View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Dispatch R49

Clathrin uncoating: Auxilin comes to life Sandra K. Lemmon

The DnaJ protein auxilin has been extensively studied fission of the vesicle has occurred, the clathrin coat must in vitro as a cofactor for uncoating clathrin-coated be rapidly shed to enable fusion of the vesicle with its vesicles by the chaperone Hsc70. Recent studies target membrane and recycle coat components for further provide the first evidence that auxilin plays this role rounds of vesicle budding. Extensive in vitro studies have in vivo, and work on a new mammalian auxilin suggests indicated that uncoating of clathrin is mediated by the the protein may have more complex cellular functions. molecular chaperone Hsc70 and a DnaJ cofactor known as auxilin [2–4]. Until recently there were hints, but no Address: Department of Molecular Biology and Microbiology, Case Western Reserve, University School of Medicine, 10900 Euclid proof, that these factors serve this function in vivo ([5,6] Avenue, Cleveland, Ohio 44106-4960, USA. for example). Now three new studies [7–9], one published E-mail: [email protected] recently in Current Biology [7], have provided the first con- clusive evidence that auxilin is required for clathrin recy- Current Biology 2001, 11:R49–R52 cling in cells. 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. Auxilin 1 is a brain-specific protein that, on the basis of its carboxy-terminal J domain, is included in the DnaJ family Clathrin is well known for its roles in vesicle formation at of chaperone cofactors (Figure 2a). Other DnaJ proteins the plasma membrane during endocytosis, and in the have been shown to act cooperatively with molecular sorting of proteins in the trans-Golgi network for delivery chaperones of the heat-shock protein 70 (Hsp70) family, to the endosomal system. The formation of clathrin- and auxilin 1 interacts via its carboxy-terminal J domain coated vesicles is a complex process involving an array of specifically with the Hsp70 family member Hsc70. Auxilin accessory and regulatory proteins [1]. In animal cells, the 1 also has an amino-terminal segment related to the tumor AP-1 and AP-2 adaptor proteins capture membrane cargo suppressor gene product PTEN and the actin-binding into clathrin-coated vesicles, while assembly of the protein tensin, and a clathrin-binding domain. A second clathrin triskelion — with its three heavy and three light auxilin, auxilin 2, is expressed more broadly and was chains — into polyhedral lattices is thought to drive mem- identified as cyclin G-associated kinase (GAK), although brane deformation during budding (Figure 1). Once the significance of this interaction is still not understood

Figure 1

The clathrin-coated vesicle cycle. Adaptor proteins (APs) bind to the membrane, Coat assemblyBud formation Fission 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 Priming with endophilin’s SH3 domain. Adaptor Clathrin release protein release also seems to be mediated by Hsc70 Hsc70, but requires an additional cytosolic Auxilin factor(s). Hsc70 may remain associated with Synaptojanin triskelions to prime them for reassembly into Endophilin lattices.

AP release Hsc70 Cytosolic factor(s) Current Biology

Clathrin Clathrin AP Receptor/ triskelion lattice complex Hsc70 Auxilin ligand R50 Current Biology Vol 11 No 2

