAK and auxilin 1 interact [6,23]. Uncoating of clathrin by Hsc70 and brain auxilin, however, does not remove adaptor proteins from coated vesicles in vitro ([24], for example). Nor does overproduction of auxilin 2 in cells, which leads to aggregation of clathrin in the cytosol but does not perturb AP-2
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localization [6]. As GAK can phosphorylate adaptor protein µ chains [6], which bind directly to receptor cargo, it may regulate adaptor protein disassembly by releasing this interaction. Might auxilin have other cellular functions? In nonneuronal cells, GAK/auxilin 2 is found in association with clathrin-coated structures, primarily the trans-Golgi network, but a significant proportion of GAK colocalizes with actin focal adhesions, possibly mediated by its tensin domain [12]. The clathrin-coated vesicles that accumulate in synaptojanin-deficient mice are found suspended in an actin-rich matrix in the endocytic zone of the nerve synapse [18]. Although this could be a result of pleiotrophic effects of synaptojanin deficiency, auxilin might tether clathrin-coated vesicles to actin, or even be involved in regulating disassembly of the actin cytoskeleton, which might be required for uncoating or movement of the vesicle into the cell. While the targets of the kinase domain of GAK are not known, two GAK kinase domain homologues, Prk1 and Ark1p, are important for organization of cortical actin patches and endocytosis in yeast [25,26]. Identification of the substrates of auxilin 2/GAK’s kinase activity should illuminate the importance of these connections to the actin cytoskeleton, and will be an active area of future research as ‘auxilin comes to life’.
12. 13. 14. 15. 16. 17. 18.
19. 20.
21.
22.
Acknowledgements I would like to thank Ernst Ungewickell, Dan Gelperin and Kenneth Henry for helpful discussions and suggestions for this manuscript. S.K.L. is supported from NIH grant R01-GM55796.
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References 1. Brodin L, Low P, Shupliakov O: Sequential steps in clathrinmediated synaptic vesicle endocytosis. Curr Opin Neurobiol 2000, 10:312-320. 2. Prasad K, Barouch W, Greene L, Eisenberg E: A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase. J Biol Chem 1993, 268:23758-23761. 3. Ungewickell E, Ungewickell H, Holstein SE, Lindner R, Prasad K, Barouch W, Martin B, Greene LE, Eisenberg E: Role of auxilin in uncoating clathrin-coated vesicles. Nature 1995, 378:632-635. 4. Holstein SEH, Ungewickell H, Ungewickell E: Mechanism of clathrin basket dissociation: Separate functions of protein domains of the DnaJ homologue auxilin. J Cell Biol 1996, 135:925-937. 5. Honing S, Kreimer G, Robenek H, Jockusch BM: Receptor-mediated endocytosis is sensitive to antibodies against the uncoating ATPase (hsc70). J Cell Sci 1994, 107:1185-1196. 6. Umeda A, Meyerholz A, Ungewickell E: Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation. Eur J Cell Biol 2000, 79:336-342. 7. Gall WE, Higginbotham MA, Chen C-Y, Ingram MF, Cyr DM, Graham TR: The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast. Curr Biol 2000, 10:1349-1358. 8. Greener T, Grant B, Zhang Y, Wu X, Greene LE, Hirsh D, Eisenberg E: Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo. Nat Cell Biol 2001, in press. 9. Pichvaee B, Costaguta G, Yeung BG, Ryazantsev S, Greener T, Greene L, Eisenberg E, McCaffery JM, Payne GS: A yeast DNA J protein required for clathrin-coated vesicle uncoating in vivo. Nat Cell Biol 2000, 2:958-963. 10. Kanaoka Y, Kimura SH, Okazaki I, Ikeda M, Nojima H: GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain. FEBS Lett 1997, 402:73-80. 11. Kimura SH, Tsuruga H, Yabuta N, Endo Y, Nojima H: Structure,
25. 26.
