ARTICLES Nature Cell Biology

ARTICLES nature cell biology

AMPH-l/P\mphiphysin/Binl functions with RME-l/Ehdl in endocytic recycling

RME-lIEHDI (receptor r ediated endocytosis/EpsI5 homology-domain containing I) family proteins are key residents of the recycling endosome, which are required for endosome-to-plasma membrane transport in Caenorhabditis elegans and mammals. Recent studies suggest similarities between the RME-lIEHD proteins and the Dynamin GTPase superfamily of mechanochemical pinchases, which promote membrane fission. Here we show that endogenous C. elegans AMPH-I, the only C. elegans member of the Amphiphysin/BIN. family of BAR (Binl-Amphiphysin-RvsI6Ip/167p}-domain-containing proteins, colocalizes with RME-I on recycling endosomes in vivo, that amph-l-deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-I Asn-Pro-Phe(Asp/Glu) sequences to the RME-I EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BINI (also known as Amphiphysin 2) in EHDI-regulated endocytic recycling. In vitro, we find that purified recombinant AMPH-I-RME-I complexes produce short, coated membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-I and RME-I cooperatively regulate endocytic recycling, probably through functions required for the production of cargo carriers that exit the recycling endosome for the cell surface.

Previous work has shown th It RME-l and mammalian mRme-l /EHD 1 Asn-Pro-Phe-containing partner proteins!2,13. Given the high degree are ATPases that require ATP binding for homo-oligomerization and of overall similarity among RME-l /EHD family members (about 65% function in vivo!-4. Like RME-l and mRme-l/EHDl, the vertebrate- sequence identity) and their recently identified similarity to Dynamin, specific paralogues EHD2, EHD3 and EHD4 also function in endocytic it has been suggested that all RME-l/EHD family proteins could have

transport, but at different steps'-'. Although not apparent from the pri- Dynamin-like properties in promoting membrane fission4,9, In particu- mary sequence, recent structural analysis of EHD2 has revealed that its lar, because recycling receptors accumulate in endosomes in the absence central ATP-binding 'G-domain' resembles the GTP-binding domain ofRME-1 or EHD1 (refs 1-3), RME-1/EHD1 could function as the

of the large GTPase Dynamin9, a protein that mediates membrane fis- fission machinery for tubules emanating from endosomes, promoting sion through its ability to constrict vesicle neckslO,!!, Additional similari- the release of transport carriers during receptor recycling events. ties between EHD2 and Dynamin have also been found, including the ability of EHD2 to assemble into spiral rings around acidic liposomes RESULTS when bound to a non-hydrolysable ATP analogue9• It has been further Identification of RME-I-interacting proteins shown that EHD2 ATPase activity is stimulated upon lipid binding and To gain greater insight into the role of RME-1 at the recycling endosome, oligomerization, reminiscer t of the assembly-stimulated GAP activity we sought functional interactors that might aid RME-1 in the recycling characteristic of Dynamin9. process. Sequence alignment of several known binding partners of the However, other than the G-domain, RME-l/EHD family proteins mammalian EHD proteins has suggested that RME-l/EHD family are distinct from establishec Dynamin superfamily members. RME-l / EH-domains prefer Asn-Pro-Phe target sequences found in multiples EHD family proteins lack pleckstrin homology (PH) domains. Rather, and/or followed by acidic residues (reviewed in ref. 13). The acidic resi- the primary lipid-binding region ofEHD2 is helical, forming a unique dues following an Asn- Pro- Phe sequence could potentially neutralize the scissor-like interface in the EHD2 dimer9• RME-l/EHD family pro- unique positive surface charge near the Asn- Pro- Phe binding pocket of teins also lack proline-rich domains. Instead, RME-l/EHD family RME-l/EHD family EH-domains!3,!4. proteins contain a carboxy-terminal Eps15-homology (EH) domain, Using bioinformatic searches of the predicted C. elegans proteome, we which is a different type of peptide-binding interface, known to target identified 839 predicted worm proteins containing at least one Asn-Pro- Phe

!Department of Molecular Biolog/ and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA. 2Department of Pathology and Laboratory Medicine, UMDNJ, Piscataway, NJ 08854, USA. 30£ partment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha NE 68198, USA. 'Correspondence should be addressed to B.D.G. (e-mail: [email protected].

••• ~ 200 839 N OF proteins ~ 1S0 ••• ~ 57 multi NPF _. NPF(O/E) proteins :~ 100 13 ••• 50 RNAi candijate interactors 0;'" in a GFP--RME-1 strain 9 0 Vector "'- ••• -iij AIv1PH-1 CD RME-1(+) + + + + 309 363 Vector rBAR t--N°F(O/E)-NPF(O• /E) • AMPH-1(+) Q3 + ~~ e' AMPH-1(F309A) + ~ AMPH-1 (F363A) + tm1060 deletion - AMPH-1(F309A, F363A) +

d S s ~ ~ GST + GST - + GST-AMPH-1 + GST-RIv1E-1 (EH) - HA-RME-1(+) + + HA-AMPH-1 (+) + + HA-AMPH-1 (F309l1" F363A) - Anti-HAl ....••

Antl-AMPH-1 •• GST-AMPH-1 GST-EH Ponceau Ponceal GST GST

GST s GST-RME- (EH) ~ GST-EH01 (EH) GST - GST-EpsH (EH) GST-RME-1 (EH) - GST -Intersectin (EH a+b) + HA-AMPH-1(+) - HA-AMPH-1(+) + + + + + + HA-AMPH-1 + (031 G-312A, 0364-366A) IM,(~ •• 54 Anti-AMPH-1 [~,_~~~~~~~_~~-~.~_~.~~ .. Anti-AMPH-1 1- -- ~~;~(~~~=I GST-EH Ponceau AA';GSTI~--.....-.-••-#-.!!-'-·~-••·••••-•.•-.-I~

