Mixed Lineage Kinase 2 Interacts with Clathrin and Influences Clathrin-coated Vesicle Trafficking
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m204626200
ISSN1083-351X
AutoresShiva Akbarzadeh, Hong Ji, David F. Frecklington, Nelly Marmy-Conus, Yee‐Foong Mok, Leanne Bowes, Lisa Devereux, Martha E. Linsenmeyer, Richard J. Simpson, Donna S. Dorow,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMixed lineage kinase 2 (MLK2) is a protein kinase that signals in the stress-activated Jun N-terminal kinase signal transduction pathway. We used immunoprecipitation and mass spectrometric analysis to identify MLK2-binding proteins in cell lines with inducible expression of green fluorescent protein-tagged MLK2. Here we report the identification of clathrin as a binding partner for MLK2 in both cultured cells and mammalian brain. We demonstrate that clathrin binding requires a motif (LLDMD) located near the MLK2 C terminus, which is similar to “clathrin box” motifs important for binding of clathrin coat assembly and accessory proteins to the clathrin heavy chain. A C-terminal fragment of MLK2 containing this motif binds strongly to clathrin, and mutation of the LLDMD sequence to LAAAD completely abrogates clathrin binding. We isolated clathrin-coated vesicles from green fluorescent protein-MLK2-expressing cells and from mouse brain lysates and found that MLK2 is enriched along with clathrin in these vesicles. In addition, we demonstrated that endogenous MLK2 co-immunoprecipitates with clathrin heavy chain from the vesicle-enriched fraction of mouse brain lysate. Furthermore, overexpression of MLK2 in cultured cells inhibits accumulation of labeled transferrin in recycling endosomes during receptor-mediated endocytosis. These findings suggest a role for MLK2 and the stress-signaling pathway at sites of clathrin activity in vesicle formation or trafficking. Mixed lineage kinase 2 (MLK2) is a protein kinase that signals in the stress-activated Jun N-terminal kinase signal transduction pathway. We used immunoprecipitation and mass spectrometric analysis to identify MLK2-binding proteins in cell lines with inducible expression of green fluorescent protein-tagged MLK2. Here we report the identification of clathrin as a binding partner for MLK2 in both cultured cells and mammalian brain. We demonstrate that clathrin binding requires a motif (LLDMD) located near the MLK2 C terminus, which is similar to “clathrin box” motifs important for binding of clathrin coat assembly and accessory proteins to the clathrin heavy chain. A C-terminal fragment of MLK2 containing this motif binds strongly to clathrin, and mutation of the LLDMD sequence to LAAAD completely abrogates clathrin binding. We isolated clathrin-coated vesicles from green fluorescent protein-MLK2-expressing cells and from mouse brain lysates and found that MLK2 is enriched along with clathrin in these vesicles. In addition, we demonstrated that endogenous MLK2 co-immunoprecipitates with clathrin heavy chain from the vesicle-enriched fraction of mouse brain lysate. Furthermore, overexpression of MLK2 in cultured cells inhibits accumulation of labeled transferrin in recycling endosomes during receptor-mediated endocytosis. These findings suggest a role for MLK2 and the stress-signaling pathway at sites of clathrin activity in vesicle formation or trafficking. mixed lineage kinase Jun N-terminal kinase dual leucine zipper bearing kinase Cdc42/Rac interactive-binding motif clathrin heavy chain JNK interacting protein huntingtin interacting protein clathrin-coated vesicle Src homology assembly proteins amino acids green fluorescent protein dominant-negative Dulbecco's modified Eagle's medium mitogen-activated protein 1,4-piperazinediethanesulfonic acid mass spectrometry 4-morpholineethanesulfonic acid The mixed lineage kinases (MLKs)1 (1Dorow D.S Devereux D. Dietzsch E. deKretser T.A. Eur. J. Biochem. 