expression, and chromosomal localization of human GAK. Genomics 1997, 44:179-187. Greener T, Zhao X, Nojima H, Eisenberg E, Greene LE: Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells. J Biol Chem 2000, 275:1365-1370. Jiang R, Gao B, Prasad K, Greene LE, Eisenberg E: Hsc70 chaperones clathrin and primes it to interact with vesicle membranes. J Biol Chem 2000, 275:8439-8447. Chen C-Y, Graham TR: An arf1-∆ synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport. Genetics 1998, 150:577-589. Schekman R, Orci L: Coat proteins and vesicle budding. Science 1996, 271:1526-1533. Morris SA, Mann A, Ungewickell E: Analysis of 100–180 kDa phosphoproteins in clathrin-coated vesicles form bovine brain. J Biol Chem 1990, 265:3354-3357. Wilde A, Brodsky FM: In vivo phosphorylation of adaptors regulates their interaction with clathrin. J Cell Biol 1996, 135:635-645. Cremona O, Di Paolo G, Wenk MR, Luthi A, Kim WT, Takei K, Daniell L, Nemoto Y, Shears SB, Flavell RA, et al.: Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 1999, 99:179-188. Harris TW, Hartwieg E, Horvitz HR, Jorgensen EM: Mutations in synaptojanin disrupt synaptic vesicle recycling. J Cell Biol 2000, 150:589-600. Gad H, Ringstad N, Low P, Kjaerulff O, Gustafsson J, Wenk M, Di Paolo G, Nemoto Y, Crun J, Ellisman MH, et al.: Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 2000, 27:301-312. Bensen ES, Costaguta G, Payne GS: Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network. Genetics 2000, 154:83-97. Maehama T, Dixon JE: PTEN: a tumour suppressor that functions as a phospholipid phosphatase. Trends Cell Biol 1999, 9:125-128. Owen DJ, Vallis Y, Noble ME, Hunter JB, Dafforn TR, Evans PR, McMahon HT: A structural explanation for the binding of multiple ligands by the α-adaptin appendage domain. Cell 1999, 97:805-815. Hannan LA, Newmyer SL, Schmid SL: ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: a dual role for hsc70. Mol Biol Cell 1998, 9:2217-2229. Zeng G, Cai M: Regulation of the actin cytoskeleton organization in yeast by a novel serine/threonine kinase Prk1p. J Cell Biol 1999, 144:71-82. Cope MJ, Yang S, Shang C, Drubin DG: Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast. J Cell Biol 1999, 144:1203-1218.
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Clathrin uncoating: Auxilin comes to life Sandra K. Lemmon
The DnaJ protein auxilin has been extensively studied in vitro as a cofactor for uncoating clathrin-coated vesicles by the chaperone Hsc70. Recent studies provide the first evidence that auxilin plays this role in vivo, and work on a new mammalian auxilin suggests the protein may have more complex cellular functions. Address: Department of Molecular Biology and Microbiology, Case Western Reserve, University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4960, USA. E-mail: [email protected] Current Biology 2001, 11:R49–R52 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Clathrin is well known for its roles in vesicle formation at the plasma membrane during endocytosis, and in the sorting of proteins in the trans-Golgi network for delivery to the endosomal system. The formation of clathrincoated vesicles is a complex process involving an array of accessory and regulatory proteins [1]. In animal cells, the AP-1 and AP-2 adaptor proteins capture membrane cargo into clathrin-coated vesicles, while assembly of the clathrin triskelion — with its three heavy and three light chains — into polyhedral lattices is thought to drive membrane deformation during budding (Figure 1). Once
fission of the vesicle has occurred, the clathrin coat must be rapidly shed to enable fusion of the vesicle with its target membrane and recycle coat components for further rounds of vesicle budding. Extensive in vitro studies have indicated that uncoating of clathrin is mediated by the molecular chaperone Hsc70 and a DnaJ cofactor known as auxilin [2–4]. Until recently there were hints, but no proof, that these factors serve this function in vivo ([5,6] for example). Now three new studies [7–9], one published recently in Current Biology [7], have provided the first conclusive evidence that auxilin is required for clathrin recycling in cells. Auxilin 1 is a brain-specific protein that, on the basis of its carboxy-terminal J domain, is included in the DnaJ family of chaperone cofactors (Figure 2a). Other DnaJ proteins have been shown to act cooperatively with molecular chaperones of the heat-shock protein 70 (Hsp70) family, and auxilin 1 interacts via its carboxy-terminal J domain specifically with the Hsp70 family member Hsc70. Auxilin 1 also has an amino-terminal segment related to the tumor suppressor gene product PTEN and the actin-binding protein tensin, and a clathrin-binding domain. A second auxilin, auxilin 2, is expressed more broadly and was identified as cyclin G-associated kinase (GAK), although the significance of this interaction is still not understood
Figure 1 The clathrin-coated vesicle cycle. Adaptor proteins (APs) bind to the membrane, capturing receptor cargo, and clathrin assembles driving invagination of the vesicle. After budding, auxilin recruits Hsc70 to the vesicle for uncoating of clathrin. This step may be regulated by activation of the phosphoinositide phosphatase activity of synaptojanin, which may involve interaction with endophilin’s SH3 domain. Adaptor protein release also seems to be mediated by Hsc70, but requires an additional cytosolic factor(s). Hsc70 may remain associated with triskelions to prime them for reassembly into lattices.