Figure 1 AM PH-1 physically interacts with RME-l. (a) A flowchart (top) showing was probed with an anti-AM PH-1 antibody. Input lanes contained 50% target the steps involved in identifying lI,MPH-1 as an RME-1 EH-domain-interacting protein. The lower panel shows equivalent loading of bait GST fusion proteins. protein. Bioinformatic searches of the C. elegans proteome identified multi-Asn- (d) Glutathione beads loaded with recombinant GST or GST-AMPH-1 (fUll Pro-Phe (NPF)- and NPF(D/E)-containing candidates, which were assayed for length) were incubated with in vitro-expressed HA-RME-1 (full length). Bound effects on GFP-RME-1 subcellu ar localization after RNAi-mediated depletion, proteins were analysed by a western blot probed with an anti-HA antibody. Input leading to the identification of A VlPH-1, a BAR and SH3 domain-containing lanes contain 10% of the target protein HA-RME-l. The lower panel shows protein. A diagram of the C. eleg3ns amph-l gene (bottom) indicating 5' and equivalent loading of bait GST fusion proteins. (e) Glutathione beads loaded 3' untranslated regions (dark grey boxes), exons (light grey boxes), introns with recombinant GST, GST-RME- 1 (442-576), GST-mRme-lIEHDI (EH (connecting lines) and the location of the amph-l(tm1060J deletion. (b) RME-1 domain), GST-Eps15 (EH domain 2) or GST-Intersectin (EH domains a + b) (residues 447-555) was expressed as bait in a yeast reporter strain. AMPH-1 were incubated with in vitro-expressed target protein HA-AMPH-H+). Bound (residues 230-394) and its mutlnt forms were expressed as prey in the same proteins were ana lysed by western blotting with an anti-AMPH-1 antibody. Input yeast cells. Interaction between bait and prey was assayed by quantitative lane contains 10% target protein. The lower panel shows equivalent loading of j3-galactosidase (j3-Gal) assays (Supplementary Information, Table SI). Mutation bait GST fusion proteins. (f) Glutathione beads loaded with recombinant GST or of either Asn-Pro-Phe motif to k; n-Pro-Ala was sufficient to significantly disrupt GST-RME-1 (442-576) were incubated with in vitro-expressed target proteins the interaction. The y-axis represents j3-Gal activity in Miller units. Data are from HA-AMPH-H+) or HA-AMPH-HD310-312A, D364-366A). Bound proteins two independent experiments. (c) Glutathione beads loaded with recombinant were analysed by western blotting with an anti-AM PH-1 antibody. Input lanes GSTor GST-RME-1 (442-576) Nere incubated with in vitro-expressed HA- contained 50% of the target protein. The lower panel shows equivalent loading AMPH-H+) or HA-AMPH-HF309A, F363A). A western blot for bound proteins of bait GST fusion proteins. sequence (Fig.1a and Method;). Of these predicted proteins, 74 contained animals expressing GFP- tagged RME-1. We reasoned that knockdown of multiple Asn- Pro- Phe sequen~es and/or Asn- Pro- Phe sequences followed a physiologically relevant RME-1 binding partner could alter RME-1 sub- by acidic Asp/Glu stretches. To determine which of these 74 candidate cellular localization and/or recycling endosome morphology. Among the

RME-1 interactors might be physiologically relevant, we performed RNA small number of candidate interactors whose RNAi- mediated knockdown interference (RNAi)-mediatec knockdown of each candidate in transgenic affected RME-1localization we noted AMPH -1, the only C. elegans member

NATURE CELL BIOLOGY VOLUME 11 I NUMBER 12 I DECEMBER 2009 © 2009 Macmillan Publishers Limited. All rights reserved. Figure 2 AMPH-1 co-localizes with RME-1 and SDPN-1 on recycling colocalizes significantly with the recycling endosome marker SDPN-1-GFP endosomes. Wild-type N2 worns or various GFP-Iabelled transgenic worms (green). Arrowheads indicate structures labelled by both AMPH-1 and were synchronized and grown :0 the adult stage before decapitation by SDPN-l. (c-.<") Endogenous AMPH-1 (red) does not colocalize microsurgery. The extruded werm intestines were fixed and immunostained significantly with clathrin (GFP-CHC-1; green). Arrowheads indicate with antibodies for visualizatie n of endogenous protein localization. (a-a' ') structures labelled by AMPH-1 and arrows indicate structures labelled Endogenous AMPH-1 labelled with a rabbit polyclonal anti-AMPH-1 by GFP-CHC-l. (d-d") Endogenous AMPH-1 (red) does not colocalize antibody (red) co-localizes ext~nsively with endogenous RME-1 labelled with the early endosome marker GFP-RAB-5 (green). Arrowheads indicate with a mouse monoclonal anti )ody (green). Arrowheads indicate structures structures labelled by AMPH-1 and arrows indicate structures labelled by labelled by both AMPH-1 and RME-l. (b-b") Endogenous AMPH-1 (red) GFP-RAB-5. Scale bars, 10 f'm. of the Amphiphysin/BIN1 family ofBAR- and SH3 (Src-homology domain Physical interaction between RME-l and AMPH-l 3) domain-containing protein, (Fig.la), This was especially intriguing given To determine whether AMPH-1 can physically bind to RME-1, we the known interactions of mal nmalian Amphiphysin with DynaInin in pre- performed yeast two-hybrid based tests and CST pull down analysis, synaptic membranes of the ntTVOUSsystem1S,16. AMPH -1 interacted with the RME-1 EH domain in the yeast two- hybrid

70 60 Q; .0 50 E ::J 40 C co U 30 C ::J Cl. 20 10 wr 35 • 30 Q; .0 25 E ::J C 20 21 u 15 c ::J Cl. 10 5 O.

@ ! co 150' 21 u C ::J Cl.

o 1.-_ _ wr

Figure 3 C. elegans amph-l mu:ants show abnormal trafficking of recycling l(tml060) mutants. (g, h) Confocal micrographs of intenstinally expressed cargo and morphologically abno'mal recycling endosomes. (a~) hTAG-GFP,a GFP-RME-1Iocalization in wild-type and amph-l mutant worms. Arrowheads cargo protein internalized indep~ndently of c1athrin, accumulates intracellularly indicate GFP-RME-1 signal. (j) Quantification of GFP-RME-1 signals as in amph-l mutants. (a, b) Intes' ines of live intact animals showing hTAG-GFP in g and h with respect to averagenumber of labelled structures per unit localization in wild-type and amoh-l mutant worms. (c) Quantification area. *P= 1.1424 x 10-'. (j-I) SDPN-1-GFP-labelled recyclingendosomes of hTAC-GFPsignals as in a anc b with respect to the averagenumber of are both reduced in number and appear larger in the amph-l(tml060) labelled structures per unit area. *P= 1.2 x 10-12. (d-f) hTfR-GFP, a clathrin- mutant intestines (k) relative to those in wild-type intestines (jl. Arrowheads dependent cargo protein, accurTiulatesintracellularly in amph-l mutants. (d, e) indicate SDPN-1-GFP-labelled structures. (I) Quantification of SDPN-1-GFP Intestines of live intact animals 5howinghTfR-GFP localization in wild-type signals as in j and k with respect to the averagearea of labelled structures. and amph-l mutant worms. (f) (~uantification of hTfR-GFP signals as in d *P= 2.4 x 10-<;.Data in e, f, iand I represent mean ± s.d. and n = 30 sampled and e with respect to the averag'l number of labelled structures per unit area. fields (six animals of each genotype were sampled from five different regions

*P= 9.7 x 10-22• Arrowheads in a--e indicate punctate and tubular signals. (g-il their intestine). Pvalues were determined using the one-tailed Student's t-test. Lossof GFP-RME-1 labelling of basolateral recycling endosomes in the amph- Scale bar, 10 ~m. wr, wild-type.

system (Fig. Ib). The intercetion was abolished when either or both protein (Fig. Ie; Supplementary Information, Fig. SIb), AMPH-I also AMPH-1 Asn- Pro- Phe sequ~nces were altered by substituting alanine for bound strongly to the EH -domain of mammalian mRme-l/EHD 1in this Phe 309 and/or Phe 363 (Fig. lb). In the pulldown experiment, full-length assay (Fig, Ie). Little or no binding of AMPH-I to the EH-domains of

