Correspondence to: Margaret S. Robinson, University of Cambridge, CIMR, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, England. Tel:44-1223-330163 Fax:44-1223-762322 E-mail:msr12{at}mole.bio.cam.ac.uk.
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Abstract |
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The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the -adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both
-adaptin and
-adaptin, and
-synergin, an alternatively spliced protein with an apparent molecular mass of ~110190 kD, which only interacts with
-adaptin.
-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of
-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the
-adaptin binding site. In cells expressing
-adaptin with the
-adaptin ear, a construct that goes mainly to the plasma membrane, much of the
-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that
-synergin links the AP-1 complex to another protein or proteins.
Key Words:
AP-1, -adaptin, clathrin, TGN, EH domain
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Introduction |
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THE coats on clathrin-coated vesicles are assembled from two components, clathrin and adaptor complexes or APs, both of which must be recruited from the cytosol onto the appropriate membrane. Two adaptor complexes have been identified on clathrin-coated vesicles, AP-1, which is associated with the TGN, and AP-2, which is associated with the plasma membrane. Both complexes are heterotetramers, consisting of two large subunits or adaptins ( and ß1 in the case of AP-1, a and ß2 in the case of AP-2), a medium-sized subunit (µ1 or µ2), and a small subunit (
sigma}">1 or
sigma}">2) (
A number of additional peripheral membrane proteins are now known to be involved in clathrin-coated vesicle formation at the plasma membrane, and most of these have been shown to bind either directly or indirectly to the COOH-terminal ear domain of the -adaptin subunit of the AP-2 complex. These include amphiphysin, which binds directly to the
-adaptin ear (
-adaptin ear (
-adaptin ear (
Clathrin-coated vesicle formation is likely to be just as complex at the TGN as at the plasma membrane. However, so far no accessory molecules have been identified that interact either directly or indirectly with the AP-1 complex. Part of the reason may be that many of the proteins described above were originally purified and characterized as abundant components of nerve terminals, where AP-2 and clathrin are also concentrated because of the enormous amount of endocytosis that must take place in the nerve terminal to recycle synaptic vesicle components. In this study, we set out to identify novel AP-1 binding partners, specifically those that interact with the -adaptin subunit. We were particularly interested in proteins that might participate in the recruitment of AP-1 onto the TGN membrane. Experiments making use of chimeras between
- and
-adaptin have shown that these subunits contain at least some of the targeting information (
-adaptin as bait. Here we report the discovery of a novel
-adaptin binding partner, which interacts with the COOH-terminal ear domain and may act as an adaptor adaptor, connecting the AP-1 complex to other proteins in the same way that amphiphysin, Eps15, and epsin are thought to connect the AP-2 complex to proteins such as dynamin and synaptojanin.
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Materials and Methods |
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Library Screening
A Matchmaker rat brain cDNA library in pGAD10 was purchased from CLONTECH Laboratories and screened according to the manufacturer's instructions. The construct used as bait was full-length -adaptin in the vector pGBT9 (
-adaptin plasmid, and then reassayed for ß-galactosidase activity. Only those colonies that tested negative were analyzed further.
Plasmid DNA was isolated from eight representative colonies, transformed into Escherichia coli TG2 cells, and analyzed by sequencing and Southern blotting (
The constructs obtained from the library screen were all tested to determine whether they interacted with any of the other adaptor subunits (-adaptin as well as
-adaptin. To determine whether the binding sites for p34 on the two adaptins mapped to their NH2-terminal domains, both constructs were digested with ApaI, which cuts just upstream from the hinge, end repaired, and digested with EcoRI, which cuts at the 5' end, and ligated into pGBT9 digested with EcoRI and SmaI. The resulting constructs were cotransformed with the p34 clone (in pGAD424) into host cells, and the colonies were assayed for ß-galactosidase activity.
To obtain the complete coding sequence of -synergin, a probe was prepared from the clone obtained in the two-hybrid library screen by random priming and was used to screen a rat brain cDNA library in
gt10 (CLONTECH Laboratories). Library screening, subcloning, and characterization of the clones were all carried out as previously described (
-synergin (accession number AC004099), which enabled us to confirm the EST sequences and to identify the exon-intron boundaries, including the alternative splice sites.
