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• W2012021856 abstract "The discovery of overlapping hot spots of dynamin (Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., and Ramaswami, M. (1996) J. Neurosci. 16, 5443–5456) and the heterotetrameric adaptor 2 complex (Gonzalez-Gaitan, M., and Jäckle, H. (1997)Cell 88, 767–776) in Drosophilanerve terminals led to the concept of zones of active endocytosis close to sites of active exocytosis. The proline-rich domain of Drosophiladynamin was used to identify and purify a third component of the endocytosis zones. Dap160 (dynamin-associated protein 160 kDa) is a membrane-associated, dynamin-binding protein of 160 kDa that has four putative src homology 3 domains and an Eps15 homology domain, motifs frequently found in proteins associated with endocytosis. The binding capacities of the four putative src homology 3 domains were examined individually and in combination and shown to bind known proteins that contained proline-rich domains. Each binding site, however, was different in its preference for binding partners. We suggest that Dap160 is a scaffolding protein that helps anchor proteins required for endocytosis at sites where they are needed in the Drosophilanerve terminal. The discovery of overlapping hot spots of dynamin (Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., and Ramaswami, M. (1996) J. Neurosci. 16, 5443–5456) and the heterotetrameric adaptor 2 complex (Gonzalez-Gaitan, M., and Jäckle, H. (1997)Cell 88, 767–776) in Drosophilanerve terminals led to the concept of zones of active endocytosis close to sites of active exocytosis. The proline-rich domain of Drosophiladynamin was used to identify and purify a third component of the endocytosis zones. Dap160 (dynamin-associated protein 160 kDa) is a membrane-associated, dynamin-binding protein of 160 kDa that has four putative src homology 3 domains and an Eps15 homology domain, motifs frequently found in proteins associated with endocytosis. The binding capacities of the four putative src homology 3 domains were examined individually and in combination and shown to bind known proteins that contained proline-rich domains. Each binding site, however, was different in its preference for binding partners. We suggest that Dap160 is a scaffolding protein that helps anchor proteins required for endocytosis at sites where they are needed in the Drosophilanerve terminal. Endocytosis of receptors such as the transferrin receptor from the plasma membrane is constitutive, whereas internalization of receptors such as the β-adrenergic receptor is stimulated by ligand binding (3Mellman I. Curr. Opin. Cell Biol. 1996; 8: 497-498Crossref PubMed Scopus (50) Google Scholar). A third type of endocytosis, compensatory endocytosis, recovers the extra membrane added to the plasma membrane when exocytosis is stimulated in a regulated secretory cell. All three types of endocytosis appear to use a similar coating mechanism that involves the heterotetrameric adaptor 2 complex (AP2), 1The abbreviations used are: AP2, adaptor protein 2; GST, glutathione S-transferase; Ddyn,Drosophila dynamin; PRD, proline-rich domain; SH3, src homology 3 domain; EH, Eps15 homology domain; PCR, polymerase chain reaction; bp, base pair; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; RACE, rapid amplification of cDNA ends; oligo, oligonucleotide(s); DAP, dynamin-associated protein; csp, cysteine string protein. clathrin and dynamin (4Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (674) Google Scholar). One apparent difference between the three is that compensatory endocytosis is faster than the other two (5Betz W.J. Wu L.-G. Curr. Biol. 1995; 5: 1098-1101Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A possible explanation of the efficiency of compensatory endocytosis is that the machinery of endocytosis is not free in the cytoplasm but is concentrated in the active zones, the sites of exocytosis of synaptic vesicles. The first indication of this was the discovery in Drosophila nerve terminals that dynamin, a large GTPase known to be necessary for synaptic vesicle biogenesis, is restricted to presynaptic sites that coincide with clusters of synaptic vesicles around active zones (1Estes P.S. Roos J. van der Bliek A. Kelly R.B. Krishnan K.S. Ramaswami M. J. Neurosci. 