AMPH -1 from wild -type WO] m lysates or in vitro-translated AMPH -1 (+) other endocytic proteins, Eps 15 or Interseetin, was observed, indicating was efficiently isolated by im nobilized GST - RME-1 EH -domain fusion the specificity of the interaction (Fig. Ie). Furthermore, in vitro-translated

Figure 4 AMPH-1function in endocytic recycling requires interaction recycling cargo hTfR-GFP in amph-l(tml060) mutants. (e) Quantification with RME-l. (a--e) AMPH-l(+) and the interaction-deficient AMPH- of the hTfR-GFP-labelied structures in the worms with respect to l(F309A, F363A) mutant wele expressed as mCherry fusion proteins average number of labelled structures per unit area. *P= 9.7 x 10-22, in amph-l(tml060) mutant iltestines. Synchronized adult worms were ** P = 9.4 X 10-22 (one-tailed Student's t-test). Data are mean ± s.d., imaged by confocal microscopy (a-d). AMPH-l(+) (e), but not AMPH- n = 30 sampled fields (six animals of each genotype sampled from five l(F309A, F363A) (d), could rescue the abnormal accumulation of different regions of their intestine). Scale bar, 10 f1m.

AMPH -1(F309A, F363A), \\0 hich lacks intact Asn- Pro- Phe motifs, failed endosome (Fig. 2b-b' ')17.Anti-AMPH-I staining failed to colocalize to bind to the GST-RME-I EH-domain fusion protein (Fig. 1c), and with markers for clathrin-coated pits (GFP-tagged CHC-I Iclathrin) 18, alanine substitution for the first three aspartic acid residues following each early endosomes (GFP- RAB-5; ref. 19), late endosomes (GFP- RAB-7; Asn-Pro-Phe motif reduced binding (Fig. If). Full-length in vitro-trans- ref. 19) or the Golgi (mannosidase-GFP)19, indicating that AMPH-I lated RME-I could be isolated by immobilized full-length GST-AMPH-1 is specifically enriched on recycling endosomes (Fig. 2c-c' " d-d' '; fusion protein (Fig.Id). We conclude that AMPH-I binds directly to Supplementary Information, Fig. S3). We also observed extensive colo- RME-I, and that this interaction requires the RME-I EH domain and calization of mCherry- RME-I and AMPH -I-GFP in living transgenic the two Asn- Pro- Phe(Asp/C lu) motifs present within AMPH -I. animals, supporting the data derived from fixed tissue (Supplementary Information, Fig. S2a-a' '). AMPH-l is enriched on rE'cyciing endosomes The lack of AMPH -I association with clathrin-coated pits is consistent RME-I is ubiquitously expressed in C. elegans, and is highly enriched with the absence of a consensus CLAP (clathrin and AP2 adaptor binding) on tubulo-vesicular basolateral recycling endosomes in the polarized domain in C. elegansAMPH-i. In fact, the CLAP domain is specific to cer- intestinal epithelium I. In tra nsgenic animals, AMPH -I-GFP under tlle tain isoforms of vertebrate Amphiphysins and is not found in any known control of amph-l genomic DNA regulatory sequences was similarly invertebrate Amphiphysin family member20. In Drosophila melanogaster, widely expressed (Supplementary Information, Fig. SIc-j). Affinity- dAmph has been shown to lack Dynamin (encoded by Shibire)-binding purified anti-AMPH -I antibodies detected a single band of the expected ability, does not colocalize with ShibirelDynamin, and does not produce size for AMPH-I in western blots of wild-type worms. This band was shibire-like phenotypes when mutated21-23.While Drosophila Shibirel absent in amph-l(tm1060) deletion-mutant worms (Supplementary Dynamin and C. elegans Dynamin (also known as DYN-I) do possess Information, Fig. SIa), establishing the specificity of the antibody. proline- rich sequences, neither Dynamin orthologue contains the Pro- X- Using immunofluorescence, we found that endogenous AMPH-I Arg- Pro- X-Arg consensus motif that is recognized by Amphiphysin SH3 colocalizes extensively with endogenous RME-I on tubulovesicular domains in vertebrates24.Notably, Drosophila Amphiphysin does contain basolateral recycling endos mes (Fig. 2a-a' '), and on more weakly an Asn- Pro- Phe sequence followed by acidic residues, suggesting potential labelled sub-apical structur ~sof the worm intestine, possibly the api- interaction with an RME-l/EHD family member. cal recycling en do somes (Sllpplementary Information, Fig. S2c-c' '). Although there was signi:lcant overlap of RME-I and AMPH-I amph-l-null mutants are defective in endocytic recycling labelling on the recycling f ndosome, the two proteins did not label To determine the function of AMPH -I in vivo, we cl1aracterized the phe- the endosome identically. This may reflect differences in the degree notype of amph-l(tm1060)-null mutants. To detect recycling defects, we of association with specific endosomal subdomains, or temporal dif- first focused on the previously validated model recycling cargo proteins ferences in the assembly or disassembly kinetics of the two proteins. hTtR-GFP (human transferrin receptor) and hTac-GFP (IL2-receptor Endogenous AMPH-I also colocalized with basolateral GFP-tagged alpha chain)25.26h.TtR is internalized through clathrin-dependent mecl1- SDPN-I (a C. elegans homologue of synaptic dynamin binding pro- anisms, and hTac is internalized by clathrin -independent mechanisms27. tein, also known as pacsin), another marker for the C. elegans recycling Both of these model recycling cargo proteins depend on RME-I/EHD I