Northern Blotting
A rat multiple tissue Northern blot was purchased from CLONTECH Laboratories and probed according to the manufacturer's instructions. The probe for p34 was the insert from one of the clones from the two-hybrid screen, containing the full coding sequence, whereas the probe for -synergin was the insert from the single clone identified in the two-hybrid screen, which encodes the NH2-terminal portion of the protein (see below). Both inserts were labeled with [32P]dCTP by random priming (
Antibody Production
Antibodies were raised against a GST--synergin fusion protein, using the expression vector pGEX3X (Pharmacia). PCR was used to introduce SmaI and EcoRI sites into the insert from the original rat
-synergin clone (corresponding to amino acids 168786 of the full-length protein, but missing amino acids 197274, presumably because the protein is alternatively spliced) so that it could be expressed in the appropriate reading frame. The construct was soluble and was purified on GSH-Sepharose (Pharmacia) according to the manufacturer's instructions. Immunization and affinity purification of the resulting antisera were carried out as previously described (
Immunoprecipitation and Western Blotting
Immunoprecipitation of coat proteins from rat liver cytosol was carried out under nondenaturing conditions as previously described (-adaptin, and anti
-adaptin (
-synergin described above. Western blotting was carried out as previously described (
-synergin was used at 1:500.
Immunofluorescence
Madin Darby bovine kidney cells were grown on multiwell test slides, fixed with methanol and acetone, and prepared for immunofluorescence as previously described (-synergin in cells expressing the chimeric adaptin
, stably transfected Rat1 cells (
-adaptin mAb100/3 (Sigma Chemical Co.) (
-adaptin in the MDBK cells and with the chimeric construct in the transfected Rat1 cells, together with affinity-purified rabbit anti
-synergin, followed by fluorescein-labeled donkey antirabbit IgG and Texas red-labeled sheep antimouse IgG, both obtained from Amersham. The slides were examined in a Zeiss Axioplan fluorescence microscope.
Identification of Binding Sites
The binding sites on -adaptin for
-synergin and on
-synergin for
-adaptin were identified using both the yeast two-hybrid system (described above) and GST pulldown experiments. The
-adaptin ear GST fusion protein has already been described (
-synergin GST fusion proteins were constructed from the clone isolated in the original two-hybrid library screen. The construct GST-
s1 contains the rat sequence corresponding to amino acids 168517 of the human sequence (but missing amino acids 197274, presumably because of alternative splicing), the construct GST-
s2 contains the rat sequence corresponding to amino acids 385661 of the human sequence, and the construct GST-
s3 contains the rat sequence corresponding to amino acids 518786 of the human sequence. The construct GST-EH contains the EH domain and several amino acids on either side of it, corresponding to amino acids 188390 of the human sequence (but missing amino acids 197274). All of the constructs were soluble and were prepared as previously described (
A blot overlay assay was used to demonstrate that g-adaptin and -synergin bind directly to each other. First, the insert from the GST-
s3 construct was ligated in-frame into the vector pQE30 to introduce a histidine tag at the NH2 terminus (Qiagen). Expression and purification of the resulting His-
s3 construct were carried out as instructed by the manufacturer. A control construct, His-DHFR (supplied with the kit), was also expressed and purified. Equivalent amounts of the two His-tagged fusion proteins were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane, and the blot was blocked with 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 0.5% BSA, 3 µM reduced glutathione for 30 min. This buffer was used in all the following steps. The blot was incubated with 10 nM GST or
-ear-GST for 45 min, washed for 30 min, and then labeled as described above using anti-GST followed by 125Iprotein A.
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Results |
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Isolation of Clones Expressing -Adaptin Interacting Proteins
Full-length mouse -adaptin cDNA was used as bait to screen a yeast two-hybrid library containing inserts derived from rat brain cDNA. Out of ~6 x 106 transformants, 62 clones were isolated that exhibited a strong and specific interaction between the g-adaptin construct and the library construct. To determine the identity of each of the 62 clones, both colony blotting and sequencing were carried out. The results are shown in Table 1.
