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• W2065559987 abstract "TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabinin vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton. TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabinin vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton. Many membrane proteins undergo complex trafficking itineraries within eukaryotic cells, cycling between intracellular compartments and the plasma membrane during such processes as nutrient uptake, biosynthetic protein sorting, intracellular signaling, and cell movement. In recent years, considerable efforts have been made to identify the individual components of these processes. Despite rapid progress in identifying the molecular components of the endocytic machinery (1Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (674) Google Scholar), components of endoplasmic reticulum-to-Golgi transport pathways (2Rothman J.E. Wieland F.T. Science. 1997; 272: 227-234Crossref Scopus (1023) Google Scholar), and components of the exocytic machinery (3Hsu S.-C. Hazuka C.D. Foletti D.L. Scheller R.H. Trends Cell Biol. 1999; 9: 150-153Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), little is known about the molecular machinery involved in post-TGN 1The abbreviations used are: TGNtrans-Golgi networkGFPgreen fluorescent proteinGSTglutathione S-transferasePDZPSD95/discs large/ZO-1PCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisTrxthioredoxin1The abbreviations used are: TGNtrans-Golgi networkGFPgreen fluorescent proteinGSTglutathione S-transferasePDZPSD95/discs large/ZO-1PCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisTrxthioredoxin protein trafficking. trans-Golgi network green fluorescent protein glutathione S-transferase PSD95/discs large/ZO-1 polymerase chain reaction polyacrylamide gel electrophoresis thioredoxin trans-Golgi network green fluorescent protein glutathione S-transferase PSD95/discs large/ZO-1 polymerase chain reaction polyacrylamide gel electrophoresis thioredoxin The type I membrane protein, TGN38, cycles between the TGN and cell surface via endosomal intermediates (4Luzio J.P. Brake B. Banting G. Howell K.E. Braghetta P. Stanley K.K. Biochem. J. 1990; 270: 97-102Crossref PubMed Scopus (258) Google Scholar, 5Reaves B. Wilde A. Banting G. Biochem. J. 1992; 283: 313-316Crossref PubMed Scopus (30) Google Scholar, 6Reaves B. Horn M. Banting G. Mol. Biol. Cell. 1993; 4: 93-105Crossref PubMed Scopus (108) Google Scholar, 7Ponnambalam S. Rabouille C. Luzio J.P. Nilsson T. Warren G. J. Cell Biol. 1994; 125: 253-268Crossref PubMed Scopus (120) Google Scholar). Internalization from the plasma membrane and subsequent traffic back to the TGN is directed by the motif -SDYQRL- within the cytosolic domain of the protein (6Reaves B. Horn M. Banting G. Mol. Biol. Cell. 1993; 4: 93-105Crossref PubMed Scopus (108) Google Scholar,8Bos K. Wraight C. Stanley K.K. EMBO J. 1993; 12: 2219-2228Crossref PubMed Scopus (193) Google Scholar, 9Wong S.H. Hong W. J. Biol. Chem. 1993; 268: 22853-22862Abstract Full Text PDF PubMed Google Scholar, 10Humphrey J.S. Peters P.J. Yuan L.C. Bonifacino J.S. J. Cell Biol. 1993; 120: 1123-1135Crossref PubMed Scopus (201) Google Scholar). We and others (11Ohno H. Stewart J. Fournier M.-C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (825) Google Scholar, 12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) have shown that this protein is internalized from the plasma membrane in a clathrin-dependent manner through direct interaction with the μ2 subunit of the clathrin adaptor complex, AP2. Biochemical data have been obtained showing that formation of secretory vesicles at the TGN requires a protein complex that coimmunoprecipitates with TGN38 (13Jones S.M. Crosby J.R. Salamero J. Howell K.E. J. Cell Biol. 1993; 122: 775-788Crossref PubMed Scopus (108) Google Scholar). This complex consists of a 62-kDa subunit with homology to phosphatidylinositol 3-kinase regulatory subunits and a 25-kDa GTPase in addition to TGN38 (13Jones S.M. Crosby J.R. Salamero J. Howell K.E. J. Cell Biol. 1993; 122: 775-788Crossref PubMed Scopus (108) Google Scholar, 14Jones S.M. Howell K.E. J. Cell Biol. 1997; 139: 339-349Crossref PubMed Scopus (77) Google Scholar). The small GTPase has been suggested to be Rab6 (13Jones S.M. Crosby J.R. Salamero J. Howell K.E. J. Cell Biol. 1993; 122: 775-788Crossref