HA-BIN1 + + IP T T HA HA I~(K) Anti-Myc[M •••,,;--w' Anti-HAl • .~46

Figure 5 RNAi-mediated depletion of human Binl impairs transferrin and merged image in f"demonstrate colocalization of HA-BINl isoform 10 recycling and disrupts mRme-l/EHD1-positive recycling endosome tubules. with Myc-EHD1-positive tubules. (g, h) mRme-lIEHD1-positive tubules are (a-d) Representative fields are ;hown for mock-treated HeLa cells or HeLa disrupted by Binl-depletion. HeLa cells grown on coverslips were mock- cells subjected to Binl RNAi after 5 min of Alexa-488 transferrin uptake treated or subjected to Binl RNAi. Endogenous EHDl was visualized by (a, c) and 30 min chase in complete medium (recycling; b, d). (e) The confocal microscopy after immunostaining with an affinity-purified polyclonal average cell fluorescence was determined from confocal micrographs of rabbit anti-EHDl antibody, followed by staining with Alexa Fluor 488 anti- 80 HeLa cells, mock-treated or SUbjected to Binl RNAi, after Alexa-488 rabbit secondary antibodies. Note the loss of tubular EHDl labelling after transferrin uptake followed by:: 0 min chase (Supplementary Information, Binl depletion. (j) mRme-l/EHDl physically associates with BINl isoform Table S2). Data are mean ± s.e. '11. from three independent experiments, 10. HeLa cells were transfected with Myc-EHDl or co-transfected with n = 80 cells per experimental c )ndition. P = 1.06 X 10-35 (one-tailed Myc-EHDl and 2xHA-BINl isoform 10. Cells were lysed and Iysates were Student's t-testl. (f-f") BINl a1d mRme-lIEHDl colocalize on intracellular immunoprecipitated with goat anti-HA antibody-conjugated agarose beads. tubules. HeLa cells transfected with 2xHA-BINl isoform 10 and Myc-EHDl Unbound protein was removed by successive washes. Bound proteins were were visualized by confocal microscopy after immunostaining with primary eluted with Laemmli sample buffer, separated by SDS-PAGE, and analysed antibodies, mouse anti-HA (f) a1d rabbit anti-EHDl (f'), followed by staining by western blotting with an anti-Myc antibody. Input lane contains 1% of the with appropriate fluorochrome-labelled secondary antibodies. The insets lysate used in the binding assay. T denotes total lysate. Scale bars, 10 f1m. for their recycling in mamm, Jian and worm cells, accumulating in recy- loss of most tubular labelling and an increase in diffuse GFP- RME-l cling endosomes in the absl nee of RME-l/EHDI function2,3,17,'9, The labelling, Only a small number of GFP- RME-l-labelled punctate struc- number of hTac-GFP and rTtR-GFP intracellular puncta in amph-l tures remained (Fig. 3g-i; Supplementary Information, Fig, S5), The mutants increased by more t1an lO-fold, compared with those in wild- loss ofRME-l from recycling endosomes confirmed the amph-l (RNAi) type control worms (Fig, 3a- f), These results indicate a defect in baso- results from the initial screen, suggesting that AMPH-l functions, at lateral recycling in the intesti: lal epithelia of amph-l mutants that is very least in part, in recruiting RME-l to the recycling endosome membrane, similar to that found in rme-l mutants, We also found that loss of amph-l disrupted another recycling endosome marker SDPN-I-GFP, causing greatly reduced SDPN-I-GFP Specific disruption of recycling endosomes in amph-l mutants puncta number and gross enlargement of remaining labelled structures Further analysis showed tha RME-llabelling ofbasolateral recycling (Fig, 3j-I), This phenotype was very similar to one we found previ- endosomes was profoundly abnormal in amph-l-mutant animals, with ously in rme-l mutantsl?, further indicating that AMPH-l functions

pelletl~_.

Supematant

RME-1 + + + + + + AMPH-1 + + + + + +

f E 200 -S ~ -0 150 .~

"3 100 .'"0 ~ 50 ~'"rn > '" 0 ~ j. •.... •.... •.... ~ ;i- i'~~ ~ Q:';{ 9 " E -S 2,000 " ~ '§> 1,500 .!J1 ~ 1,000 .0

; 500 ~rn ~~ 0

u c'" .-~-E 'Oc c- o- ·c~ ()~ t).~ , '" £cIDa. "rn'O- ~ ~

Figure 6 Membrane binding ane tubulation by AMPH-1 and RME-1 in vitro. the average width measurement from several representative points along the (a) Coomassie-stained gels of sLpematant and pellet fractions from liposome tubule was calculated. Error bars represent mean ± s.d., n = 15 representative co-sedimentation assays are shewn. Binding reactions were performed in tubules per experimental condition. *P= 0.0074, **P= 1.29 x 10-13. (g) the absence or presence of 100 Yo phosphatidylserine (ptdSer), or 100% Quantification of tubule lengths. The length from the edge of the liposome phosphatidylcholine (ptdChl) li~osomes (0.33 mg ml-1, average diameter body to end of the tubule was measured for every tubule on 15 electron 0.4 flm). Liposomes were incubated with 1 mM of ATP-y-S and 1 flM of full- micrographs per experimental condition. Error bars represent mean ± s.d., length AMPH-1 or RME-1 protem, or equimolar quantities of both proteins, n = 15 representative tubules per experimental condition, * P = 1.7 X 10-7, as indicated. (b-e') Electron mi,;rographs of negatively stained ptdSer **P= 3.2 X 10-". (h) Quantification of inter-striation distance, Error bars liposomes, prepared as above (but used at 0.05 mg ml-') in the presence of represent mean ± s.d., n = 15 representative tubules per experimental 1 mM of ATP-y-S and 2.5 flM of GST (b, b'), AMPH-1 (c-c') or RME-1 (d, d'), condition. The average distance between every successive ring-like striation or equimolar quantities of AMPH-1 and RME-1 (e, e'), (f) Quantification of was calculated for each condition. *P = 0.0031. Pvalues were determined tubule widths. For each experimental condition, the width was measured using the one-tailed Student's t-test. Full scans of blots in a can be seen in for every tubule on 15 electron r icrographs. For tubules with uneven width, Supplementary Information, Fig. S8a, b.

Pellet 1 I~(K1

Supernatant I~~~······~·~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I RME-l + + + - + + - + + + AMPH-l + + - + + - + + + +

f 9 1,800 80, 1,600 E 70 ' E .s .s 1,400 .<= 60 ' .<= 15 0, 1,200 .~ 50, c ~ 1,000 > 10 ' « 200

0- 0 RME-l AMPH-l RME-l + RME-l AMPH-l RME-l + AMPH-l AMPH-l

Figure 7 In vitro membrane binding and tubulation by AMPH-l and RME-l and images in b'~' are enlarged regions from panels b--e. (f) Quantification to Ptdlns-containing liposomes. (a) Coomassie-stained gels of supernatant of tubule widths. For each experimental condition, the width was measured and pellet fractions from liposome co-sedimentation assaysare shown. for every tubule on 10 electron micrographs. For tubules with uneven width, Binding reactions were performed in the absence or presence of liposomes the average measurement from several representative points along the tubule (0.33 mg ml-l, 4 11maverage diameter) containing 80% phosphatidylcholine was calculated. Error bars represent mean ± s.d., n = 10 representative (ptdChl), 10% phosphatidylser ne (PtdSer) and 10% of unphosphorylated tubules. The asterisk indicates a significant difference in the one-tailed ptdlns, (Ptdlns(4)P) or ptdlns(,~,5)P2' Liposomes were incubated with Student's T-test: *P = 0.01, *** P =0.004. (g) Quantification of tubule 1 mM of ATP-y-S and 111Mof fUll-length AMPH-l, RME-l or equimolar lengths. The length from the edge of the liposome body to end of the tubule quantities of both proteins, as indicated. (b-e') Electron micrographs of was measured for every tubule on 10 electron micrographs per experimental negatively stained Ptdlns(4,5)F 21iposomes, prepared as above, (but used condition. Error bars represent mean ± s.d., n = 10 representative tubules 6 at 0.05 mg ml-1), in the presen:e of 1 mM of ATP-y-S and 2.5 11Mof for each condition. *P= 0.005, **P= 1.8 x 10- , ***P= 3.8 X 10-4. P GST (b, b'), RME-l-l (c, c'), AMPH-l (d, d') or equimolar quantities of values were determined using the one-tailed Student's t-test. Full scans of AMPH-l and RME-l (e, e'). Images in b--e are at x50,000 magnification, blots in a can be seen in Supplementary Information, Fig. S8c d. with RME-l in vivo. We ncted, however, that some amph-l pheno- other BAR-domain adaptor proteins, or residual RME-l activity in the types seemed weaker than rne-l phenotypes, such as in the accumu- absence of AMPH-l. lation of fluid-filled vacuoles in the intestine, and in the transport of We also examined amph-l mutants for effects on early endosomes. yolk proteins from the intes :ine to the oocyte (data not shown), This This was particularly important because mutants known to affect might indicate some functi,mal redundancy between AMPH-l and an earlier recycling step, from the early endosome to the recycling