1996; 16: 5443-5456Crossref PubMed Google Scholar). Dynamin concentrations at the plasma membrane are even more obvious (1Estes P.S. Roos J. van der Bliek A. Kelly R.B. Krishnan K.S. Ramaswami M. J. Neurosci. 1996; 16: 5443-5456Crossref PubMed Google Scholar) when Drosophila shibirets1 mutants, temperature-sensitive in dynamin function, are depleted of synaptic vesicles by stimulating exocytosis at nonpermissive temperatures (6Koenig J.H. Ikeda K. J. Neurosci. 1989; 11: 3844-3860Crossref Google Scholar). The idea that these hot spots are indeed loci of intense endocytosis received strong support when it was found that AP2 co-localized with dynamin at these spots (2Gonzalez-Gaitan M. Jäckle H. Cell. 1997; 88: 767-776Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). These authors reported that AP2 distribution was even more tightly localized to the hot spots than dynamin. It thus seems plausible that Drosophila neurons have specialized sites on the plasma membrane at or close to the sites of exocytosis, where the endocytotic machinery accumulates. One way to understand how such an endocytotic machine might operate is to identify the other components of the complex, particularly those that might anchor it to the plasma membrane. In mammalian cells the C-terminal, proline-rich domain (PRD) of dynamin is involved in membrane anchoring since removing it inhibits the association of dynamin with the plasma membrane (7Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 8Shpetner H.S. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Consistent with a role for the dynamin PRD in anchoring is the observation that the src homology 3 (SH3) domain of amphiphysin helps anchor dynamin via its PRD to neuronal plasma membranes in lamprey nerve terminals (9Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (404) Google Scholar). To find howDrosophila dynamin might be anchored at endocytotic hot spots, it seemed reasonable to look for a protein onDrosophila membranes to which the proline-rich domain ofDrosophila dynamin might bind. By using this approach, a new peripheral membrane protein has been identified that can be released from the membranes in a complex withDrosophila dynamin. Sequencing revealed that the protein is multimodular with four contiguous SH3 domains and an Eps15 homology (EH) domain. The SH3 domains were shown to be capable of binding dynamin and other PRD-containing proteins. Each SH3 domain, however, had a different repertoire of binding partners. EH domain-containing proteins have been implicated in receptor and fluid phase endocytosis in yeast (10Tang H.-Y. Munn A. Cai M. Mol. Cell. Biol. 1997; 17: 4294-4304Crossref PubMed Scopus (120) Google Scholar). Furthermore, the progenitor of the family, Eps15, binds AP2 (11Iannolo G. Salcini A.E. Gaidarov I. Goodman Jr., O.B. Carpenter G. Pelicci P.G. DiFiore P.P. Keen J.H. Cancer Res. 1997; 57: 240-245PubMed Google Scholar, 12Tebar F. Sorkina T. Sorkin A. Ericsson M. Kirchhausen T. J. Biol. Chem. 1996; 271: 28727-28730Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) and clusters near the neck of clathrin-coated pits (12Tebar F. Sorkina T. Sorkin A. Ericsson M. Kirchhausen T. J. Biol. Chem. 1996; 271: 28727-28730Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). A ligand for the EH domain in the new protein has not yet been identified. One of only two proteins thus far demonstrated to bind to a GST-EH fusion protein is the Drosophila Numb protein (13Salcini A.E. Confalonieri S. Doria M. Santoline E. Tassi E. Minenkova O. Cesareni G. Pelicci P.G. DiFiore P.P. Genes Dev. 1997; 11: 2239-2249Crossref PubMed Scopus (287) Google Scholar). Numb, in turn, is membrane-associated, possesses a phosphotyrosine-binding (PTB) domain, and associates with Numb-associated kinase (14Chien C.T. Wang S. Rothenberg M. Jan L.Y. Jan Y.N. Mol. Cell. Biol. 1998; 18: 598-607Crossref PubMed Scopus (65) Google Scholar). Thus, EH domain- and SH3 domain-containing proteins may function as either scaffolding or anchoring proteins. The multimodular