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Six of the clones were found to contain plasmids encoding sigma}">1 (
sigma}">1A), the small chain subunit of the AP-1 adaptor complex. We have previously shown that
sigma}">1 and
-adaptin interact strongly in the yeast two-hybrid system, so the isolation of a
sigma}">1-encoding plasmid confirmed that the library screen had worked (
sigma}">1B. Eleven plasmids were found to encode ß-spectrin. The significance of this interaction is at present unclear (see Discussion). The other 39 plasmids were found to encode two unknown proteins, and these cDNAs were subjected to further analysis.
The first of the two unknown cDNAs was isolated with very high frequency in the two-hybrid screen, accounting for more than half of all the clones. A representative clone with an insert of ~2.5 kb was sequenced and was found to encode a protein of 315 amino acids with a deduced size of ~34 kD (p34) (Figure 1 a). There are several mammalian ESTs in the database encoding p34, but no homologues were found that might help to establish the protein's function. Northern blotting demonstrated that p34 is expressed ubiquitously and that the mRNA has a size of ~2.75 kb (Figure 1 b). Unlike most of the other proteins identified in the screen, p34 was found to interact not only with -adaptin but also with
-adaptin in the two-hybrid system, and this interaction was mapped to the NH2-terminal domains of the two adaptins (Figure 1 c). Attempts were made to raise antisera against p34, but unfortunately the protein proved to be a very poor antigen. Thus, although two different domains were expressed as fusion proteins for antibody production, and although the resulting antisera were affinity-purified, all of the antisera labeled multiple bands on Western blots. However, one of the antisera labeled a band of around the expected size (~37 kD), and this protein could be immunoprecipitated in substoichiometric amounts with cytosolic AP-1 and AP-2, suggesting that the interactions detected in the two-hybrid system are physiologically relevant (data not shown). But because we were looking for proteins that interact specifically with the AP-1 complex, p34 was not characterized further.
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-Synergin: Sequence and Splicing
The other unknown cDNA came up with the least frequency in the library screen, accounting for only one of the clones. Northern blotting showed that the transcript is expressed ubiquitously and has a size of ~4.45.6 kb, with bands of different mobilities labeled in different tissues (Figure 2 a). The original clone contained an insert of only ~1.6 kb and the sequence appeared to be all open reading frame, so this clone was used as a probe to screen a rat brain gt10 cDNA library to try to obtain a full-length sequence. Three additional clones were isolated and sequenced (Figure 2 b). A comparison of the four sequences revealed that the mRNA is alternatively spliced, consistent with the heterogeneity seen on the Northern blot. A putative start was identified in one of the clones, but none of the clones had an in-frame stop codon at the 3' end. However, when the EST database was searched with the rat sequences, three human sequences were found, and from the corresponding cDNAs it was possible to assemble a contiguous human open reading frame. The nonredundant database was also searched with both the rat and the human sequences, and the human genomic sequence encoding the 3' end of the mRNA was found. Figure 2 c shows the genomic structure, including the alternative splice sites.
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An analysis of the open reading frame revealed that the protein contains an EH domain at amino acids 295377 (Figure 2 d and 3 b). EH domains, which bind to proteins containing the sequence NPF, have now been found in a large number of proteins, including the mammalian proteins Eps15, Ese1, and Ese2, and the yeast proteins End3p and Pan1p, all of which are involved in endocytosis (-Adaptin contains no NPF sequences, so it is likely that the novel protein serves as a linker between
-adaptin and some other unidentified protein. We propose that this protein be called
-synergin (from the Greek, synergos, meaning partner or workmate).
A schematic diagram of -synergin is shown in Figure 3 a, indicating the positions of the EH domain, some of the alternative splice sites, and the
-adaptinbinding domain (see below). Figure 3 b shows an alignment of the EH domains from
-synergin, Eps15, Ese1, End3p, and Pan1p. The EH domain of
-synergin can be seen to contain all of the highly conserved amino acids found in other, well characterized EH domains. However, apart from the EH domain,
-synergin shows no significant sequence homology to any other proteins in the database, and it does not share any of the other features found in the
-adaptin binding partner Eps15 such as a coiled-coil domain or a proline-rich region.