a PtdSer PtdChl No lipid M,(K) 65 Pellet 52

Su~,ernatant 65 52

RME-1 + + + + + + AMPH-1 + + + + + +

70 ~-o 'Sw 60 .Q'+= 2"2 50 og> Q; E 40 .Q .- E Q; :JQ. 30 Z AMPH-1 ., AMPH-1 20

-. 10 c 0 AMPH-1 RME-1 +AMPH-1

Figure 8 Nucleotide effects on RME-1- and AMPH-1-mediated liposome presence of ADP (compare with ATP-y-S in Fig, 5 d, d '), RME-1 (ADP) tubulation. (a) Coornassie-stai led gels of supernatant and pellet fractions lacks tubulation capacity and most liposomes remain spherical (open from liposome co-sedimentation assays are shown. Binding reactions arrow) in the presence of RME-1 (ADP). Striations are often visible were performed in the i1bsenCEor! presence liposomes (0.33 mg ml-', and rare short protrusions (closed arrow) are present on occasional 4 flm average diameter) containing 100% phosphatidylserine (PtdSer), liposomes. (d, d') RME-1 in its ADP-bound state can affect the tubulation or 100% phosphatidylcholine (PtdChl). Liposomes were incubated with ability of AMPH-1 as observed in an experiment containing equimolar ADP (l mM) and 1 flM of full- ength AMPH-1 or RME-1 protein, or concentrations of both proteins in the presence of ADP. (e) Quantification equimolar quantities of both proteins, as indicated. Note that RME-1 can reveals an approximately 7-fold decrease in the number of tubules in bind to PtdSer liposornes in the presence of ADP. Tubulation experiments conditions where RME-1 (ADP) is present with AMPH-1, as compared to were perforrned with each protein (2.5 flM) and ADP (l mM) incubated tubulation produced by AMPH-1 alone. Error bars represent mean ± s.d., with 100% PtdSer liposomes 10.05mg ml-'). (b, b') AMPH-1 incubated n = 10 representative fields for each condition (imaged at x3,000 with PtdSer liposornes in the ~resence of ADP (compare with ATP-y-S magnification), * P = 1.48 X 10-15 (one-tailed Student's t-testl. Full scans in Fig, 5 c, c '), (c, c ') RME-1 incubated with PtdSer liposomes in the of blots in a can be seen in Supplementary Information, Fig, S8e, f. endosome, produce a similar set of phenotypes, including loss of phenotype not found in rme-l mutants'9. Consistent with a later func- RME-1-labelled recycling mdosomes and intracellular trapping of tion for AMPH-1 at the recycling endosome to plasma membrane recycling cargo proteins, such as hTac-GFP19, The key distinguish- step, the morphology and distribution of early-endosome markers ing characteristic of an earlier block, such as that shown by rab-lO GFP-RAB-5 and GFP-RAB-lO was unperturbed in amph-l mutants mutants, is the dramatic oyer-accumulation of early endosomes, a (Supplementary Information, Fig. S4a-f).