PubMed Scopus (108) Google Scholar), but cDNA clones encoding p62 and this small GTPase have yet to be isolated. Other protein complexes that function at a post-TGN level have also been identified. A considerable amount of data has been gathered regarding the role of a number of vacuolar protein sorting mutants in yeast. This work has led to the identification of a complex of proteins, conserved in higher eukaryotes, that is involved in endosome to Golgi transport (15Seaman M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (320) Google Scholar, 16Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (556) Google Scholar). Many individual components of trafficking pathways have also been identified from yeast two-hybrid screens using integral membrane protein cytosolic domains as bait. Examples include the identification of TIP47, which binds a specific motif within the cytosolic domain of the cation-independent mannose 6-phosphate receptor directing traffic from endosomes to the TGN (17Diaz E. Pfeffer S.R. Cell. 1998; 93: 433-443Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), and P-CIP-1 (18Chen L. Johnson R.C. Milgram S.L. J. Biol. Chem. 1998; 273: 33524-33532Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), which mediates the endosomal trafficking of peptidylglycine α-amidating monooxygenase. The endoprotease furin has also been successfully used to identify novel components of the post-TGN sorting machinery. Two-hybrid screens of cDNA libraries using the cytosolic domain of furin as bait led to the identification of both PACS-1 (19Wan L. Molloy S.S. Thomas L. Liu G. Xiang Y. Rybak S.L. Thomas G. Cell. 1998; 94: 205-216Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) and the actin-binding protein ABP-280 (20Liu G. Thomas L. Warren R.A. Enns C.A. Cunningham C. Hartwig J.H. Thomas G. J. Cell Biol. 1997; 139: 1719-1733Crossref PubMed Scopus (121) Google Scholar) as molecules involved in trafficking of furin. We chose to use the cytosolic domain of TGN38 to screen a two-hybrid library to identify interacting proteins, particularly those that may be involved in post-TGN membrane traffic. By using this approach we have identified a direct interaction between TGN38 and the brain-specific F-actin binding protein neurabin-I. We show that TGN38 also interacts directly with the ubiquitously expressed isoform of neurabin-I, neurabin-II (also known as spinophilin). These interactions are highly specific to TGN38 and can be abolished by a point mutation of a serine residue within the cytosolic domain of TGN38. TGN38 preferentially binds to the dimeric form of neurabin-I in vitro. The two proteins can also be coimmunoprecipitated from PC12 cells stably transfected with expression constructs encoding GFP-tagged neurabin-I or -II. These data suggest that neurabin provides a direct link between TGN38-containing membranes and the actin cytoskeleton. All reagents were purchased from Sigma unless otherwise stated. DNA restriction and modifying enzymes were from Roche Molecular Biochemicals. The entire 33-amino acid cytosolic domain of TGN38 was cloned into the two-hybrid bait vector pBTM116 (21Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1661) Google Scholar) to generate pBTM-TGN38 (12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This plasmid was then transformed into yeast strain L40 (21Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1661) Google Scholar) as described previously (12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A 5-ml overnight culture of yeast containing the pBTM-TGN38 plasmid was grown in synthetic medium, Yc (1.2 g·liter−1 yeast nitrogen base without (NH4)2SO4, 5 g·liter−1 (NH4)2SO4, 10 g·liter−1 succinic acid, 6 g·liter−1 NaOH, 2% glucose) supplemented with 0.1 g·liter−1 adenine, arginine, cysteine, threonine, and leucine and 0.05 g·liter−1 aspartate, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, and valine (i.e.lacking tryptophan, uracil, histidine, and lysine). This was then used to inoculate a 100-ml culture of the same medium that was then grown overnight and used to inoculate a 1000-ml culture of YPAD (YEPD (20 g·liter−1 peptone, 10 g·liter−1 yeast extract, pH 5.8, 2% glucose) with 40 μg·ml−1 adenine) that had been prewarmed to 30 °C. Cells were grown for 3 h, pelleted, washed in 500 ml of 1× TE (10 mm Tris·HCl, pH 7.5, 1 mm EDTA), and finally resuspended in 20 ml