protein we describe has, therefore, the capacity to bind simultaneously to several different proteins at once, and to keep them on a membrane. We refer to the protein we have isolated as dynamin-associated protein 160 kDa (Dap160). By immunofluorescence, Dap160 was restricted to the synaptic nerve terminal, or boutons, of Drosophilaneuromuscular junctions. In addition it was found in spots that were associated with active zones in the resting nerve terminal and in membrane-associated “hot spots” after depletion of vesicle content by stimulating Drosophila shibirets1mutants at the nonpermissive temperature. The observed distribution of Dap160 is indistinguishable from that reported for dynamin (1Estes P.S. Roos J. van der Bliek A. Kelly R.B. Krishnan K.S. Ramaswami M. J. Neurosci. 1996; 16: 5443-5456Crossref PubMed Google Scholar) and AP2 (2Gonzalez-Gaitan M. Jäckle H. Cell. 1997; 88: 767-776Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Thus, Dap160 has the properties expected of a membrane-associated scaffolding protein that helps hold proteins required for endocytosis close to where they are needed both before and during the recovery of synaptic vesicle membrane proteins. In so doing it could enhance the efficiency or the specificity of compensatory endocytosis. MBP-Ddyn(Δ241) fusion protein for anti-dynamin-specific antibodies was produced as follows. A 1.9-kilobase pair EcoRV-HpaI fragment from pBS(Ddyn3) (Alexander van der Bliek, UCLA School of Medicine), encodingDrosophila dynamin lacking the N-terminal 241 amino acids, was subcloned into the XmnI site of pMAL-c2 (New England Biolabs, Beverly, MA), generating pJR2. GST-p160(JR120) fusion protein for anti-Dap160-specific antibodies was produced by using a 768-bpEcoRI fragment from pJR74 subcloned into theEcoRI site of pGEX-2T (Amersham Pharmacia Biotech), generating pJR73. MBP-Ddyn fusion protein was generated for affinity purification of anti-dynamin-specific polyclonal antibodies. The following oligos were used to generate a full-length clone ofDrosophila dynamin by polymerase chain reaction (PCR): 5′-GGG GAA TTC ATG GAT AGT TTA ATT AC-3′ and 5′-GGG AAT TCA TCC CCT AGA TAC ATA TG-3′. The PCR product was digested with EcoRI and subcloned into pBluescript (Stratagene, La Jolla, CA), generating pJR5. The 2.5-kilobase pair EcoRI fragment was then excised from pJR5 and subcloned into the EcoRI site of pMAL-c2, generating pJR7. GST-Ddyn(PRD) fusion protein for overlay analysis was constructed by PCR from pBS(Ddyn3) to generate a fragment encoding the proline-rich domain of Drosophila dynamin (amino acids 750–836). The two oligos used for PCR were 5′-GTA CCG TGC TTG CAA GG-3′ and 5′-GGG AAT TCA TCC CCT AGA TAC ATA TG-3′. The resulting PCR fragment was subcloned into pTA (Invitrogen, Carlsbad, CA) generating plasmid pTA(750–836). pTA(750–836) was then digested withEcoRI, and the 276-bp fragment containing the sequence encoding the proline-rich domain was subcloned into theEcoRI site of pGEX-5X (Amersham Pharmacia Biotech), generating pJR70. GST-SH3(ABCD), GST-SH3(BCD), GST-SH3(CD), GST-SH3(A), GST-SH3(B), GST-SH3(C), and GST-SH3(D) were constructed by PCR from Dap160–2 cDNA using a similar subcloning scheme to that described for GST-Ddyn(PRD). Oligos used in PCR were as follows: for GST-SH3(ABCD), 5′-AGT GCT TGG GAG GAG AC-3′ (primer JR125) and 5′-AAA TAA ACA CTA AAG TGG-3′ (primer JR128); for GST-SH3(BCD), 5′-CCC CGG TTG ATG CTC AA-3′ (primer JR129) and primer JR128; for GST-SH3(CD), 5′-GAT CAG GGA ATG CGT GC-3′ (primer JR135) and primer JR128; for GST-SH3(A), primer JR125 and 5′-GCC ACT TGA GCA TCA AC-3′; for GST-SH3(B), primer JR129 and 5′-GCA CGC ATT CCC TGA TC-3′; for GST-SH3(C), primer JR135 and 5′-ATT TTG TGC TTT GTA CG-3′; and for GST-SH3(D), 5′-CGT ACA AAG CAC AAA AT-3′ and primer JR128. PCR products were subcloned into pT-Adv (CLONTECH, Palo Alto, CA), digested withEcoRI, and EcoRI fragments were subcloned into either pGEX-2T or pGEX-3X (Amersham Pharmacia Biotech). GST control fusion protein was generated by expression of the plasmid pGEX-3X. GST-grb2 was generously provided by Dr. Joseph Schlessinger (New York Uuniversity School of Medicine). GST-Amph(SH3) was kindly provided by Dr. Pietro De Camilli (Yale University, New Haven, CT). Expression of GST fusion proteins followed the recommendation of the manufacturer. Fusion protein containing lysates were generated as described by Kleidet al. (15Kleid D.G. Yansura D. Small B. Dowbenko D. Moore D.M. Grubman M.J. McKercher P.D. Morgan D.O. Robertson B.H. Bachrach H.L. Science. 