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-Synergin: Association with AP-1 In Vivo
To learn more about the function of -synergin, the original clone identified in the two-hybrid library screen was expressed as a fusion protein for antibody production. Figure 4 shows a Western blot of equal protein loadings of homogenate from both brain and liver as well as various subcellular fractions from liver probed with the affinity-purified antibody. Two bands are labeled in the brain of ~110 and ~150 kD, whereas in the liver, a single band is labeled of ~190 kD. This is consistent with the Northern blot (Figure 2 a) in which a single band was labeled in liver, whereas two bands were labeled in brain, indicating that the different protein species might represent different spliced variants, although we cannot rule out the possibility that the differences might also be due to proteolysis.
-Synergin is found in both a high speed supernatant and membrane-containing pellet, indicating that it is peripherally associated with membranes. It is somewhat enriched in a TGN-enriched fraction from liver and it is strongly enriched in liver clathrin-coated vesicles.
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The association between -synergin and
-adaptin was confirmed by immunofluorescence microscopy. Double labeling of MDBK cells with anti
-synergin and anti
-adaptin revealed a striking degree of colocalization of the two proteins (Figure 5, a and b). Next, we investigated whether the membrane association of g-synergin is affected by the drug brefeldin A (BFA). This drug causes ARF to dissociate rapidly from membranes (
-synergin (c) and
-adaptin (d). Both proteins can be seen to have redistributed to the cytoplasm. To examine the behavior of the two proteins upon BFA washout, we treated cells with the drug for 30 min, and then allowed them to recover for 2 min (Figure 5e and Figure f). Both proteins can be seen to have reassociated with the membrane, which now has a more tubular appearance as a result of the BFA treatment. Thus,
-synergin, like the AP-1 adaptor complex, appears to associate with the TGN membrane in an ARF-dependent manner.
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The immunofluorescence data demonstrate that -synergin is associated with AP-1 on TGN membranes. To find out whether the two proteins are also associated in the cytosol, immunoprecipitations were carried out under nondenaturing conditions (Figure 6). Rat liver cytosol was immunoprecipitated with anti
-adaptin followed by protein ASepharose, and Western blots were probed with anti
-synergin or anti
-adaptin. As a control, cytosol was also immunoprecipitated with antia-adaptin. Cytosolic
-synergin was found to coprecipitate with
-adaptin but not with
-adaptin. Thus,
-synergin is associated with AP-1 in the cytosol as well as on membranes.
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Identification of Binding Domains
To identify the domain on -adaptin that binds to
-synergin and the domain on
-synergin that binds to
-adaptin, two approaches were used: yeast two-hybrid analysis and GST pulldown experiments. The NH2-terminal domain construct of
-adaptin, which was found to interact with p34 in the two-hybrid system (Figure 1 c), did not interact with
-synergin nor did a
-adaptin construct with the a-adaptin ear, indicating that g-synergin binds to the ear domain of
-adaptin (data not shown). This was confirmed using GST fusion proteins to isolate binding partners in rat liver cytosol, followed by Western blotting and probing with anti
-synergin. Figure 7 a shows that GST fused to the
-adaptin ear binds
-synergin, whereas GST alone or GST fused to the
-adaptin ear do not.
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The same general strategy was used to identify the domain of -synergin that binds to
-adaptin. Four sets of constructs were made from the original
-synergin clone isolated in the two-hybrid library screen: one containing just the EH domain (GST-EH); one containing the NH2-terminal half, including the EH domain (GST
s-1, corresponding to amino acids 168517 of the human sequence but missing amino acids 197274, presumably because of alternative splicing); one containing the middle portion of the protein (GST
s-2, amino acids 385661); and one containing the COOH-terminal half (GST
s-3, amino acids 518786). Only the construct containing the more COOH-terminal portion of g-synergin (GST
s-3) bound
-adaptin in the GST pulldown experiments (Figure 7 b), and this interaction was confirmed using the two-hybrid system (data not shown).