of 0.5× TE (5 mm Tris·HCl, pH 7.5, 0.5 mm EDTA) containing 100 mm lithium acetate. To this, 1.0 ml of 10 mg·ml−1 denatured and sheared salmon sperm DNA was added with 0.5 mg of library plasmid DNA (a rat brain cDNA library (kindly provided by Dr. Jeremy Henley, University of Bristol) consisting of both random-primed and oligo(dT)-primed cDNA from RNA of 100 pooled rat brains from 10- to 12-week-old Harlan Sprague-Dawley males, representing approximately 2,000,000 independent clones (CLONTECH, Basingstoke, UK)). After mixing, 140 ml of 1× TE containing 100 mm lithium acetate and 40% polyethylene glycol 3350 were added. This was thoroughly mixed and placed at 30 °C for 30 min, transferred to a sterile 2-liter beaker, and 17.6 ml of dimethyl sulfoxide added. This was then heat-shocked for 6 min at 42 °C with occasional mixing. Cells were then washed in a further 500 ml of 1× TE and resuspended in 1000 ml of YEPD. After a 1-h incubation at 30 °C with gentle mixing, the cells were washed in synthetic medium (Yc), lacking leucine, tryptophan, lysine, and uracil, before incubating overnight in 100 ml of the same. Cells were finally washed twice in 1× TE and resuspended in 10 ml of 1× TE before plating on selection medium (Yc containing 20 g·liter−1 agar, lacking tryptophan, leucine, histidine, lysine, and uracil). The entire transformation was split evenly between eight 22 × 22-cm square Petri dishes. Aliquots were also plated to determine transformation efficiency onto plates lacking leucine, lysine, tryptophan, and uracil. After 8 days growth at 30 °C, colonies were picked to duplicate plates with or without histidine and assayed as described in the Yeast Protocols Handbook (CLONTECH, Basingstoke, UK). Colonies that were scored double-positive for both histidine selection and β-galactosidase were amplified in liquid culture, plasmid DNA-extracted, and library plasmids isolated by transformation into HB101 Escherichia coli. Plasmids were isolated by miniprep purification (Qiagen, Crawley, UK) and isolated library plasmids retransformed to L40 with the negative control plasmid, pLexA-Lamin. Those which were negative for interaction with lamin were analyzed as follows. Plasmids were sequenced using vector-specific primers (insert-screening amplimers, CLONTECH) using the DNA sequencing service within the Department of Biochemistry, University of Bristol. DNA sequences were used to search the non-redundant GenBankTM data base using the BLAST search algorithm available over the Internet from the European Bioinformatics Institute. Plasmids containing full-length cDNAs of neurabin-I and neurabin-II were obtained from Professor Yoshimi Takai (ERATO Biotimer Project, Kobe, Japan) and Professor Paul Greengard (The Rockefeller University, New York). The PstI fragment of neurabin-I containing the majority of the coiled coil domain (amino acids 719–1023) was subcloned to pRSETc (Invitrogen, Groningen, Netherlands) and expressed as a hexahistidine-tagged fusion protein inE. coli strain BLRDE3 (Novagen, Cambridge, UK) according to recommended protocols (Invitrogen). A fragment of neurabin-II (BamHI-EcoRI fragment containing amino acids 543–814) containing the majority of neurabin-II coiled coil regions was similarly expressed. A neurabin-II construct equivalent to the neurabin-I clone identified in the library screen (amino acids 483–817) was generated by PCR and cloned into pGAD10 (CLONTECH). Recombinant TGN38 cytosolic domain fusion proteins were generated as described previously (12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Other TGN38 constructs used in this study have also been previously described (12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 22Stephens D.J. Banting G. Biochem. J. 1998; 335: 567-572Crossref PubMed Scopus (43) Google Scholar). Other two-hybrid constructs were generated by PCR according to standard protocols. The full coding sequence of neurabin-I was amplified by PCR and cloned into pEGFP-C1 (CLONTECH) according to standard protocols. The BglII restriction fragment of neurabin-II (amino acids 1–801, lacking the carboxyl-terminal 16 amino acids of neurabin-II) was subcloned to pEGFP-N3 (CLONTECH) generating an in-frame fusion with enhanced GFP (it was not found possible to amplify the full coding region of neurabin-II by PCR, presumably due to the high GC content of the 5′ end of the cDNA). Histidine-tagged