1981; 214: 1125-1128Crossref PubMed Scopus (309) Google Scholar). Fusion proteins were bound to glutathione-agarose (Sigma), eluted in glutathione elution buffer (100 mm glutathione, 50 mm Tris-HCl, pH 8.0, 120 mm NaCl), concentrated in a CentriPrep 10 concentrator (Amicon, Beverly, MA), and dialyzed against PBS. Fusion protein concentration was determined using the BCA reagent system (Pierce) and then subsequently diluted to 50% glycerol and stored at −20 °C. Polyclonal antibodies to Drosophiladynamin and Dap160 were raised against purified recombinant fusion protein. pJR2 was expressed and sent to Immuno-Dynamics, Inc. (La Jolla, CA) for antibody production, which generated rabbit polyclonal antibodies Ab2074 and Ab2075. pJR73 was expressed and sent to Alpha Diagnostic Inc. (San Antonio, TX) for antibody production, which generated rabbit polyclonal antibodies Ab1703 and Ab1704. Antibodies were affinity purified by the method of Smith and Fisher (16Smith D.E. Fisher P.A. J. Cell Biol. 1984; 99: 20-28Crossref PubMed Scopus (445) Google Scholar). To affinity purify anti-dynamin antibodies from Ab2074 sera, MBP-Ddyn was overexpressed in bacteria and recovered as a sarkosyl extract (17Grieco F. Hay J.M. Hull R. BioTechniques. 1992; 13: 856-857PubMed Google Scholar). To affinity purify anti-Dap160 antibodies from Ab1703 sera, GST-p160(JR120) was overexpressed in bacteria and purified as described above. Sarkosyl extract containing MBP-Ddyn or purified recombinant GST-p160(JR120) fusion protein was resolved on a 5–15% SDS-PAGE preparative mini-gel and transferred to nitrocellulose using standard methods (18Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The nitrocellulose bearing the fusion protein was excised, blocked in PBS, 0.05% Tween 20, 1% BSA, washed with PBS, 0.05% Tween 20, 0.1% BSA, and then incubated with sera overnight at 4 °C. The antibody-bound nitrocellulose was washed three times with PBS, 0.05% Tween 20, 0.1% BSA and then eluted with 300-μl aliquots of 5 mm glycine, pH 2.3, 500 mm NaCl, 0.5% Tween 20, 100 mg/ml BSA. The eluates were immediately neutralized with 15.75 μl of 1 m Na2HPO4. To affinity purify anti-GST antibodies, GST was overexpressed in bacteria. Soluble recombinant protein was purified as described for GST-p160(JR120) above. Affinity purification proceeded as described above, using Ab1703 as the sera containing the anti-GST antibodies. Anti-amphiphysin I (CD5) antibody was the generous gift of Dr. Pietro De Camilli (Yale University, New Haven, CT). Anti-amphiphysin II-(1874) and anti-endophilin-(1903) antibodies were the generous gift of Dr. Peter McPherson (McGill University, Montreal). Anti-csp monoclonal antibody was kindly provided by Dr. Konrad Zinsmaier (University of Pennsylvania School of Medicine, Philadelphia), and anti-SAP-47 and anti-synapsin mAbs were the generous gifts of Dr. Erich Buchner (Biozentrum der Universität Würzburg, Germany). Lastly, anti-clathrin polyclonal antisera was the generous gift of Dr. Mani Ramaswami (University of Arizona, Tucson, AZ). Samples for overlay analysis were prepared by resolving 20 μg of protein on 5–15% SDS-PAGE gels. The gels were electroblotted onto nitrocellulose and briefly stained with 0.2% Ponceau S. For single sample overlay analysis, each lane of the blot was excised, blocked with 5% nonfat dry milk powder in PBS, 0.05% Tween 20 (BLOTTO), and incubated with 40 μg/ml fusion protein overnight at 4 °C. Fusion protein binding was determined by incubating the overlays with affinity purified anti-GST antibodies and detected with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Cappel/ICN, Aurora, OH) and developed with ECL detection system (Amersham Pharmacia Biotech) according to the manufacturer's directions. All procedures were conducted on ice or at 4 °C. Heads from 10 