The GST pulldown experiments were carried out using whole cytosol, and, thus, they cannot distinguish between a direct interaction between -synergin and
-adaptin and an indirect one, mediated by another protein or proteins. The ability of the two proteins to interact in the yeast two-hybrid system strongly suggests that the interaction is direct, but to prove this formally, we also carried out Western blot overlay experiments, a technique that has been used to demonstrate that the
-adaptin ear domain binds directly to proteins such as amphiphysin and epsin (
-synergin that contains the
-adaptinbinding domain, amino acids 518786, was expressed as a histidine-tagged construct (His-
s3) and purified on a nickel affinity column. A control histidine-tagged construct was also expressed and purified (His-control). The two constructs were subjected to SDS-PAGE, blotted, and probed either with GST alone followed by anti-GST, with GST-
ear followed by anti-GST, or with anti
-synergin. Figure 7 c shows that the GST-
ear construct, but not GST alone, binds to the His-
s3 band on the Western blot. Thus, the interaction between
-adaptin and
-synergin must be a direct one. The binding site on
-synergin for
-adaptin is indicated in Figure 3 a.
-Synergin Follows AP-1 onto the Membrane
We have previously shown that the COOH-terminal ear domains of - and
-adaptin contain weak targeting signals for recruitment onto the TGN and plasma membranes, respectively (
-adaptin COOH-terminal domain, coassembles with the three subunits normally found in the AP-2 complex and is mainly associated with the plasma membrane, although a small fraction is seen on the TGN. Similarly, a construct containing mostly
-adaptin, but with the
-adaptin COOH-terminal domain, coassembles with the subunits normally found in the AP-1 complex and is mainly associated with the TGN, although a small fraction is seen on the plasma membrane.
We and others have long been interested in identifying the membrane docking sites for coat proteins, and although it is clear that -synergin cannot be the only docking site for AP-1, since the a-adaptin chimera with the
-adaptin ear goes mainly to the plasma membrane, it is possible that it might participate in AP-1 recruitment. Alternatively,
-synergin may be localized to the TGN because of its association with AP-1 rather than vice versa. To distinguish between these two possibilities, i.e., to determine whether
-synergin leads AP-1 onto the TGN or follows it there, we examined the distribution of
-synergin in cells expressing a chimera consisting of the
-adaptin NH2-terminal domain with the
-adaptin hinge and ear (agg). If
-synergin helps to recruit AP-1, we would expect its distribution to be unchanged in such cells, However, if AP-1 recruits g-synergin, we would expect some of the
-synergin to be rerouted to the plasma membrane. Figure 8 clearly shows that the latter is the case. In the transfected cell expressing the chimeric adaptin, much of the
-synergin labeling shows the characteristic punctate plasma membrane pattern (b), colocalizing with the chimera (a). Thus,
-synergin follows AP-1 onto the appropriate membrane rather than leading it there.
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Discussion |
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The yeast two-hybrid system has proved to be a powerful way of investigating proteinprotein interactions that may be difficult to study by more conventional biochemical methods. Among the advantages of the two-hybrid system are that it can detect interactions that may occur only transiently in the cell, and that it can be used not only to identify but also to clone the binding partners of a protein of interest. Its disadvantages are that it sometimes fails to pick up proteinprotein interactions that normally occur in the cell, while at the same time revealing interactions that may occur in the two-hybrid system, but not under more physiological conditions. In the present study, we have used this approach to search for -adaptin binding partners and have cloned cDNAs encoding five different proteins:
sigma}">1A,
sigma}">1B, ß-spectrin, p34, and
-synergin.
The cloning of s1A acts as a positive control, since we previously showed that it interacts strongly with -adaptin in the two-hybrid system (
sigma}">1B also binds to
-adaptin is consistent with the findings of
. Northern blotting reveals that both isoforms of s1 are expressed ubiquitously (
sigma}">1B but not
sigma}">1A by immunizing and cross-absorbing with different fusion proteins, so far we have not succeeded, presumably because of the high degree of homology between the two proteins. Thus, at present we do not know whether there are any functional differences between the two
sigma}">1 isoforms.