1993; 213: 701-710Crossref PubMed Scopus (90) Google Scholar) are a family of protein kinases that activate the stress-activated protein kinase cascade leading to phosphorylation of transcription factor c-Jun by the Jun N-terminal kinase (JNK) (2Hirai S. Izawa M. Osada S. Spyrou G. Ohno S. Oncogene. 1996; 12: 641-650PubMed Google Scholar, 3Fan G. Merritt S.E. Kortenjann M. Shaw P.E. Holzman L.B. J. Biol. Chem. 1996; 271: 24788-24793Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Like other mitogen-activated protein kinases, JNK is the final member of a three-kinase cascade in which sequential phosphorylation events transmit signals from upstream activators to the nucleus to initiate gene transcription (4Davis R.J. Biochem. Soc. Symp. 1999; 64: 1-12PubMed Google Scholar). JNKs are activated by dual phosphorylation on threonine and tyrosine by MAP kinase kinases (MAP2Ks), MKK4 and MKK7, that are in turn activated by a number of MAP3Ks including MLK family members (2Hirai S. Izawa M. Osada S. Spyrou G. Ohno S. Oncogene. 1996; 12: 641-650PubMed Google Scholar, 3Fan G. Merritt S.E. Kortenjann M. Shaw P.E. Holzman L.B. J. Biol. Chem. 1996; 271: 24788-24793Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 5Cuenda A.I. Dorow D.S. Biochem. J. 1998; 333: 11-15Crossref PubMed Scopus (57) Google Scholar). The three sequential kinases in a given cascade form a signaling module that responds to a specific set of stimuli to initiate cellular events. In addition to transcription factors, JNKs phosphorylate a diverse range of proteins (6Shanavas A. Papasozomenos S.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14139-14144Crossref PubMed Scopus (20) Google Scholar, 7Fernyhough P. Gallagher A. Averill S.A. Priestley J.V. Hounsom L. Patel J. Tomlinson D.R. Diabetes. 1999; 48: 881-889Crossref PubMed Scopus (150) Google Scholar, 8Fan M. Goodwin M., Vu, T. Brantley-Finley C. Gaarde W.A. Chambers T.C. J. Biol. Chem. 2000; 275: 29980-29985Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar) and play significant roles in many cellular functions including proliferation and differentiation, inflammatory and immune responses, cell cycle arrest, and apoptosis (4Davis R.J. Biochem. Soc. Symp. 1999; 64: 1-12PubMed Google Scholar, 9Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar, 10Leppa S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (443) Google Scholar). In mammalian cells, there are three closely related MLK family members (MLKs 1–3) (1Dorow D.S Devereux D. Dietzsch E. deKretser T.A. Eur. J. Biochem. 1993; 213: 701-710Crossref PubMed Scopus (90) Google Scholar, 11Dorow D.S. Devereux L., Tu, G.-F. Price G. Nicholl J.K. Sutherland G.R. Simpson R.J. Eur. J. Biochem. 1995; 234: 492-500Crossref PubMed Scopus (52) Google Scholar, 12Ing Y.L. Leung I.W.L. Heng H.H.Q. Tsui L.-C. Lassam N.J. Oncogene. 1994; 9: 1745-1750PubMed Google Scholar) as well as two more distantly related members (DLK/ZPK and LZK) (13Holzman L.B. Merritt S.E. Fan G. J. Biol. Chem. 1994; 269: 30808-30817Abstract Full Text PDF PubMed Google Scholar, 14Reddy U.R. Pleasure D. Biochem. Biophys. Res. Commun. 