Figure 2

Domain structures of auxilins, and sequence (a) Clathrin alignment of the DnaJ domains. (a) The Kinase PTEN/tensin binding J 1311 domain arrangement of human GAK/Auxilin 2 GAK/Auxilin 2 (BAA22623); bovine auxilin 1 (SP Q27974); 910 C. elegans auxilin (AAG02478) and yeast Auxilin 1 Swa2p/Aux1p (YDR320C) are shown with 784 the kinase, PTEN/tensin, clathrin-binding and C. elegans auxilin DnaJ (J) regions indicated. The TPR domain is TPR 668 only found in yeast auxilin, and all but the Swa2p/Aux1p worm auxilin contain ‘DPF’ AP-2-binding motifs (black dots). (b) Alignment of carboxy-terminal DnaJ domains from the four (b) GAK/Auxilin 2 WIEGKERNIRALLSTLHTVL-WDGESRWTPVGMADLVAPEQVKKHYRRAV sequences indicated above. Identities with Auxilin 1 WIEGKERNIRALLSTMHTVL-WAGETKWKPVGMADLVTPEQVKKVYRKAV C.e. auxilin WTQGKERNIRALLGSLHNVL-WEGADRWNQPSMGDLLTPDQIKKHYRKAC three or four matches are blocked. Asterisks Swa2p/Aux1p WKDGKDDDIRHLLANLSSLLTWCN---WKDVSMQDLVMPKRVKITYMKAV and periods underneath indicate identities and * **. .** ** . .* * * * **. * .* * .* conserved residues, respectively. The black line above the alignment notes the conserved GAK/Auxilin 2 LAVHPDKAAGQPYEQ---HAKMIFMELNDAWSEFENQGSRPLF 1311 sequence HPDK that is important for J domain Auxilin 1 LVVHPDKATGQPYEQ---YAKMIFMELNDAWSEFENQGQKPLY 910 stimulation of Hsc70 ATPase activity. C.e. auxilin LVVHPDKLTGSPHLS---LAKMAFTELNDAYSKYQNDPAAM 784 Swa2p/Aux1p AKTHPDKIPESLSLENKMIAENIFSTLSIAWDKFKLQNDIN 668 **** * * * * . Current Biology

[10,11]. In addition to the regions of the protein that The most notable feature of Swa2p is its carboxy-terminal are similar in sequence to auxilin 1, GAK/auxilin 2 DnaJ domain, which has greatest sequence similarity — has an amino-terminal serine/threonine kinase domain approximately 40% identity — to the J domains of animal (Figure 2a). Only the clathrin-binding and DnaJ regions of auxilins (Figure 2). This suggested that Swa2p might be the two auxilins, however, are required for uncoating the yeast clathrin uncoating factor. This same sequence activity in vitro [4,6,12]. similarity was also recognized in an analysis of the yeast genome sequence by Pichvaee et al. [9], who refer to the According to current models, auxilin binds assembled gene as AUX1. Swa2p/Aux1p has no other homology to clathrin, recruits ATP-activated Hsc70 via its J domain, auxilin or GAK, although it does contain a tetratricopep- and stimulates the