In our previous study in which we investigated interactions between neighboring adaptor subunits using the two-hybrid system, we found that -adaptin binds not only to
sigma}">1, but also to ß1 and (to a lesser extent) to ß2 (
-adaptin and ß1 or ß2 is not strong enough to produce a signal (data not shown). Another potential
-adaptin binding partner is p75, which can be cross-linked to membrane-associated
-adaptin (
-adaptin does not occur when the two are expressed as fusion proteins in yeast (e.g., if p75 only interacts with
-adaptin when it is incorporated into the AP-1 complex). Alternatively, if p75 is an integral membrane protein, the presence of a transmembrane domain may prevent it from entering the nucleus, which is where it must be to be detected by the two-hybrid system.
The cloning of ß-spectrin was unexpected and it is not yet clear whether its interaction with -adaptin is physiologically relevant. Although spectrin was initially assumed to be associated only with the plasma membrane, a number of immunofluorescence studies using certain antibodies against erythrocyte ß-spectrin have indicated that an isoform of the protein is associated with the Golgi apparatus, and recently a novel member of the ß-spectrin family, bIII spectrin, was cloned and localized to the Golgi region of the cell (
-adaptin binding partner is not ßIII spectrin but ßII spectrin, which has been localized to the plasma membrane. Future studies should show whether
-adaptin can bind to ßIII spectrin as well as to ßII spectrin, and whether the two proteins can associate with each other in the cell.
The protein that came up most frequently in the screen, p34, is unusual in that it interacts with both -adaptin and
-adaptin. This interaction was mapped to the NH2-terminal domains of the two adaptins, which is where
and
show the most homology, although even here they are only 32% identical. Another clue as to the function of p34 comes from the observation that it can be coimmunoprecipitated with soluble adaptor complexes, both AP-1 and AP-2, although it is not enriched in purified clathrin-coated vesicles. This suggests that p34 may play some sort of chaperone role. For instance, it could help to prevent the soluble adaptors from coassembling with soluble clathrin, or it could participate in uncoating by helping to remove the adaptors from the coated vesicle. Another possibility is that p34 may aid in the recruitment of soluble adaptors onto the membrane. However, it is clear that it cannot be involved in the specificity of adaptor recruitment, since it appears to interact equally well with both adaptor complexes.
Potentially the most interesting of the proteins that we isolated is -synergin. This protein colocalizes with AP-1 by immunofluorescence, and it can be coimmunoprecipitated with cytosolic AP-1. It binds specifically to the COOH-terminal ear domain of
-adaptin, the same domain that, on a-adaptin, binds to at least three different partners. The COOH-terminal ear domains of both
and
have also been implicated in the recruitment of the AP-1 and AP-2 complexes onto their respective membranes, although the major targeting information appears to reside in the adaptor heads. However,
-synergin is a peripheral membrane protein, not an integral membrane protein; its sensitivity to BFA indicates that it associates with the TGN in an ARF-dependent manner, and we have previously shown that the only soluble proteins required for AP-1 recruitment are the AP-1 itself and ARF-1 (
-adaptin with the
-adaptin ear, a construct that goes mainly to the plasma membrane, a substantial amount of the
-synergin also goes to the plasma membrane. These observations indicate that
-synergin is recruited onto the membrane through its interaction with AP-1 rather than vice versa, and, thus, that it does not play any part in targeting the AP-1 complex to the appropriate membrane.
What, then, is the function of -synergin? The presence of an EH domain indicates that, like Eps15 (the first EH domain-containing protein to be characterized) it is an adaptor for an adaptor. Eps15 interacts with the ear domain of the
-adaptin subunit of the AP-2 complex (
-synergin, its adaptin binding site is distinct from its EH domain. The
-adaptin binding site on Eps15 is quite large, comprising over 100 amino acids and including multiple repeats of the tripeptide DPF (
-adaptin binding site on g-synergin shows no homology to the
-adaptin binding site on Eps15, and no DPF sequences are present in
-synergin. However, it may be relevant that there are five repeats of the sequence DDFXD/EF, three of which (at positions 668673, 689694, and 774779) are within amino acids 518786, which we mapped as the
-adaptin binding site (Figure 3 a, the repeats are marked D). The other two copies of this sequence are outside of this region (456461 and 10221027); however, preliminary evidence suggests that the true
-adaptin binding site may encompass more than amino acids 518786. Although only the construct containing this sequence interacted with the
-adaptin ear domain in GST pulldown experiments, when interactions between
-adaptin and
-synergin were assayed using the two-hybrid system, clones containing amino acids 168517 and 385661 as well as 518786, but not the clone containing the EH domain alone, produced positive results (data not shown). We now intend to investigate whether the DDFXD/EF sequence is required for g-adaptin binding.