and GST-tagged recombinant fusion proteins were expressed in BLRDE3E. coli (Novagen) and purified according to standard protocols using Talon resin (CLONTECH) or glutathione-Sepharose (Sigma), respectively. Surface plasmon resonance analyses were performed on a BIAcore 1000 biosensor using phosphate-buffered saline as running buffer. Manufacturer's recommended immobilization protocols for histidine-tagged and GST-tagged fusion proteins were followed. Data were aligned using BIAcore evaluation software and prepared for publication using Microsoft Excel (Microsoft, Reading, UK). Purified recombinant hexahistidine-tagged neurabin-I (coiled coil region) was separated by SDS-PAGE on an 8% gel, transferred to nitrocellulose (Schleicher & Schuell), and blocked overnight in TBS containing 0.05% Tween 20 with 10% dried skimmed milk (blocking buffer). The blot was then incubated with 50 μg·ml−1 Trx-TGN38 (a thioredoxin fusion of the entire cytosolic domain of TGN38, Ref. 12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), washed (3 × 10 min) in TBS containing 0.05% Tween 20, and incubated with a 1:1000 dilution of an anti-thioredoxin polyclonal antibody (raised in rabbit using purified recombinant thioredoxin as immunogen, Ref. 23Crump C.M. Williams J.L. Stephens D.J. Banting G. J. Biol. Chem. 1998; 273: 28073-28077Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The blot was washed again and incubated with a 1:10000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody (Sigma). Immunoreactive bands were visualized by chemiluminescence (Western blotting Kit, Roche Molecular Biochemicals). PC12 cells (Ref. 24Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4859) Google Scholar; a kind gift of Dr. Frank Gunn-Moore, University of Bristol) were cultured on plastic dishes in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 5% horse serum, penicillin, and streptomycin. Cells were transfected using Lipofectin (Life Technologies Inc.) according to standard protocols. Stable cell lines were selected using 400 μg·ml−1 G418 (Life Technologies Inc.) and maintained in 200 μg·ml−1 G418. For all cell imaging applications, cells were grown on collagen-coated glass coverslips. Immunofluorescence staining was performed according to established protocols (described in Ref. 25Roquemore E.P. Banting G. Mol. Biol. Cell. 1998; 9: 2125-2144Crossref PubMed Scopus (36) Google Scholar). The following antibodies were used: monoclonal anti-TGN38, clone 2F7.1 (26Horn M. Banting G. Biochem. J. 1994; 301: 69-73Crossref PubMed Scopus (65) Google Scholar, Affinity Bioreagents, Golden, CO), anti- mannosidase-II (clone 53FC3, see Refs.27Baron M.D. Garoff H. J. Biol. Chem. 1990; 265: 19928-19931Abstract Full Text PDF PubMed Google Scholar and 28Burke B. Griffiths G. Reggio H. Louvard D. Warren G. EMBO J. 1982; 1: 1621-1628Crossref PubMed Scopus (156) Google Scholar). Primary antibodies were detected by incubation with 1:1000 dilutions of either Alexa-488- or Alexa-594-conjugated anti-mouse or anti-rabbit secondary antibodies (Molecular Probes, Cambridge, UK). Rhodamine-conjugated phalloidin was kindly provided by Professor Jeremy Tavaré (University of Bristol). Stably transfected PC12 cells expressing GFP-neurabin-II were grown to approximately 70% confluence in T75 culture flasks, lysed in RIPA-containing protease inhibitors as described previously (25Roquemore E.P. Banting G. Mol. Biol. Cell. 1998; 9: 2125-2144Crossref PubMed Scopus (36) Google Scholar) supplemented with 20 μg·ml−1 latrunculin B (Calbiochem). Cleared supernatants were incubated with 2 μl each of polyclonal anti-TGN38 antibodies, 1918 and G29 (29Wilde A. Reaves B. Banting G. FEBS Lett. 1992; 313: 235-238Crossref PubMed Scopus (28) Google Scholar), or 4 μl of G29 preimmune serum, prebound to Gammabind (Amersham Pharmacia Biotech). After 2 h mixing at 4 °C, immunoprecipitates were washed six times (5 min each) in RIPA supplemented with protease inhibitors as for cell lysis. After boiling in SDS sample buffer, samples were separated on 6% polyacrylamide gels, transferred to nitrocellulose, and probed with 1:2000 anti-GFP polyclonal antibody (CLONTECH). Blots were developed as described above. Binding was competed by including 30 or 100 μmTrx-TGN38 (a fusion protein including the entire cytosolic domain of TGN38 (12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar)) as indicated. The entire cytosolic domain of TGN38 was used as bait in a screen of 6.5 × 106 clones of a rat brain cDNA library. This represented greater than 3-fold redundancy since the library contained 2 × 106 independent clones (see “Experimental Procedures”). 