1994; 205: 1494-1495Crossref PubMed Scopus (10) Google Scholar, 15Sakuma H. Ikeda A. Oka S. Kozutsumi Y. Zanetta J.P. Kawasaki T. J. Biol. Chem. 1997; 272: 28622-28629Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). MLKs 1–3 each contain a set of highly conserved structural domains associated with protein interactions and signal transduction including an Src homology 3 (SH3) domain (16Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1438) Google Scholar), a kinase catalytic domain, two leucine zipper (LeuZip) motifs (17Landschultz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2538) Google Scholar), a basic peptide, and a Cdc42/Rac interactive binding (CRIB) motif (18Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). In addition, each has a unique N-terminal peptide and a large C-terminal domain rich in serine, threonine, and proline but with reduced amino acid similarity among the three proteins. The more distantly related members retain much of the overall MLK family structure but lack an SH3 domain and share a lower level of amino acid conservation with MLKs 1–3. Although all are activators of the stress-signaling pathway, little is known about their modes of activation, and their physiological roles in mammalian cells are just beginning to be revealed. One of the MLK proteins, MLK2, is expressed at high levels in human brain, skeletal muscle, and testis (11Dorow D.S. Devereux L., Tu, G.-F. Price G. Nicholl J.K. Sutherland G.R. Simpson R.J. Eur. J. Biochem. 1995; 234: 492-500Crossref PubMed Scopus (52) Google Scholar, 19Phelan D.R. Loveland K.L. Devereux L. Dorow D.S. Mol. Reprod. Dev. 1998; 52: 135-140Crossref Scopus (13) Google Scholar); however, it is present at low levels in many human epithelial cell lines, and searches of EST data bases reveal a wide distribution in embryonic and adult human tissues. Past studies (20Nagata K.-I. Puls A. Futter C. Aspenstrom P. Schaefer E. Nakata T. Hirokawa N. Hall A. EMBO J. 1998; 17: 149-158Crossref PubMed Scopus (233) Google Scholar, 21Rasmussen R.K., Ji, H. Eddes J.S. Moritz R.L. Reid G.E. Simpson R.J. Dorow D.S. Electrophoresis. 1998; 19: 809-817Crossref PubMed Scopus (20) Google Scholar, 22Savinainen A. Garcia E.P. Dorow D. Marshall J. Liu Y.F. J. Biol. Chem. 2001; 276: 11382-11386Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 23Rasmussen R.K. Rusak J. Price G. Robinson P.J. Simpson R.J. Dorow D.S. Biochem. J. 1998; 335: 119-124Crossref PubMed Scopus (23) Google Scholar) have identified MLK2 interactions with proteins involved in regulation of membrane and cytoskeletal changes during cellular activation, cell contact, and vesicle formation. These include tubulin (21Rasmussen R.K., Ji, H. Eddes J.S. Moritz R.L. Reid G.E. Simpson R.J. Dorow D.S. Electrophoresis. 1998; 19: 809-817Crossref PubMed Scopus (20) Google Scholar) and microtubule motor proteins (20Nagata K.-I. Puls A. Futter C. Aspenstrom P. Schaefer E. Nakata T. Hirokawa N. Hall A. EMBO J. 1998; 17: 149-158Crossref PubMed Scopus (233) Google Scholar) as well as actin-regulating GTPases, Cdc42, and Rac that bind the MLK2-CRIB motif in a GTP-dependent manner (18Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 20Nagata K.-I. Puls A. Futter C. Aspenstrom P. Schaefer E. Nakata T. Hirokawa N. Hall A. EMBO J. 1998; 17: 149-158Crossref PubMed Scopus (233) Google Scholar). MLK2 also binds to PSD95 (22Savinainen A. Garcia E.P. Dorow D. Marshall J. Liu Y.F. J. Biol. Chem. 2001; 276: 11382-11386Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), a member of the membrane-associated guanylate kinase protein family involved in formation of multiprotein signaling complexes at synapses, cellular junctions, and polarized membrane domains (24Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (275) Google Scholar). PSD-95 anchors MLK2 to a complex with the kainate receptor, GluR6, in neuronal cells (22Savinainen A. Garcia E.P. Dorow D. Marshall J. Liu Y.F. J. Biol. Chem. 2001; 276: 11382-11386Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and