chaperone’s ATPase activity [4,13]. tide repeat (TPR) region (Figure 2), other cases of which Hsc70 then disrupts clathrin–clathrin interactions, leading have been shown to facilitate protein interactions with to release of the clathrin coat. Hsc70 remains bound to Hsc chaperones. disassembled clathrin and may prime triskelions for reassembly [13] (Figure 1), while auxilin is recycled and The DnaJ domain homology spurred both groups [7,9] on promotes multiple rounds of uncoating. More recent to determine whether Swa2p/Aux1p really is a yeast studies, using dominant-negative mutant forms of Hsc70, auxilin. Indeed, Swa2p/Aux1p has an amino-terminal support the view that Hsc70 has a role in regulating clathrin-binding domain [7,9], and stimulates the ATPase clathrin disassembly and the clathrin-coated-vesicle cycle activity of yeast Hsc70 in vitro [7]. Furthermore, in intact cells (S. Newmeyer and S. Schmid, personal com- Swa2p/Aux1p can recruit mammalian Hsc70 to pre-assem- munication). But what about auxilin? bled bovine clathrin baskets, co-assemble with clathrin into coats and stimulate efficient Hsc70-dependent Two studies [7,9] supporting the inference from the in depolymerization of clathrin lattices [9]. These results vitro experiments that auxilin cooperates with Hsc70 indicate that Swa2p/Aux1p has the in vitro biochemical in vivo to promote uncoating have been carried out with properties of auxilin. budding yeast, Saccharomyces cerevisiae. Graham and colleagues [7,14] identified yeast auxilin, which they call The two groups [7,9] next tested whether the yeast Swa2p, in a genetic screen designed to find factors auxilin-like protein plays a role in uncoating of clathrin required for ADP-ribosylation factor (ARF)-dependent in vivo, by generating Swa2p/Aux1p-deficient yeast. They pathways. ARF is a small GTP-binding protein involved found [7,9] that loss of auxilin results in a phenotype in recruitment of AP-1 and coatomer (COPI) to Golgi similar to that caused by clathrin deficiency: slowed membranes [15]. Clathrin-mediated vesicular transport growth, delayed endocytic delivery to the vacuole of the a- was implicated early on in their screen, as swa5 turned out factor receptor Ste3p, and inefficient maturation of the to be an allele of the clathrin heavy chain gene vacuolar protein carboxypeptidase Y, which is normally CHC1 [14]. sorted from the trans-Golgi network to the vacuole. Most Dispatch R51