How much of the -adaptin and
-synergin in the cell are associated with each other? Western blots of AP-1 immunoprecipitated from cytosol under nondenaturing conditions show that
-synergin coprecipitates (Figure 6); however, under conditions where AP-1 subunits can be seen by Coomassie blue staining, no
-synergin band can be seen, indicating that the interaction is substoichiometric (Sowerby, P.J., and M.S. Robinson, unpublished observation). When we immunoprecipitate
-synergin under nondenaturing conditions, we are unable to detect any
-adaptin by Western blotting (Sowerby, P.J., and M.S. Robinson, unpublished observations). This is presumably because the anti
-synergin antibody, which was raised against the portion of the protein that we isolated in the two-hybrid screen, binds to the same site on
-synergin as
-adaptin. Thus, it is clear that although some of the AP-1 and
-synergin are associated with each other in the cytosol, there are also unoccupied pools of both proteins. At present, we do not know whether the
-synergin and AP-1 that are associated with each other are stably bound, or whether the interaction is more dynamic.
In addition to its association with -adaptin, we would predict that
-synergin has at least one additional binding partner, which would interact with its EH domain. So far, attempts to screen a yeast two-hybrid library with the EH domain of
-synergin have been unsuccessful, nor have we identified any candidates by GST pulldown or blot overlay experiments using the
-synergin EH domain. However, when we have carried out GST pulldown experiments with the
-adaptin ear construct, we find that other proteins come down in addition to
-synergin (Liu, W.W., P.J. Sowerby, and M.S. Robinson, unpublished observations), which could interact either directly with
-adaptin or indirectly, via
-synergin or another
ear binding partner. We now intend to identify and characterize these proteins, to see whether they are related to any of the AP-2 interacting proteins, or whether they contain NPF sequences. One potential (indirect)
-adaptin binding partner might be an isoform or homologue of dynamin, such as dynamin 2, which has been implicated in trafficking from the TGN (
ear and the
ear (
Some of the proteins associated either directly or indirectly with AP-2, including Eps15 (-adaptin binding portion of Eps15 is a potent inhibitor of clathrin-mediated endocytosis (
-synergin. Preliminary experiments in which we transiently transfected cells with a truncated form of
-synergin, consisting of amino acids 168786 (the original two-hybrid clone), indicate that this construct is toxic to cells, so we are now developing inducible systems. These studies should help to define both the role of g-synergin and the role of the AP-1 pathway in general. Although there is abundant evidence that AP-1 is involved in the trafficking of newly synthesized lysosomal proteins from the TGN to an endosomal or prelysosomal compartment, it may have other functions as well. AP-1 has been localized not only to the TGN, but also to early/recycling endosomes (
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Footnotes |
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L.J. Page and P.J. Sowerby contributed equally to this work.
Dr. Page's present address is Imperial Cancer Research Fund, Lincoln's Inn Field, London WC2A 3PX.
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Acknowledgements |
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We thank Tom Chappell for his valuable suggestions while this study was being carried out and for comments on the manuscript, David Owen for the a-adaptin ear GST fusion protein, Sharon Tooze for letting one of us (L.J. Page) moonlight on this project, and Paul Luzio, John Kilmartin, David Owen, and members of the Robinson lab for reading the manuscript and for helpful discussions.
This work was supported by grants from the Wellcome Trust and the Medical Research Council.
Submitted: 2 March 1999
Revised: 6 July 1999
Accepted: 12 July 1999
1.used in this paper: BFA, brefeldin A; EH, Eps15 homology; EST, expressed sequence tag; GST, glutathione-S-transferase
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