Coat assembly
Bud formation
Fission
Priming
Clathrin release Hsc70 Auxilin Synaptojanin Endophilin
AP release Hsc70 Cytosolic factor(s) Clathrin triskelion
Clathrin lattice
AP complex
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Figure 2
(a) Kinase
PTEN/tensin
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J 1311 GAK/Auxilin 2 910
Auxilin 1
784 C. elegans auxilin TPR
668 Swa2p/Aux1p
(b)
GAK/Auxilin 2 Auxilin 1 C.e. auxilin Swa2p/Aux1p
WIEGKERNIRALLSTLHTVL-WDGESRWTPVGMADLVAPEQVKKHYRRAV WIEGKERNIRALLSTMHTVL-WAGETKWKPVGMADLVTPEQVKKVYRKAV WTQGKERNIRALLGSLHNVL-WEGADRWNQPSMGDLLTPDQIKKHYRKAC WKDGKDDDIRHLLANLSSLLTWCN---WKDVSMQDLVMPKRVKITYMKAV * **. .** ** . .* * * * **. * .* * .*
GAK/Auxilin 2 Auxilin 1 C.e. auxilin Swa2p/Aux1p
LAVHPDKAAGQPYEQ---HAKMIFMELNDAWSEFENQGSRPLF LVVHPDKATGQPYEQ---YAKMIFMELNDAWSEFENQGQKPLY LVVHPDKLTGSPHLS---LAKMAFTELNDAYSKYQNDPAAM AKTHPDKIPESLSLENKMIAENIFSTLSIAWDKFKLQNDIN **** * * * * .
[10,11]. In addition to the regions of the protein that are similar in sequence to auxilin 1, GAK/auxilin 2 has an amino-terminal serine/threonine kinase domain (Figure 2a). Only the clathrin-binding and DnaJ regions of the two auxilins, however, are required for uncoating activity in vitro [4,6,12]. According to current models, auxilin binds assembled clathrin, recruits ATP-activated Hsc70 via its J domain, and stimulates the chaperone’s ATPase activity [4,13]. Hsc70 then disrupts clathrin–clathrin interactions, leading to release of the clathrin coat. Hsc70 remains bound to disassembled clathrin and may prime triskelions for reassembly [13] (Figure 1), while auxilin is recycled and promotes multiple rounds of uncoating. More recent studies, using dominant-negative mutant forms of Hsc70, support the view that Hsc70 has a role in regulating clathrin disassembly and the clathrin-coated-vesicle cycle in intact cells (S. Newmeyer and S. Schmid, personal communication). But what about auxilin? Two studies [7,9] supporting the inference from the in vitro experiments that auxilin cooperates with Hsc70 in vivo to promote uncoating have been carried out with budding yeast, Saccharomyces cerevisiae. Graham and colleagues [7,14] identified yeast auxilin, which they call Swa2p, in a genetic screen designed to find factors required for ADP-ribosylation factor (ARF)-dependent pathways. ARF is a small GTP-binding protein involved in recruitment of AP-1 and coatomer (COPI) to Golgi membranes [15]. Clathrin-mediated vesicular transport was implicated early on in their screen, as swa5 turned out to be an allele of the clathrin heavy chain gene CHC1 [14].
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Domain structures of auxilins, and sequence alignment of the DnaJ domains. (a) The domain arrangement of human GAK/Auxilin 2 (BAA22623); bovine auxilin 1 (SP Q27974); C. elegans auxilin (AAG02478) and yeast Swa2p/Aux1p (YDR320C) are shown with the kinase, PTEN/tensin, clathrin-binding and DnaJ (J) regions indicated. The TPR domain is only found in yeast auxilin, and all but the worm auxilin contain ‘DPF’ AP-2-binding motifs (black dots). (b) Alignment of carboxy-terminal DnaJ domains from the four sequences indicated above. Identities with three or four matches are blocked. Asterisks and periods underneath indicate identities and conserved residues, respectively. The black line above the alignment notes the conserved sequence HPDK that is important for J domain stimulation of Hsc70 ATPase activity.