ml packed wild-type (OR) adult flies, flash-frozen in liquid nitrogen and stored at −80 °C, were isolated using a molecular sieve (Fisher) and kept cold with liquid nitrogen. The heads were then ground using a mortar and pestle and subsequently homogenized in 2 ml of either Hepes buffer (10 mm Hepes, pH 7.4, 1 mm EGTA/0.1 mm MgCl2) or Buffer A (10 mm Hepes, pH 7.4, 150 mm NaCl, 1 mm EGTA, 0.1 mm MgCl2) + protease inhibitors (leupeptin, 5 mg/ml, antipain, 2 mg/ml, benzamidine, 10 mg/ml, aprotinin, 10 mg/ml, pepstatin, 5 mg/ml, chymostatin, 1 mg/ml, and 1 mm phenylmethylsulfonyl fluoride) with 20 strokes using a Teflon Dounce homogenizer. The homogenate was centrifuged at 1000 × g in an Eppendorf microcentrifuge for 20 min, resulting in an S1 and P1 fraction. The S1 fraction was then centrifuged at 25,000 × g in a Beckman TL100 tabletop ultracentrifuge for 30 min, resulting in an S2 and a P2 fractions. The S2 fraction was centrifuged at 208,000 × g in a Beckman TL100 tabletop ultracentrifuge for 60 min, resulting in an S3 and a P3 fraction. To generate rat brain cytosol, rat brains (PelFreeze, Rogers, AR) were diced and homogenized in Buffer A. The resulting homogenate was then fractionated as described above. Further fractionation of the cytosol was achieved by charging 50 mg of cytosol on a MonoQ 5/5 anion exchange FPLC column (Amersham Pharmacia Biotech) and eluting the bound proteins with a 20-ml gradient (Buffer 1, Hepes buffer; Buffer 2, Hepes buffer, 1 m NaCl). 5 mg of DrosophilaS3 extract was resolved on a 24-ml Superdex 200 FPLC column (Amersham Pharmacia Biotech) using either a Bio-Rad BioLogic workstation or Amersham Pharmacia Biotech LC501 FPLC. Protein samples were eluted from the column with Buffer A at a flow rate of 0.35 ml/min, and 0.5-ml fractions were collected. 30-μl samples from each fraction beginning with the void volume fraction were then analyzed by SDS-PAGE, visualized with SYPRO Orange protein stain (Bio-Rad), and either Western blotted or processed for overlay analysis as described above. Superdex 200 gel filtration fractions immunopositive for dynamin and Dap160 were diluted to 1 ml with IP Buffer (50 mm Tris, pH 7.4, 0.1 mmEDTA, 0.5% Tween 20). 25 μg of affinity purified anti-dynamin-specific polyclonal antibodies or equivalent preimmune antibodies were added. Immunoprecipitation reactions were incubated overnight at 4 °C with tumbling end over end. Immunoprecipitations were processed by addition of 30 μl of a 50% protein A-Sepharose slurry, incubation at 4 °C with tumbling end over end for 2 h, and collected by centrifugation at 1000 × g for 5 min. Beads were washed three times with 1 ml of IP Buffer and eluted with SDS-PAGE sample buffer. Eluates were analyzed by resolving on a 5–15% gradient SDS-PAGE gel and immunoblotted using anti-Dap160 and anti-dynamin-specific antibodies. Heads from 2.5 liters of adultDrosophila Oregon-R flies were isolated. This resulted in 40 g of packed fly heads. The heads were ground in a mortar and pestle and homogenized in 500 ml of Hepes buffer. The extract was then centrifuged at 100,000 × g for 1 h. The pellet was extracted with 500 ml of Buffer A. Triton X-100 was added to 1%, and the extract was incubated for 1 h at 4 °C and then centrifuged at 100,000 × g. The resulting supernatant was precipitated with 30% ammonium sulfate. The 30% ammonium sulfate pellet was resuspended in 15 ml of Buffer A and charged on a 250-ml Superose 6 (Amersham Pharmacia Biotech) column and eluted with Buffer A. Three-ml fractions were collected and analyzed for the presence of Dap160 by the overlay assay. Dap160-positive fractions were then charged onto a DEAE-Sepharose column in Buffer A, washed extensively, and eluted with a Buffer A, 400 mm NaCl isocratic buffer flow. The DEAE eluate was concentrated, and 5-mg aliquots were charged onto a Superdex 200 FPLC column and eluted with Buffer A. Dap160-positive fractions, as determined by the overlay assay, were then charged onto a MonoQ FPLC column and eluted with a Buffer A → Buffer A, 1 m NaCl gradient. Dap160-positive fractions were pooled and resolved on a 5–15% SDS-PAGE gel and stained with SYPRO Orange, and the Dap160 band was excised from the gel. This band was then sent to the University of Michigan PCSF facility for protein microsequencing. mRNA from adult Drosophila(OR) heads was isolated using TRI-zol reagent (MBP, San Diego). First-strand cDNA was generated (45Doyle K. Promega Protocols and Applications Guide. 