expression of a dominant-negative MLK2 mutant (DN-MLK2) inhibits kainic acid signaling in this cellular system. Furthermore, the MLK2-SH3 domain binds to and activates the GTPase, dynamin (23Rasmussen R.K. Rusak J. Price G. Robinson P.J. Simpson R.J. Dorow D.S. Biochem. J. 1998; 335: 119-124Crossref PubMed Scopus (23) Google Scholar), that is involved in vesicle formation during receptor-mediated endocytosis, neurotransmission, and Golgi transport (reviewed in Ref. 25Danino D. Hinshaw J.E. Curr. Opin. Cell Biol. 2001; 13: 454-460Crossref PubMed Scopus (160) Google Scholar). Interestingly, Cdc42 and Rac also regulate endocytic traffic (Refs. 26Garrett W.S. Chen L.M. Kroschewski R. Ebersold M. Turley S. Trombetta S. Galan J.E. Mellman I. Cell. 2000; 102: 325-334Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar and 27Lamaze C. Chuang T.H. Terlecky L.J. Bokoch G.M. Schmid S.L. Nature. 1996; 382: 177-179Crossref PubMed Scopus (331) Google Scholar reviewed in Ref. 28Ellis S. Mellor H. Trends Cell Biol. 2000; 10: 85-88Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), most likely through actin rearrangement at sites of clathrin-coated vesicle formation (29Yin H.A. Stull J.T. J. Biol. Chem. 1999; 274: 32529-32530Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Further interaction studies reveal that the three members of the MLK to JNK signaling module, MLKs 2, 3, or DLK, together with MKK7 and JNK, bind a set of scaffold proteins, JNK-interacting proteins (JIPs) (30Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (409) Google Scholar). The JIP scaffolding interactions serve to modulate, and possibly direct, signaling within the MLK/JNK cascade (30Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (409) Google Scholar, 31Tawadros T. Formenton A. Dudler J. Thompson N. Nicod P. Leisinger H.J. Waeber G. Haefliger J.A. J. Cell Sci. 2002; 115: 385-393PubMed Google Scholar). JIPs also bind apoER2, the neuronal receptor for reelin (32Stockinger W. Brandes C. Fasching D. Hermann M. Gotthardt M. Herz J. Schneider W.J. Nimpf J. J. Biol. Chem. 2000; 275: 25625-25632Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), a molecule required for correct positioning of neurons during development. In addition, JIPs bind kinesin motor proteins, and a complex of kinesin, JIP, DLK, and apoER2 can be precipitated by addition of stabilized microtubules to a high speed supernatant fraction of rat brain lysate (33Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (497) Google Scholar). These results led to the suggestion that the DLK-MKK7-JNK signaling module is pre-assembled onto JIP together with an apoER2-containing vesicle and that the entire complex is transported along microtubules to potential sites of activity. A separate study of the JIP-DLK interaction revealed that JIP binding holds DLK in an inactive monomeric form and that upon release from JIP, DLK dimerizes, thereby becoming active (34Nihalani D. Meyer D. Pajni S. Holzman L.B. EMBO J. 2001; 20: 3447-3458Crossref PubMed Scopus (87) Google Scholar). Thus, vesicle transport and receptor signaling activities can be coordinated through scaffold interactions of the MLK-JNK signaling module with JIP. However, the exact functional outcome of this coordination remains to be defined. Recently, we established a role for MLK2 in stress-related apoptosis in neuronal cells. Thus, MLK2 activation of the JNK pathway is required for HN33 neuronal cell apoptosis in response to expression of glutamine-expanded huntingtin, the mutant product of the Huntington's disease gene (35Liu Y.F. Dorow D. Marshall J. J. Biol. Chem. 2000; 275: 