Current Biology
The most notable feature of Swa2p is its carboxy-terminal DnaJ domain, which has greatest sequence similarity — approximately 40% identity — to the J domains of animal auxilins (Figure 2). This suggested that Swa2p might be the yeast clathrin uncoating factor. This same sequence similarity was also recognized in an analysis of the yeast genome sequence by Pichvaee et al. [9], who refer to the gene as AUX1. Swa2p/Aux1p has no other homology to auxilin or GAK, although it does contain a tetratricopeptide repeat (TPR) region (Figure 2), other cases of which have been shown to facilitate protein interactions with Hsc chaperones. The DnaJ domain homology spurred both groups [7,9] on to determine whether Swa2p/Aux1p really is a yeast auxilin. Indeed, Swa2p/Aux1p has an amino-terminal clathrin-binding domain [7,9], and stimulates the ATPase activity of yeast Hsc70 in vitro [7]. Furthermore, Swa2p/Aux1p can recruit mammalian Hsc70 to pre-assembled bovine clathrin baskets, co-assemble with clathrin into coats and stimulate efficient Hsc70-dependent depolymerization of clathrin lattices [9]. These results indicate that Swa2p/Aux1p has the in vitro biochemical properties of auxilin. The two groups [7,9] next tested whether the yeast auxilin-like protein plays a role in uncoating of clathrin in vivo, by generating Swa2p/Aux1p-deficient yeast. They found [7,9] that loss of auxilin results in a phenotype similar to that caused by clathrin deficiency: slowed growth, delayed endocytic delivery to the vacuole of the afactor receptor Ste3p, and inefficient maturation of the vacuolar protein carboxypeptidase Y, which is normally sorted from the trans-Golgi network to the vacuole. Most
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importantly, swa2-∆/aux1-∆ mutants secrete a precursor form of the mating pheromone α-factor, the final processing of which is dependent upon a group of proteases localized in the trans-Golgi network [7]. This phenotype is a hallmark of a clathrin defect, which causes the escape of trans-Golgi network membrane proteins to the cell surface in yeast. Auxilin-deficient cells were found [7,9] also to exhibit a major loss of triskelia from the cytosol, and to have significantly more membrane-associated or assembled clathrin than normal cells. The absence of auxilin was associated with a significant shift of clathrin, the Golgi clathrin adaptor protein AP-1, and the cargo proteins procarboxypeptidase Y and Ste3p into clathrin-coated vesicle fractions [9]. Finally, 50–100 nm vesicles coated with clathrin were found to accumulate in auxilin-depleted, but not in normal cells [9], further supporting the conclusion that auxilin has a role in the uncoating of clathrin in vivo. Evidence that auxilin has a similar function in a metazoan organism has been obtained by Greener et al. [8], who identified an auxilin homologue, CeAUX, of the nematode worm Caenorhabditis elegans (Figure 2). This group used RNA-mediated interference (RNAi) to reduce the auxilin level in vivo. They found that internalization of a yolk protein into growing oocytes was severely perturbed in the auxilin-deficient worms, and progeny arrested development during early larval stages. Many somatic cell types producing a form of clathrin heavy chain tagged with the green fluorescent protein (GFP) displayed increased punctate fluorescence, perhaps reflecting increased association of clathrin with vesicles or membranes; alternatively, the auxilin defect might have caused a compensatory increase in the level of clathrin expression. Indeed the production of GFP–clathrin from a transgene appeared to increase the ability of auxilin-deficient larvae to survive past early stages, suggesting that overproduction of clathrin might compensate for the inability to recycle clathrin efficiently. The conclusion that auxilin deficiency affects clathrin recycling was further supported by analysis using the fluorescence recovery after photobleaching (FRAP) technique: fluorescent GFP–clathrin puncta in cells from auxilin-deficient animals showed little recovery after photobleaching, compared to wild-type worms. Now that in vitro observations of clathrin uncoating by auxilin/Hsc70 has been validated by in vivo evidence, a number of questions remain. First, how is uncoating regulated? What prevents premature lattice release prior to fission of the coated vesicle from the membrane? The clathrin light chain might be involved, as suggested in earlier studies, even though it is not required for uncoating in vitro [3]. Perhaps vesicle acidification upon pinching off from the membrane serves as a switch for uncoating. Another possibility is that phosphorylation