3rd Ed. 1996: 184Google Scholar) using oligo(dT) primer and SuperScriptII RT (Life Technologies, Inc.) as per the manufacturer's suggestions. 1 ng of cDNA template was then used for PCR. From the peptide microsequencing data, the following degenerate oligos were designed. Peptide KIPVTLPQEW generated 5′-AAR ATH CCN GTN ACN YTN CCN CAR GAR TGG-3′ and was labeled 25F. The reverse complement corresponding to peptide IKEQNAKLPQ generated 5′-YTG NGG NAR YTT NGC RTT YTG YTC YTT DAT-3′ and was labeled 4050 RC. Polymerase chain reaction generated a 768-bp PCR product whose translation product contained both peptide sequences from the protein microsequencing project. Additional sequencing data were generated by PCR using a λgt11 expression library constructed by Ito et al. (19Ito N. Salvaterra P. Itakura K. Drosophila Information Service. 1985; 69: 81Google Scholar) as template and oligo JR116 (5′-CTC TGC TTG ACC CTT AA-3′) and the 5′ λgt11 sequencing primer. The full-length sequence was generated by using the 3′- and 5′-RACE kit (Life Technologies, Inc.). For 5′-RACE, oligo JR118 (5′-TAA GAG ACG ACA GCA GGC T-3′) was used first in conjunction with the abridged anchor primer in PCR round 1 and then with the AUAP primer in PCR round 2. For 3′-RACE, oligo JR108 (5′-AAA GCA GAA GGC ACA CA-3′) was used with the 3′ anchor primer in PCR round 1 and then oligo JR123 (5′-GCC CAC AAG CAG TTA AT-3′) was used with the AUAP primer in PCR round 2. All PCR was performed with the EXPAND High Fidelity PCR kit (BMB, Indianapolis, IN), and PCR products were subsequently subcloned into the pT-Adv cloning vector (CLONTECH) and sequenced using an ABI model 373A DNA sequencing machine by Yi Zhang in the UCSF/HRI sequencing facility. A full-length clone was generated by PCR from DrosophilacDNA using oligo JR131 (5′-noncoding region) and oligo JR139 (3′-noncoding region) and sequenced to confirm the sequence of Dap160. Third instar larval neuromuscular preparations were generated as described (1Estes P.S. Roos J. van der Bliek A. Kelly R.B. Krishnan K.S. Ramaswami M. J. Neurosci. 1996; 16: 5443-5456Crossref PubMed Google Scholar) for antibody staining and protein localization studies using a Leica TCS NT laser confocal microscope. Briefly, wild-type (Oregon-R) orshibirets1 crawling third instar larvae were pinned dorsal side up and filleted in calcium-free saline (130 mm NaCl, 36 mm sucrose, 5 mm KCl, 5 mm Hepes, pH 7.3, 4 mm MgCl2, 0.5 mm EGTA). Viscera were removed, allowing visualization of type 1b synaptic boutons innervating the ventral longitudinal muscles 6 and 7. shibirets1-depleted nerve terminals were stimulated in high K+ saline (75 mm NaCl, 36 mm sucrose, 60 mm KCl, 5 mm Hepes, pH 7.3, 2 mm MgCl2, 2 mmCaCl2) at 30 °C for 10 min. After stimulation, the high K+ saline was replaced briefly with prewarmed calcium-free saline prior to fixation. Double-labeling experiments were performed using either affinity purified anti-dynamin or anti-Dap160 polyclonal antibodies in conjunction with anti-csp or anti-SAP-47 monoclonal antibodies. Secondary antibodies used in this study were goat anti-rabbit Texas Red (Cappel/ICN) and goat anti-mouse-fluorescein isothiocyanate (Cappel/ICN). Images were acquired using the Leica TCS software package. All images were viewed with a 100 × 1.4NA lens and the zoom set to 4. Z series were taken where the focal plane was stepped by 0.25 μm between images. When a protein such as dynamin is known to have a role in synaptic vesicle recycling, the fusion protein overlay (or far-Western) technique can be used to identify additional interacting proteins that may mediate recycling. The identification of binding partners for proteins enriched in the mammalian nerve terminal has focused on SH3-containing proteins, such as Grb2 and amphiphysin, that bind proline-rich domains present in synaptojanin and dynamin (20McPherson P.S. Czernik A.J. Chilcote T.J. Onofri R. Benefenati R. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (152) Google Scholar, 21McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar, 22David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (351) Google Scholar, 23de Heuvel E. Bell A.W. Ramjaun A.R. Wong K. Sossin W.S. McPherson P.S. J. Biol. Chem. 1997; 272: 8710-8716Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 24Ringstad N. Nemoto Y. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8569-8574Crossref PubMed Scopus (328) Google Scholar, 25Micheva K.D. Kay B.K. McPherson P.S. J. Biol. Chem. 1997; 272: 27239-27245Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 26Nemoto Y. Arribas M. Haffner C. De Camilli P. J. Biol. Chem. 1997; 272: 30817-30821Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). A subset of the SH3-containing proteins identified by a polyproline peptide phage display screen (27Sparks A.B. Hoffman N.G. McConnell S.J. Fowlkes D.M. Kay B.K. Nat. Biotechnol. 1996; 14: 741-744Crossref PubMed Scopus (215) Google Scholar) has been shown to bind to synaptojanin and is implicated in binding to dynamin (23de Heuvel E. Bell A.W. Ramjaun A.R. Wong K. Sossin W.S. McPherson P.S. J. Biol. Chem. 1997; 272: 8710-8716Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 24Ringstad N. Nemoto Y. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8569-8574Crossref PubMed Scopus (328) Google Scholar). We have used the overlay technique to identify dynamin-binding proteins in Drosophila, hoping to take advantage of its favorable genetics. To detect proteins that contain SH3 domains, the proline-rich domain of Drosophila dynamin (Ddyn(PRD)) was fused to GST. Initial characterization of the GST-Ddyn(PRD) fusion is shown in Fig. 1 A. As a positive control, GST-Amph(SH3) (28David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar) overlays on rat brain cytosol identified two proteins, a 145-kDa protein and a 100-kDa protein (lane 2). These two proteins are predicted to be synaptojanin and dynamin, as reported by David et al. (22David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (351) Google Scholar). GST-Ddyn(PRD) interacted with three major proteins in rat brain cytosol of 120, 55, and 40 kDa (lane 3). A minor band was also seen at 80 kDa. The mobilities of the four dynamin PRD interacting proteins closely resemble the migration pattern of four proteins recently characterized by McPherson and colleagues (23de Heuvel E. Bell A.W. Ramjaun A.R. Wong K. Sossin W.S. McPherson P.S. J. Biol. Chem. 1997; 272: 8710-8716Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) as synaptojanin-binding proteins. To confirm the identity of these four proteins, rat brain cytosol was fractionated on a MonoQ anion exchange column, and fractions were assayed by fusion protein overlays and by Western blotting using antibodies directed against three of the synaptojanin-binding proteins. The 120- and 80-kDa proteins co-migrate with amphiphysin I and amphiphysin II, respectively (Fig. 1 B, lanes 1–4). Assays on the MonoQ fraction containing the 55- and 40-kDa dynamin-interacting proteins support the conclusion that endophilin (SH3p4), a 40-kDa protein shown to interact with synaptojanin and dynamin (23de Heuvel E. Bell A.W. Ramjaun A.R. Wong K. Sossin W.S. McPherson P.S. J. Biol. Chem. 1997; 272: 8710-8716Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 24Ringstad N. Nemoto Y. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8569-8574Crossref PubMed Scopus (328) Google Scholar, 25Micheva K.D. Kay B.K. McPherson P.S. J. Biol. Chem. 1997; 272: 27239-27245Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), is one of the four dynamin-interacting proteins identified by the GST-Ddyn(PRD) reagent (Fig. 1 B,lanes 5 and 6). The identity of the 55-kDa protein is currently being characterized and will be reported on in a separate study. The interaction between the SH3 dom" @default.
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• W2012021856 title "Dap160, a Neural-specific Eps15 Homology and Multiple SH3 Domain-containing Protein That Interacts with DrosophilaDynamin" @default.
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