NATURE CEll BIOLOGY VOLUME 11 I NUMBER 12 I DECEMBER 2009 © 2009 Macmillan Publishers Limited, All rights reserved, Finally, because mammalian amphiphysin has been associated with of all forms of Amph2 in HeLa cells as determined by western blot- plasma membrane clathrin· coated pits16.28-3o,we investigated whether ting (Supplementary Information, Fig. S6a). Consistent with the pre- amph-i mutants alter the morphology or distribution of functional GFP- viously published results from knockout MEFs, we found that HeLa tagged Dynamin or clathri:1. We found that DYN-I-GFP and GFP- cells depleted of Amph2 are not defective in transferrin uptake. Rather, CHC-Ilocalization was unal :ered in amph-i mutants, consistent with the uptake was increased in BINI- knockdown cells after 7 min oftransfer- proposal that C. elegans AM PH -1 does not interact with Dynamin, and rin uptake as measured using FACS (fluorescence-activated cell sorting; similar to previous findings in Drosophila (Supplementary Information, mock-treated 348 ± 118 a.u., Bini RNAi 662 ± 325 a.u., averaged from Fig. S4g_I)21-23.Thus, we did not find any evidence that worm AMPH -1 is three independent experiments) or visualized by microscopy (Fig. Sa, c). involved in recruiting DYN -.IDynamin to the clathrin-coated pit during Consistent with a role for BINI in recycling from the endocytic recycling endocytic uptake at the plasma membrane. Instead, C. elegansAMPH-I compartment, we found that transferrin accumulated in the juxtanuclear seems to be specific for RME-I-mediated recycling processes. compartment in Amph2-depleted cells (Fig. 5b, d), and recycling was delayed as measured by image quantification (Fig. 5e) as well as by FACS AMPH-l requires interaction with RME-l for recycling function analysis (Supplementary Information, Fig. S6b). Having established the effects of AMPH -1 loss on endocytic traffic, and Consistent with the idea that BINl/ Amph2 functions directly in trans- the close association of AMP] {-I with RME-I, we sought to determine the port from the endocytic recycling department, we found that BINI was importance of the AMPH-I-RME-I physical association in vivo. Thus, enriched on distinctive mRME-I/EHDI-positive tubules. This could be we tested the ability of transgenically expressed AMPH-I, with or with- visualized in cells overexpressing Myc- EHD 1 and HA- BIN 1isoform 10 out intact RME-I-interactiol1 motifs, to rescue amph-i mutant pheno- (Fig.5 f-f' '), and in untransfected cells showing the endogenous pro- types. We found that althou!:h AMPH-I(F309A, F363A) was expressed teins using anti-EHDI and anti-BINI (isoform non-specific) antibodies as efficiently as AMPH-I (+; (Supplementary Information Fig. S5a-b), (Supplementary Information, Fig. S6c-c' '). The EHDI-positive tubules only AMPH -1(+) rescued de amph-l mutant phenotypes with respect have been shown previously to be endogenous features of HeLa cells, to hTtR accumulation and I~E-I localization (Fig. 4; Supplementary emanating from the endocytic recycling compartment3. These tubular Information, Fig. SSe-g), indicating that AMPH-I requires interaction endosomes are involved in the recycling of cargo proteins from the endo- with RME-I to perform its roleas an endocytic recycling regulator. We also cytic recycling compartment to the plasma membrane3, and the absence found that AMPH -1 protein .srnislocalized in rme-i (0) mutants. Instead ofEHDI from these structures leads to impaired recycling36. We found of an extended tubulovesicular pattern, we observed a more concentrated that depletion ofBINI/Amph2 disrupted the EHDI-positive recycling localization of endogenous AMPH-I to enlarged structures, similar to tubules (Fig. 5g, h). After depletion ofBINl/ Amph2, EHDIlocalization the abnormal localization of SDPN -1 in rme-i mutants (Supplementary was limited to a compact juxtanuclear localization, consistent with the Information, Fig. S5h-I). However, AMPH-I still appeared to be associated main body of the endocytic recycling compartment (Fig. 5h)37.Taken with the membrane in the absence of RME-I, suggesting that AMPH-I together, these results indicate that BINl/ Amph2 is important for the for- does not require RME-I for membrane recruitment. mation and/or maintenance of endocytic recycling compartment-derived tubules. Furthermore. these results indicate that the requirement for an Mammalian BINlIAmph2 functions in recycling amphiphysin family protein in the endocytic recycling pathway is an evo- Unlike C. elegans, which seems to express only one Amphiphysin iso- lutionarily conserved feature from worms to mammals. form, the mammalian genome contains two distinct Amphiphysin genes denoted Amphiphysin I and Amphiphysin 2 (also known as Bini). Reconstitution of RME-I-AMPH-l interactions in a liposome Each of these genes encodes several protein isoforms. Amphl is brain- system specific and Amph2a is brain-enriched. Both of these isoforms contain Previous reports have shown that mammalian Amphiphysin38, mam- a CLAP domain for clathrin and AP2 adaptor binding. Amphl and malian Dynamin,s.33.38,and mammalian EHD2 (ref. 9) have the capacity Amph2a form heterodimers and are thought to aid endocytic uptake at to bind to negatively charged liposomes and to deform the membrane to the synaptic clathrin-coated pit, at least in part through interaction with produce tubules1S,38,9In. mammalian HeLa cells, recycling endosomes are DynaminlS.16.28.29.31-33Other. hrms of Amph2 (also known as BINI) lack enriched in PtdIns( 4,5)P2 (phosphatidylinositol-4,5-bisphosphate) due clathrin-AP2 interaction ffiI)dules. One specific isoform functions in to the activation ofPI(5)K by Arf6 on these endosomes39 and PtdIns(4) muscle development34, whereas other isoforms, such as BINI isoform 10, P (phosphatidylinositol-4-phosphate) has also been recently identified have no known function. In fact, it has been shown that Amph2-knockout as a component of these endosomes36. As a first step towards determin- MEF cells are not defective in endocytic uptake of transferrin, rather they ing the membrane-binding capacity of worm RME-I and AMPH -1, we show an approximate twofold increase in transferrin uptake3s. tested the interaction of purified recombinant RME-I and AMPH-I Although the known BIN 1isoforms lack Asn- Pro- Phe sequences, and with liposomes in a sedimentation assay. We found that both RME-I and so may not bind to mammalian RME-I/EHD proteins directly, we con- AMPH -1 pelleted with acidic phosphatidylserine (PtdSer) liposomes, or sidered the possibility that, similar to AMPH -1 in C. elegans,mammalian with a more physiologicalliposome composition containing 10% PtdSer Amph2 might have a role in endocytic recycling. In fact, we observed and 10% PtdIns. PtdIns(4,5)P2 or PtdIns( 4)P (Figs 6a, 7a). Interestingly, weak co- immunoprecipitation of Myc- EHD 1 with HA- BINI isoform the binding to PtdIns( 4.5)P2 -, PtdIns( 4)P- and PtdIns-containing lipo- 10 in transfected cells (Fig. 5i). As a direct measure of the importance of somes seemed enhanced when both proteins were added to the reaction, BINI in endocytic transport, we followed fluorescently labelled transfer- compared with adding the proteins individually (Fig. 7a). We did not rin transport in BINI-deplel ed HeLa cells using previously described observe binding of either RME-I or AMPH -1 to neutral phosphatidyl- pulse-chase assays27. BINI-knockdown strongly reduced expression choline (PtdChl) liposomes (Figs 6a. 7a).

NATURE CEll BIOLOGY VOLUME 11 I NUMBER 12 I DECEMBER 2009 © 2009 Macmillan Publishers Limited. All rights reserved. We observed equivalent binding of RME-l to PtdSer liposomes in while keeping RME-l levels constant (2.5 flM), produced many short the presence of ADP or the non-hydrolysable ATP analogue ATP-y-S, PtdSer tubules that were similar in length to those produced by the pro- indicating that, under these binding conditions, RME-l association with teins at a 1:1 stoichiometry. This suggests that AMPH-l and RME-l liposomes was independent ofits nucleotide state (Figs 6a, 8a). tend to assemble into a coat of a particular length, rather than one that We also examined the morphology of liposomes incubated with varies in length depending on their relative available concentrations. RME-l and/or AMPH-l using electron microscopy. In the presence of (Supplementary Information, Fig. S7f, g). ATP-y-S, RME-l tubulates l'tdSer liposomes, with an average diameter of 400 nm, into approxima1.ely l20-nm wide tubules with an average RME-l nucleotide state is critical for tubulation length of 1 IlIIl (Fig. 6d, d', f, g). The tubules were decorated with ring- Finally we investigated the effects of nucleotide state on the in vitro reac- like striations reminiscent of ;tructures formed by mammalian Dynamin tion. We found that RME-l (ADP) lacked most tubulation activity in this and EHD2 on liposomes. The rings probably represent highly ordered assay, even though it still bound to PtdSer-containing liposomes (Fig. 8). RME-l (ATP-y-S) oligomer:; wrapped around the tubules, with regions Furthermore, we found that RME-l (ADP) together with AMPH-l of close and looser ring packillg. We found that in the presence of ATPyS, added to PtdSer liposome preparations led to an approximately 7-fold RME-l tubulated PtdIns( 4,5) P2' PtdSer and PtdChl-containing liposomes decrease in tubulation capacity, including loss of most of the thin tubules into longer, but narrower, tu")ules than those produced from liposomes characteristic of generation by AMPH-l alone. This suggests that RME-l containing only PtdSer (compare Fig. 6d, d', f, g with Fig. 7c, c', f, g). in the inactive ADP-bound state interacts with AMPH-l and prevents In profIle, these tubules had an irregular spike-like, coated appearance. AMPH-l-mediated tubulation. Our RME-l tubulation results are dif- Frequently, we also observec structures resembling constrictions along ferent from those reported for mammalian EHD2, the most diverged the length of the tubule, imparting a beads-on-a-string appearance to member of this protein family. EHD2 was found to be insensitive to the tubulating liposomes. nucleotide conditions in tubulating PtdSer-containing liposomes9. In the absence ofRME- L AMPH -1 tubulated PtdSer-containing lipo- Although under more stringent binding conditions, with liposomes of somes, with an average diameter of 400 nm, into 50-nm wide tubules lower PtdSer composition, EHD2liposome tubulation was also depend- with an average length of 1.1 IlIIl (Fig. 6c, c', f, g), which was much nar- ent on nucleotide state9• rower than the tubules forml~d in the presence of RME-l alone. Similar