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regulates the uncoating process in vivo. Kinases are known to be associated with clathrin-coated vesicles ([16], for example), and both yeast and mammalian auxilins are heavily phosphorylated [7,16]. The kinase domain in GAK/auxilin 2 might play some regulatory role in coat disassembly. Phosphorylation of the large subunits of the adaptor proteins AP-1 and AP-2 decreases their affinity for clathrin [17], and the consequent reduction in the interactions between the adaptor proteins and clathrin ‘legs’ could shift the equilibrium in favour of lattice disassembly. It seems unlikely, however, that the kinase domain of GAK has this regulatory role, as it exhibits no kinase activity towards the AP-1 and AP-2 large chains in vitro [6]. Synaptojanin 1, a polyphosphoinositide phosphatase that acts on the phosphoinositide lipids PI(4,5)P2 and PI(3,4,5)P3, has been implicated as a potential regulator of clathrin-coated vesicle uncoating in vivo. Synaptojanin is highly enriched in synapses and binds to a number of proteins involved in endocytosis [1]. Recent studies [18,19] have shown that synaptojanin 1 knockout mice and C. elegans unc-26 synaptojanin mutants have increased numbers of clathrin-coated vesicles in nerve endings, consistent with a defect in uncoating after synaptic vesicle endocytosis. Endophilin, a protein with a Src homology 3 (SH3) domain that binds synaptojanin, may also be important for clathrin uncoating, as inhibition of endophilin–synaptojanin interaction in vivo also leads to accumulation of clathrin-coated vesicles in the synapse [20]. Mutation of Inp53p, one of three yeast synaptojanins, was found to exacerbate the trans-Golgi network defects caused by a temperature-sensitive mutation of clathrin heavy chain [21], though it is not yet known whether this leads to accumulation of clathrin-coated vesicles. Finally, the mammalian auxilin domain related to PTEN is intriguing as, like synaptojanin, PTEN is a phosphoinositide phosphatase [22]. Auxilin lacks amino acids required for phosphatase activity, but binding of phosphoinositides might directly regulate auxilin function. Another important issue is the mechanism by which the heterotetrameric adaptor proteins, AP-1 and AP-2, dissociate from clathrin-coated vesicles once the outer triskelion lattice has disassembled. The adaptor protein shell would still present a barrier for subsequent vesicle fusion, and adaptor proteins would need to be recycled for further rounds of clathrin-coated vesicle formation (Figure 1). Mammalian and yeast auxilins contain ‘DPF’ motifs (Figure 2), which are known to bind to the appendage domain of the AP-2 α chain and, as predicted, GAK and auxilin 1 interact [6,23]. Uncoating of clathrin by Hsc70 and brain auxilin, however, does not remove adaptor proteins from coated vesicles in vitro ([24], for example). Nor does overproduction of auxilin 2 in cells, which leads to aggregation of clathrin in the cytosol but does not perturb AP-2
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localization [6]. As GAK can phosphorylate adaptor protein µ chains [6], which bind directly to receptor cargo, it may regulate adaptor protein disassembly by releasing this interaction. Might auxilin have other cellular functions? In nonneuronal cells, GAK/auxilin 2 is found in association with clathrin-coated structures, primarily the trans-Golgi network, but a significant proportion of GAK colocalizes with actin focal adhesions, possibly mediated by its tensin domain [12]. The clathrin-coated vesicles that accumulate in synaptojanin-deficient mice are found suspended in an actin-rich matrix in the endocytic zone of the nerve synapse [18]. Although this could be a result of pleiotrophic effects of synaptojanin deficiency, auxilin might tether clathrin-coated vesicles to actin, or even be involved in regulating disassembly of the actin cytoskeleton, which might be required for uncoating or movement of the vesicle into the cell. While the targets of the kinase domain of GAK are not known, two GAK kinase domain homologues, Prk1 and Ark1p, are important for organization of cortical actin patches and endocytosis in yeast [25,26]. Identification of the substrates of auxilin 2/GAK’s kinase activity should illuminate the importance of these connections to the actin cytoskeleton, and will be an active area of future research as ‘auxilin comes to life’.
12. 13. 14. 15. 16. 17. 18.
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Acknowledgements I would like to thank Ernst Ungewickell, Dan Gelperin and Kenneth Henry for helpful discussions and suggestions for this manuscript. S.K.L. is supported from NIH grant R01-GM55796.