64 clones that showed positive interaction with TGN38 by both histidine autotrophy and expression of β-galactosidase activity were isolated. Of these 64 clones, three represented a partial clone of the F-actin binding protein, neurabin-I (30Nakanishi H. Obaishi H. Satoh A. Wada M. Mandai K. Satoh K. Nishioka H. Matsuura Y. Mizoguchi A. Takai Y. J. Cell Biol. 1997; 139: 951-961Crossref PubMed Scopus (162) Google Scholar). These clones (numbers 11, 17, and 118) were identical, showing 100% identity to amino acids 488–1095 of the published sequence of neurabin-I. This region of sequence includes the PSD-95/discs large/ZO-1 (PDZ) domain of neurabin-I as well as the entire carboxyl-terminal coiled coil region but is missing the amino-terminal actin binding domain. PDZ domains are known to interact with the extreme cytosolic domain of transmembrane receptors (31Saras J. Heldin C.H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (222) Google Scholar). However, a PDZ domain containing construct of neurabin-I (amino acids 488–718) showed no interaction with the cytosolic domain of TGN38 in our two-hybrid system, leading us to the conclusion that the PDZ domain of neurabin-I does not bind to TGN38. We also tested the ability of the full coding region of neurabin-I to interact with TGN38. Upon prolonged incubation in either growth or β-galactosidase assays, an interaction could be detected; the data showed that this interaction was significantly lower in affinity to that of the partial clone isolated from the library screen. We went on to characterize this interaction in more detail, screening library clone 118 against a number of TGN38 cytosolic domain constructs as well as a number of other receptor cytosolic domain constructs (all cloned as bait in pBTM116). Fig. 1 shows that as well as a strong positive interaction with the wild-type cytosolic domain of TGN38 (Fig. 1 , B1), clone 118 showed a positive interaction with TGN38 in which the last four amino acids (NLKL) had been deleted (Fig. 1, E1). This further suggests that the interaction is not mediated through the PDZ domain of neurabin-I, since PDZ domains are believed to bind to the terminal residues of membrane protein cytosolic domains (31Saras J. Heldin C.H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (222) Google Scholar). Neurabin-I was found to interact with a TGN38 construct in which the critical tyrosine residue of the internalization motif was mutated to alanine (Y333A). Mutation of serine 331 of TGN38 to alanine (S331A, Fig. 1, C1) completely abolished binding of neurabin-I (clone 118, Fig. 1, D1), an effect also seen for the interaction between TGN38 and the μ2 subunit of the AP2 clathrin adaptor (12Stephens D.J. Crump C.M. Clarke A.R. Banting G. J. Biol. Chem. 1997; 272: 14104-14109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Together, these data suggest that neurabin and μ2 may have distinct but overlapping binding sites on the TGN38 cytosolic domain. The S331A mutant protein is functionally expressed in this system, since it interacts with another clone we have isolated in a different library screen. 2C. M. Crump, D. J. Stephens, and G. Banting, unpublished observations. In addition we also see no interaction between TGN38 and neurabin-I in assays using TGN38 constructs in which serine 331 is mutated to aspartate (not shown). The specificity of interaction between TGN38 and neurabin-I is further demonstrated by the lack of detectable interaction in the two-hybrid system of clone 118 with the cytosolic domains of CD63, TrkB, Lgp120, Simian immunodeficiency virus envelope, or the polymeric immunoglobulin receptor (Fig. 1, row 2). A diagrammatic representation of the TGN38-neurabin interaction is shown in Fig. 2. Given the recent characterization of neurabin-II (spinophilin, Refs. 32Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (388) Google Scholar and 33Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), the ubiquitous homologue of neurabin-I, we decided to investigate the interaction of neurabin-II with TGN38. Owing to the weakly observed signal for interaction of TGN38 with the full coding sequence