, results were obtained for AMPH -1 incubated with PtdIns( 4,5)P2 PtdSer DISCUSSION and PtdChl-containing liposomes (Fig. 7d, d '). The work of Daumke et.aP and the work presented here, suggest that The physical and function,J interactions of AMPH -1 and RME-l sug- RME-l/EHD family proteins can function in membrane tubulation and gested that the two proteins '."ork together to promote recycling through potentially cause membrane fission through a self-assembly mecha- endosomal tubules. We then {ore investigated the biochemical interac- nism largely similar to that mediated by Dynamin. AMPH-l, through tion of AMPH -1 and RME-l :n membrane tubulation. Tubules generated its BAR domain, could function to initiate endosome tubule formation from PtdSer-containing liposomes incubated with AMPH -1 and RME-l and then recruit and activate RME-l ATPase activity to drive tubule in the presence of ATP-y-S wl~requalitatively different from tubules gen- fission. Noteably, AMPH-l seems to limit the assembly ofRME-l rings erated by the individual proteins (Fig. 6e, e '). The most striking differ- on membrane tubules. Recent work on the mechanism of Dynamin- ence was in the reduction of tubule length produced by AMPH-l and mediated membrane fission indicates that short Dynamin spirals are RME-l (ATP-y-S) together. S"lChtubules were about one-third the length the active agents of fission40"!. These studies found that long Dynamin of tubules produced by the individual proteins (Fig. 6f, g). In addition, assemblies constricted tubules but failed to promote fission upon GTP in the presence of AMPH-l, the apparent RME-l (ATP-y-S) spiral coat hydrolysis, whereas GTP hydrolysis and membrane release by short on the tubules was more rqular and closely spaced than in RME-l Dynamin assemblies frequently produced full fission. Thus, by limit- (ATP-y-S) tubules in the ab;ence of AMPH-l (Fig. 6h). The tubules ing RME-l spiral assembly, AMPH -1 may help to generate productive produced by AMPH-l and RME-l (ATP-y-S) together were more rigid RME-l assemblies. The AMPH -1 SH3 domain might interact with other and the decoration more pronounced in profIle than those produced by endocytic regulatory proteins and help create a hub for protein interac- the individual proteins (Fig. (ie'). Similar to our observation with 100% tions relevant for the RME-l recycling function. PtdSer-containing liposome:;, upon incubation with 10% PtdIns(4,5) Another similarity of RME-l /EHD family proteins and Dynamin

P2-containing liposomes we f,)und that a combination of AMPH -1- and is that they interact with Syndapin (also known as Pacsin), a known RME-l generated tubules that were dramatically shorter than those pro- activator of Wasp- and Arp2/3-mediated actin polymerization, sug- duced by the individual proteins (Fig. 7e, e', g). gesting that RME-l family proteins may promote actin dynamics on The physical interaction of AMPH-l and RME-l through the endosomes to promote membrane fission". Membrane-associated AMPH -1 Asn- Pro- Phe motif; seems critical for the formation of hybrid actin dynamics is thought to promote membrane fission by increasing tubules. AMPH-l(F309A, F363A) was unable to form short tubules membrane tension during Dynamin-mediated membrane constric- from PtdSer-containing lipc1somes when co-incubated with RME-l tion43• Further analysis of RME-l/EHD function is likely to provide (ATP-y-S). Rather, long thin tubules equivalent to those formed from important insights into membrane fission mechanisms that operate on AMPH -1(F309A, F363A) alone dominated the reaction (Supplementary endosomal membranes. 0 Information, Fig. S7). A few wide RME-l-type tubules were also observed but were relatively rare. METHODS The ratio of AMPH -1 to lU\1£-1 in the reaction mixture does not seem Methods and any associated references are available in the online version to control tubule length, as reducing AMPH-l by 10-fold to 250 nM of the paper at http://www.nature.com/naturecellbiology/ .