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References 1. Brodin L, Low P, Shupliakov O: Sequential steps in clathrinmediated synaptic vesicle endocytosis. Curr Opin Neurobiol 2000, 10:312-320. 2. Prasad K, Barouch W, Greene L, Eisenberg E: A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase. J Biol Chem 1993, 268:23758-23761. 3. Ungewickell E, Ungewickell H, Holstein SE, Lindner R, Prasad K, Barouch W, Martin B, Greene LE, Eisenberg E: Role of auxilin in uncoating clathrin-coated vesicles. Nature 1995, 378:632-635. 4. Holstein SEH, Ungewickell H, Ungewickell E: Mechanism of clathrin basket dissociation: Separate functions of protein domains of the DnaJ homologue auxilin. J Cell Biol 1996, 135:925-937. 5. Honing S, Kreimer G, Robenek H, Jockusch BM: Receptor-mediated endocytosis is sensitive to antibodies against the uncoating ATPase (hsc70). J Cell Sci 1994, 107:1185-1196. 6. Umeda A, Meyerholz A, Ungewickell E: Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation. Eur J Cell Biol 2000, 79:336-342. 7. Gall WE, Higginbotham MA, Chen C-Y, Ingram MF, Cyr DM, Graham TR: The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast. Curr Biol 2000, 10:1349-1358. 8. Greener T, Grant B, Zhang Y, Wu X, Greene LE, Hirsh D, Eisenberg E: Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo. Nat Cell Biol 2001, in press. 9. Pichvaee B, Costaguta G, Yeung BG, Ryazantsev S, Greener T, Greene L, Eisenberg E, McCaffery JM, Payne GS: A yeast DNA J protein required for clathrin-coated vesicle uncoating in vivo. Nat Cell Biol 2000, 2:958-963. 10. Kanaoka Y, Kimura SH, Okazaki I, Ikeda M, Nojima H: GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain. FEBS Lett 1997, 402:73-80. 11. Kimura SH, Tsuruga H, Yabuta N, Endo Y, Nojima H: Structure,
25. 26.
expression, and chromosomal localization of human GAK. Genomics 1997, 44:179-187. Greener T, Zhao X, Nojima H, Eisenberg E, Greene LE: Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells. J Biol Chem 2000, 275:1365-1370. Jiang R, Gao B, Prasad K, Greene LE, Eisenberg E: Hsc70 chaperones clathrin and primes it to interact with vesicle membranes. J Biol Chem 2000, 275:8439-8447. Chen C-Y, Graham TR: An arf1-∆ synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport. Genetics 1998, 150:577-589. Schekman R, Orci L: Coat proteins and vesicle budding. Science 1996, 271:1526-1533. Morris SA, Mann A, Ungewickell E: Analysis of 100–180 kDa phosphoproteins in clathrin-coated vesicles form bovine brain. J Biol Chem 1990, 265:3354-3357. Wilde A, Brodsky FM: In vivo phosphorylation of adaptors regulates their interaction with clathrin. J Cell Biol 1996, 135:635-645. Cremona O, Di Paolo G, Wenk MR, Luthi A, Kim WT, Takei K, Daniell L, Nemoto Y, Shears SB, Flavell RA, et al.: Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 1999, 99:179-188. Harris TW, Hartwieg E, Horvitz HR, Jorgensen EM: Mutations in synaptojanin disrupt synaptic vesicle recycling. J Cell Biol 2000, 150:589-600. Gad H, Ringstad N, Low P, Kjaerulff O, Gustafsson J, Wenk M, Di Paolo G, Nemoto Y, Crun J, Ellisman MH, et al.: Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 2000, 27:301-312. Bensen ES, Costaguta G, Payne GS: Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network. Genetics 2000, 154:83-97. Maehama T, Dixon JE: PTEN: a tumour suppressor that functions as a phospholipid phosphatase. Trends Cell Biol 1999, 9:125-128. Owen DJ, Vallis Y, Noble ME, Hunter JB, Dafforn TR, Evans PR, McMahon HT: A structural explanation for the binding of multiple ligands by the α-adaptin appendage domain. Cell 1999, 97:805-815. Hannan LA, Newmyer SL, Schmid SL: ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: a dual role for hsc70. Mol Biol Cell 1998, 9:2217-2229. Zeng G, Cai M: Regulation of the actin cytoskeleton organization in yeast by a novel serine/threonine kinase Prk1p. J Cell Biol 1999, 144:71-82. Cope MJ, Yang S, Shang C, Drubin DG: Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast. J Cell Biol 1999, 144:1203-1218.