importantly, swa2-∆/aux1-∆ mutants secrete a precursor regulates the uncoating process in vivo. Kinases are form of the mating pheromone α-factor, the final process- known to be associated with clathrin-coated vesicles ing of which is dependent upon a group of proteases local- ([16], for example), and both yeast and mammalian aux- ized in the trans-Golgi network [7]. This phenotype is a ilins are heavily phosphorylated [7,16]. The kinase hallmark of a clathrin defect, which causes the escape of domain in GAK/auxilin 2 might play some regulatory role trans-Golgi network membrane proteins to the cell surface in coat disassembly. Phosphorylation of the large subunits in yeast. of the adaptor proteins AP-1 and AP-2 decreases their affinity for clathrin [17], and the consequent reduction in Auxilin-deficient cells were found [7,9] also to exhibit a the interactions between the adaptor proteins and major loss of triskelia from the cytosol, and to have signifi- clathrin ‘legs’ could shift the equilibrium in favour of cantly more membrane-associated or assembled clathrin lattice disassembly. It seems unlikely, however, that the than normal cells. The absence of auxilin was associated kinase domain of GAK has this regulatory role, as it with a significant shift of clathrin, the Golgi clathrin exhibits no kinase activity towards the AP-1 and AP-2 adaptor protein AP-1, and the cargo proteins pro- large chains in vitro [6]. carboxypeptidase Y and Ste3p into clathrin-coated vesicle fractions [9]. Finally, 50–100 nm vesicles coated with Synaptojanin 1, a polyphosphoinositide phosphatase that clathrin were found to accumulate in auxilin-depleted, but acts on the phosphoinositide lipids PI(4,5)P2 and not in normal cells [9], further supporting the conclusion PI(3,4,5)P3, has been implicated as a potential regulator of that auxilin has a role in the uncoating of clathrin in vivo. clathrin-coated vesicle uncoating in vivo. Synaptojanin is highly enriched in synapses and binds to a number of pro- Evidence that auxilin has a similar function in a metazoan teins involved in endocytosis [1]. Recent studies [18,19] organism has been obtained by Greener et al. [8], who have shown that synaptojanin 1 knockout mice and identified an auxilin homologue, CeAUX, of the nema- C. elegans unc-26 synaptojanin mutants have increased tode worm Caenorhabditis elegans (Figure 2). This group numbers of clathrin-coated vesicles in nerve endings, used RNA-mediated interference (RNAi) to reduce the consistent with a defect in uncoating after synaptic vesicle auxilin level in vivo. They found that internalization of a endocytosis. Endophilin, a protein with a Src homology 3 yolk protein into growing oocytes was severely perturbed (SH3) domain that binds synaptojanin, may also in the auxilin-deficient worms, and progeny arrested be important for clathrin uncoating, as inhibition of development during early larval stages. Many somatic cell endophilin–synaptojanin interaction in vivo also leads to types producing a form of clathrin heavy chain tagged with accumulation of clathrin-coated vesicles in the synapse the green fluorescent protein (GFP) displayed increased [20]. Mutation of Inp53p, one of three yeast synaptojanins, punctate fluorescence, perhaps reflecting increased associ- was found to exacerbate the trans-Golgi network defects ation of clathrin with vesicles or membranes; alternatively, caused by a temperature-sensitive mutation of clathrin the auxilin defect might have caused a compensatory heavy chain [21], though it is not yet known whether this increase in the level of clathrin expression. Indeed the leads to accumulation of clathrin-coated vesicles. Finally, production of GFP–clathrin from a transgene appeared to the mammalian auxilin domain related to PTEN is increase the ability of auxilin-deficient larvae to survive intriguing as, like synaptojanin, PTEN is a phosphoinosi- past early stages, suggesting that overproduction of tide phosphatase [22]. Auxilin lacks amino acids required clathrin might compensate for the inability to recycle for phosphatase activity, but binding of phosphoinositides clathrin efficiently. The conclusion that auxilin deficiency might directly regulate auxilin function. affects clathrin recycling was further supported by analysis using the fluorescence recovery after photobleaching Another important issue is the mechanism by which the (FRAP) technique: fluorescent GFP–clathrin puncta in heterotetrameric adaptor proteins, AP-1 and AP-2, dissoci- cells from auxilin-deficient animals showed little recovery ate from clathrin-coated vesicles once the outer triskelion after photobleaching, compared to wild-type worms. lattice has disassembled. The adaptor protein shell would still present a barrier for subsequent vesicle fusion, and Now that in vitro observations of clathrin uncoating by adaptor proteins would need to be recycled for further auxilin/Hsc70 has been validated by in vivo evidence, a rounds of clathrin-coated vesicle formation (Figure 1). number of questions remain. First, how is uncoating Mammalian and yeast auxilins contain ‘DPF’ motifs regulated? What prevents premature lattice release prior (Figure 2), which are known to bind to the appendage to fission of the coated vesicle from the membrane? The domain of the AP-2 α chain and, as predicted, GAK and clathrin light chain might be involved, as suggested in auxilin 1 interact [6,23]. Uncoating of clathrin by Hsc70 and earlier studies, even though it is not required for uncoat- brain auxilin, however, does not remove adaptor proteins ing in vitro [3]. Perhaps vesicle acidification upon pinch- from coated vesicles in vitro ([24], for example). Nor does ing off from the membrane serves as a switch for overproduction of auxilin 2 in cells, which leads to aggrega- uncoating. Another possibility is that phosphorylation tion of clathrin in the cytosol but does not perturb AP-2 R52 Current Biology Vol 11 No 2