NATURE CEll BIOLOGY VOLUME 11 I NUMBER 12 I DECEMBER 2009 © 2009 Macmillan Publishers Limited. All rights reserved. 16. David, C., McPherson, P. S., Mundigl, O. & de Camilli, P, A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals. Proc. Natl Acad. Sci. USA 93, 331-335 (1996). ACKNOWLEDGEMENTS 17. Shi, A. et al. A novel requirement for C. elegans AlixlALX-1 in RME-1-mediated mem- We thank P.McPherson, Z. Zhou, B. Kay, G. Prandergast and S. Mitani for brane transport. Curf. Bioi. 17, 1913-1924 (2007). important reagents. The monoclonal against RME-1 was made at Washington 18. Sato, K. et al. Differential requirements for clathrin in receptor-mediated endocytosis University School of Medicine and funded by Grant R24RR22234 (PI M. L. Nonet). and maintenance of synaptic vesicle pools. Proc. Natl Acad. Sci. USA 106,1139-1144 We also thank C. Martin, P.Yurchenco, D. Winkelman, F. Matsumura, S. Yamashiro (2009). and S. H. deGregori for sharing their instruments, reagents and protocols; A. Shi 19. Chen, C. C. et al. RAB-10 is required for endocytic recycling in the Caenorhabditis for the construction and gift oftc e GFP-SDPN-1 strain; R. Patel and V. Starovoytov elegans intestine. Mol. Bioi. Cell 17, 1286-1297 (2006). for expert assistance with electron microscopy protocols and Z. Pan for usage of the 20. lhang, B. & lelhof, A. C. Amphiphysins, raising the BAR for synaptic vesicle recycling and membrane dynamics. Bin-Amphiphysin-Rvsp. Traffic 3, 452-460 confocal microscopy facility; O. Daumke and T. Pucadyil for discussions and advice (2002). on liposome tubulation experim€nts; P.Schweinsberg for technical assistance. 21. Razzaq,A. et al. Amphiphysin is necessaryfor organization of the excitation-contraction This work was supported by NIH Grants GM67237 to B.D.G and GM074876 to coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila. S.c., a NJCSCR Graduate Fellow/hip 06-2915-SCR-E-O, an Anne B. and James Genes Dev. 15,2967-2979 (2001). B. Leathem Fellowship, a McCallum Fund Fellowship Grant to S.P.,an American 22. lelhof, A. C. et al. Drosophila Amphiphysin is implicated in protein localization and Heart Association student fellow/,hip to M.S. and a Rutgers Summer Undergraduate membrane morphogenesis but not in synaptic vesicle endocytosis. Development 128, Research Fellowship (SURF) to K.P. 5005--5015 (20011. 23. Leventis, P. A. et al. Drosophila Amphiphysin is a post-synaptic protein required for normal locomotion but not endocytosis. Traffic 2,839-850 (2001). AUTHOR CONTRIBUTIONS 24. Grabs, D. et al. The SH3 domain of amphiphysin binds the proline-rich domain of S.P.participated in experimental ,fesign, performed the C. elegans-related dynamin at a single site that defines a new SH3 binding consensus sequence. J. Bioi experiments and the biochemical and liposome experiments (Figs 1-4, 6-8 and Chern. 272,13419-13425 (1997). Supplementary Information, Figs Sl-5 and S7) and wrote the manuscript. 25. Burack, M. A., Silverman, M. A. & Banker, G. The role of selective transport in neuronal M.S. performed the mammalian cell experiments (Fig. 5 and Supplementary protein sorting. Neuron 26,465--472 (2000). Information, Fig. S6) and helped :0 write those sections of the manuscript. K.P. 26. Naslavsky, N., Weigert, R. & Donaldson, J. G. Characterization of a nonclathrin endo- contributed to developing some strains used in the study. S.c. designed the cytic pathway, membrane cargo and lipid requirements. Mol. Bioi. CeliS, 3542-3552 mammalian cell experiments. C.M.C. designed the experiments related to liposome (2004). 27. Naslavsky,N., Weigert, R. & Donaldson, J. G. Convergenceof non-clathrin- and clathrin- biochemistry and trained S.P.to co those experiments. B.D.G. designed the derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Mol. experiments, trained S.P.in all C. degans experiments and wrote the paper. Bioi. Cell 14, 417-431 (2003). 28. Farsad, K. et al. A putative role for intramolecular regulatory mechanisms in the COMPETING FINANCIAL INTERESTS adaptor function of amphiphysin in endocytosis. Neuropharmacology 45, 787-796 The authors declare no competing financial interests. (2003). 29. McMahon, H. 1., Wigge, P.& Smith, C. Clathrin interacts specifically with amphiphysin Published online at http://www.na·ure.comlnaturecellbiology/. and is displaced by dynamin. FEBS Left. 413, 319-322 (1997). Reprints and permissions information is available online at http://npg.nature.com/ 30. Olesen, L. E. et al. Solitary and repetitive binding motifs for the AP2 complex alpha- repri n!sa nd permissions!. appendage in amphiphysin and other accessory proteins. J. Bioi. Chern. 283, 5099- 5109 (2008). 1. Grant, B. et al. Evidence that RME-1, a conserved C. elegans EH-domain protein, 31. Owen, D. J. et al. Crystal structure of the amphiphysin-2 SH3 domain and its role in functions in endocytic recycling. Nature Cell Bioi. 3, 573-579 (2001). the prevention of dynamin ring formation. EMBO J. 17, 5273-5285 (1998). 2. Lin, S. X., Grant, B., Hirsh, D. 8, Maxfield, F. R. Rme-1 regulates the distribution and 32. Slepnev, V. I. & De Camilli, P.Accessory factors in clathrin-dependent synaptic vesicle function of the endocytic recycli 19compartment in mammalian cells. Nature Cell Bioi. endocytosis. Nature Rev. Neurosci. 1, 161-172 (2000). 3,567-572 (2001). 33. Yoshida, Y. et al. The stimulatory action of amphiphysin on dynamin function is depend- 3. Caplan, S. et al. A tubular EHD: -containing compartment involved in the recycling of ent on lipid bilayer curvature. EMBO J. 23, 3483-3491 (2004). major histocompatibility compl"x class I molecules to the plasma membrane. EMBO 34. Lee, E. et al. Amphiphysin 2 (BinI) and T-tubule biogenesis in muscle. Science 297, J. 21, 2557-2567 (2002). 1193-1196 (2002). 4. Lee, D. W. et al. ATP binding re~ulates oligomerization and endosome association of 35. Muller, A. J. et al. Targeted disruption of the murine Bin1/Amphiphysin II gene does not RME-1 family proteins. J. Bioi. ~hem. 280,17213-17220 (2005). disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril 5. Naslavsky, N., Rahajeng, J., Shuma, M., Jovic, M. & Caplan, S. Interactions between formation. Mol. Cell. Bioi. 23, 4295--4306 (2003). EHD proteins and Rab11-FIP2, a role for EHD3 in early endosomal transport. Mol. 36. Jovic, M., Kieken, E, Naslavsky, N., Sorgen, P. L. & Caplan, S. Eps15 homology domain Bioi. Cell 17, 163-177 (2006). I-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinosi- 6. Sharma, M., Naslavsky, N. & Caplan, S. A role for EHD4 in the regulation of early tol-(4, 5)-bisphosphate and are required for efficient recycling. Mol. Bioi. Cell 20, endosomal transport. Traffic 9, !J95-1018 (2008). 2731-2743 (2009). 7. Shao, Y. et al. Pincher, a pinoc~'tic chaperone for nerve growth factorlTrkA signaling 37. Yamashiro, D. J., Tycko, B., Fluss, S. R. & Maxfield, F. R. Segregation of transferrin endosomes. J. Cell Bioi. 157,619-691 (2002). to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Ce1l37, 8. Guilherme, A. et a/. EHD2 and the novel EH domain binding protein EHBP1 couple 789--800 (1984). endocytosis to the actin cytoske eton. J. Biol.Chem. 279, 10593-10605 (2004). 38. Takei, K., Slepnev, V. I. & De Camilli, P. Interactions of dynamin and amphiphysin with 9. Daumke, O. et al. Architectural 2nd mechanistic insights into an EHD ATPaseinvolved liposomes. Methods Enzymol. 329, 478--486 (2001). in membrane remodelling. Natu'e 449, 923-927 (2007). 39. Brown, F.D., Rozelle, A. L., Yin, H. L., Balla, 1. & Donaldson, J. G. Phosphatidylinositol 10. Sever, S. Dynamin and endocytosis. Curro Opin. Cell Bioi. 14,463-467 (2002). 4, 5-bisphosphate and Arf6-regulated membrane traffic. J. Cell Bioi. 154, 1007-1017 11. Roux, A. & Antonny, B. The long "nd short of membrane fission. Cell 135, 1163-1165 (2001). (2008). 40. Pucadyil, 1. J. & Schmid, S. L. Real-time visualization of dynamin-catalyzed membrane 12. Salcini, A. E. et al. Binding spe,;ificity and in vivo targets of the EH domain, a novel fission and vesicle release. Cell 135, 1263-1275 (2008). protein-protein interaction module. Genes Dev. 11,2239-2249 (1997). 41. Bashkirov, P.V. et al. GTPase cycle of Dynamin is coupled to membrane squeeze and 13. Grant, B. D. & Caplan, S. Mech,nisms of EHD/RME-1 protein function in endocytic release, leading to spontaneous fission. Cell (2008). transport. Traffic (2008). 42. Braun, A. et al. EHD proteins associate with syndapin I and II and such interac- 14. Kieken, F., Jovic, M., Naslavsky, N., Caplan, S. & Sorgen, P. L. EH domain of EHDI. tions playa crucial role in endosomal recycling. Mol. Bioi. Cell 16, 3642-3658 J. Biomol. NMR 39, 323-329 (:'007). (2005). 15. Takei, K., Slepnev, V. I., Haucke, V. & De Camilli, P. Functional partnership between 43. Roux, A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin amphiphysin and dynamin in clat1rin-mediated endocytosis. Nature Cell Bioi. 1,33-39 implicates constriction and tension in membrane fission. Nature 441, 528-531 (1999). (2006).