of neurabin-I in the two-hybrid system (which we also observed for neurabin-II (not shown)), we generated a neurabin-II clone that precisely corresponded to the neurabin-I clone isolated from the library screen (neurabin-II amino acids 483–817) and tested this truncated neurabin-II construct for interaction with the same panel of receptor cytosolic domain constructs described above. The results are shown in Fig. 1 (rows 3 and 4). Exactly the same pattern of interactions is observed for neurabin-II as was seen for neurabin-I. Whereas wild-type TGN38 interacts with neurabin-II (Fig. 1,B3), mutation of serine 331 to alanine abolishes this interaction (Fig. 1, D3). Mutation of tyrosine 333 to alanine or removal of the last four amino acids of TGN38 has no effect on this interaction (Fig. 1, C3and E3). Similarly we detect no binding of neurabin-II to any of the other receptor cytosolic domains tested (Fig. 1, row 4). Since TGN38 is ubiquitously expressed (4Luzio J.P. Brake B. Banting G. Howell K.E. Braghetta P. Stanley K.K. Biochem. J. 1990; 270: 97-102Crossref PubMed Scopus (258) Google Scholar), it is of great significance that an interaction with neurabin-II is detected as this is also expressed in all tissues examined (32Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (388) Google Scholar, 33Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). To confirm that the interaction between TGN38 and neurabin is direct, we generated recombinant fusion proteins incorporating the cytosolic domain of TGN38 as a glutathione S-transferase (GST) fusion and the coiled coil domain of neurabins-I and -II as hexahistidine-tagged fusions. We reasoned from the two-hybrid data that the coiled coil domains of the neurabins were the most likely to interact with TGN38. Surface plasmon resonance experiments were performed to measure the interactions in real time. Fig. 3 A shows the results of an experiment in which the neurabin-I fusion was immobilized onto a charged Ni2+-NTA biosensor chip (0–60 s) followed by direct injection of either purified GST (gray circles) or a GST-TGN38 fusion (black circles). The data show that a robust binding of the GST-TGN38 fusion is detected with no detectable binding of GST alone. These data confirm the interaction between TGN38 and neurabin-I identified from the two-hybrid screen. As with our two-hybrid data, we went on to measure binding of the coiled coil domain of neurabin-II to the cytosolic domain of TGN38. In this experiment, represented in Fig. 3 B, we immobilized a GST-TGN38 fusion to the biosensor chip surface and flowed purified fusions of either neurabin-I (gray circles) or neurabin-II (black circles) coiled coil regions (amino acids 719–1023 and 543–814, respectively) across (note that this is the reciprocal of the previous experiment). As is evident from Fig. 3 B, we detect almost identical binding of these fusions to TGN38, whereas we detect no binding to negative control surfaces (coated with GST alone, data not shown). As a further control experiment, we attempted to measure binding of tropomyosin to the GST-TGN38 fusion. Tropomyosin contains significant regions of predicted coiled coil structure (34Whitby F.G. Kent H. Stewart F. Stewart M. Xie X. Hatch V. Cohen C. Phillips Jr., G.N. J. Mol. Biol. 1992; 227: 441-448Crossref PubMed Scopus (65) Google Scholar) similar to those of neurabin-I and -II used in these experiments. The recent identification of tropomyosin isoforms on Golgi membranes (35Heimann K. Percival J.M. Weinberger R. Gunning P. Stow J.L. J. Biol. Chem. 1999; 274: 10743-10750Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) makes this a particularly suitable negative control for these TGN38 interaction assays. However, we detected no interaction of TGN38 with tropomyosin by surface plasmon resonance (not shown). We were also able to confirm the specificity of these interactions by generating fusion proteins in which the tyrosine 333 or serine 331 residues" @default.
• W2065559987 created "2016-06-24" @default.
• W2065559987 creator A5004412496 @default.
• W2065559987 creator A5038675573 @default.
• W2065559987 date "1999-10-01" @default.
• W2065559987 modified "2023-09-29" @default.
• W2065559987 title "Direct Interaction of the trans-Golgi Network Membrane Protein, TGN38, with the F-actin Binding Protein, Neurabin" @default.
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