localization [6]. As GAK can phosphorylate adaptor protein expression, and chromosomal localization of human GAK. µ Genomics 1997, 44:179-187. chains [6], which bind directly to receptor cargo, it may 12. Greener T, Zhao X, Nojima H, Eisenberg E, Greene LE: Role of cyclin regulate adaptor protein disassembly by releasing this inter- G-associated kinase in uncoating clathrin-coated vesicles from action. non-neuronal cells. J Biol Chem 2000, 275:1365-1370. 13. Jiang R, Gao B, Prasad K, Greene LE, Eisenberg E: Hsc70 chaperones clathrin and primes it to interact with vesicle Might auxilin have other cellular functions? In non- membranes. J Biol Chem 2000, 275:8439-8447. 14. Chen C-Y, Graham TR: An arf1-∆ synthetic lethal screen identifies neuronal cells, GAK/auxilin 2 is found in association with a new clathrin heavy chain conditional allele that perturbs clathrin-coated structures, primarily the trans-Golgi vacuolar protein transport. Genetics 1998, 150:577-589. network, but a significant proportion of GAK colocalizes 15. Schekman R, Orci L: Coat proteins and vesicle budding. Science 1996, 271:1526-1533. with actin focal adhesions, possibly mediated by its tensin 16. Morris SA, Mann A, Ungewickell E: Analysis of 100–180 kDa domain [12]. The clathrin-coated vesicles that accumulate phosphoproteins in clathrin-coated vesicles form bovine brain. in synaptojanin-deficient mice are found suspended in an J Biol Chem 1990, 265:3354-3357. 17. Wilde A, Brodsky FM: In vivo phosphorylation of adaptors actin-rich matrix in the endocytic zone of the nerve regulates their interaction with clathrin. J Cell Biol 1996, synapse [18]. Although this could be a result of 135:635-645. 18. Cremona O, Di Paolo G, Wenk MR, Luthi A, Kim WT, Takei K, Daniell pleiotrophic effects of synaptojanin deficiency, auxilin L, Nemoto Y, Shears SB, Flavell RA, et al.: Essential role of might tether clathrin-coated vesicles to actin, or even be phosphoinositide metabolism in synaptic vesicle recycling. involved in regulating disassembly of the actin cytoskele- Cell 1999, 99:179-188. 19. Harris TW, Hartwieg E, Horvitz HR, Jorgensen EM: Mutations in ton, which might be required for uncoating or movement synaptojanin disrupt synaptic vesicle recycling. J Cell Biol 2000, of the vesicle into the cell. While the targets of the kinase 150:589-600. domain of GAK are not known, two GAK kinase domain 20. 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 homologues, Prk1 and Ark1p, are important for organiza- uncoating of synaptic clathrin-coated vesicles are perturbed by tion of cortical actin patches and endocytosis in yeast disruption of interactions with the SH3 domain of endophilin. Neuron 2000, 27:301-312. [25,26]. Identification of the substrates of auxilin 2/GAK’s 21. Bensen ES, Costaguta G, Payne GS: Synthetic genetic interactions kinase activity should illuminate the importance of these with temperature-sensitive clathrin in Saccharomyces cerevisiae. connections to the actin cytoskeleton, and will be an Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network. active area of future research as ‘auxilin comes to life’. Genetics 2000, 154:83-97. 22. Maehama T, Dixon JE: PTEN: a tumour suppressor that functions as a phospholipid phosphatase. 9: Acknowledgements Trends Cell Biol 1999, 125-128. 23. Owen DJ, Vallis Y, Noble ME, Hunter JB, Dafforn TR, Evans PR, I would like to thank Ernst Ungewickell, Dan Gelperin and Kenneth Henry for McMahon HT: A structural explanation for the binding of multiple helpful discussions and suggestions for this manuscript. S.K.L. is supported ligands by the α-adaptin appendage domain. Cell 1999, from NIH grant R01-GM55796. 97:805-815. 24. Hannan LA, Newmyer SL, Schmid SL: ATP- and cytosol-dependent References release of adaptor proteins from clathrin-coated vesicles: a dual 1. Brodin L, Low P, Shupliakov O: Sequential steps in clathrin- role for hsc70. Mol Biol Cell 1998, 9:2217-2229. mediated synaptic vesicle endocytosis. Curr Opin Neurobiol 2000, 25. Zeng G, Cai M: Regulation of the actin cytoskeleton organization 10:312-320. in yeast by a novel serine/threonine kinase Prk1p. J Cell Biol 2. Prasad K, Barouch W, Greene L, Eisenberg E: A protein cofactor is 1999, 144:71-82. required for uncoating of clathrin baskets by uncoating ATPase. 26. Cope MJ, Yang S, Shang C, Drubin DG: Novel protein kinases J Biol Chem 1993, 268:23758-23761. Ark1p and Prk1p associate with and regulate the cortical actin 3. Ungewickell E, Ungewickell H, Holstein SE, Lindner R, Prasad K, cytoskeleton in budding yeast. J Cell Biol 1999, 144:1203-1218. 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,