19035-19040Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The SH3 domain of MLK2 binds normal huntingtin protein, and the polyglutamine expansion associated with Huntington's disease blocks the interaction. Expression of DN-MLK2 inhibits the apoptotic response to mutated huntingtin in these cells. As MLK2 expression is not limited to neuronal cells, it is likely to have functional roles in a number of biologically relevant settings. To identify such roles for MLK2 in mammalian cells, we created cell lines with inducible expression of GFP-tagged MLK2. We report here that MLK2 binds to clathrin heavy chain (CHC) and is enriched in clathrin-coated vesicles (CCVs) in cultured cells and in murine brain. In addition, overexpression of MLK2 in cultured cells inhibits endocytic function. Taken together with previous findings of MLK2 interactions with endocytic regulators (20Nagata K.-I. Puls A. Futter C. Aspenstrom P. Schaefer E. Nakata T. Hirokawa N. Hall A. EMBO J. 1998; 17: 149-158Crossref PubMed Scopus (233) Google Scholar, 21Rasmussen R.K., Ji, H. Eddes J.S. Moritz R.L. Reid G.E. Simpson R.J. Dorow D.S. Electrophoresis. 1998; 19: 809-817Crossref PubMed Scopus (20) Google Scholar, 22Savinainen A. Garcia E.P. Dorow D. Marshall J. Liu Y.F. J. Biol. Chem. 2001; 276: 11382-11386Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 23Rasmussen R.K. Rusak J. Price G. Robinson P.J. Simpson R.J. Dorow D.S. Biochem. J. 1998; 335: 119-124Crossref PubMed Scopus (23) Google Scholar) and involvement of MLK family members in JIP-kinesin complexes with microtubules (33Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (497) Google Scholar), the MLK2-clathrin interaction suggests a role for MLK2 at sites of clathrin activity in vesicle trafficking. Dulbecco's modified Eagle's medium (DMEM), leupeptin, pepstatin, and aprotinin were from ICN. Fetal calf serum was from the Commonwealth Serum Laboratories (Parkville, Australia) and gentamycin from David Bull Laboratories (Melbourne, Australia). Polyclonal anti-MLK2 antibodies were raised in sheep against a recombinant protein containing amino acids 1–100 of human MLK2 (11Dorow D.S. Devereux L., Tu, G.-F. Price G. Nicholl J.K. Sutherland G.R. Simpson R.J. Eur. J. Biochem. 1995; 234: 492-500Crossref PubMed Scopus (52) Google Scholar) and affinity-purified using a MLK2-(1–100)-glutathione S-transferase fusion protein bound to GSH beads. For immunoblotting, anti-MLK2 antibodies were used with biotinylated rabbit anti-goat immunoglobulin secondary antibodies from Dako Australia (Botany, New South Wales, Australia). Rabbit anti-GFP antibodies and Texas Red-conjugated transferrin were from Molecular Probes (Eugene, OR); mouse anti-CHC, anti-α-adaptin (AP-2), and anti-γ-adaptin (AP-1) antibodies were from Transduction Laboratories, and Texas Red-conjugated goat anti-mouse immunoglobulin antibodies were from Sigma. Biotinylated sheep anti-mouse and goat anti-rabbit immunoglobulin antibodies, streptavidin-biotin conjugated alkaline phosphatase, and Hybond C Super nitrocellulose membranes were from Amersham Biosciences. Alkaline phosphatase substrate reagent, 5-bromo-4-chloro-3-indolylphosphate, was from Roche Molecular Biochemicals, and nitro blue tetrazolium was from Sigma. FuGENETM 6 transfection reagent was also from Roche Molecular Biochemicals, and protein A-Sepharose was from AmershamBiosciences. Vectashield mounting medium was from Vector Laboratories (Burlingame, CA). cDNAs containing the complete human MLK2 protein-coding region (nucleotides 289–3454, including an in-frame stop codon beginning at nucleotide 3151 (11Dorow D.S. Devereux L., Tu, G.-F. Price G. Nicholl J.K. Sutherland G.R. Simpson R.J. Eur. J. Biochem. 1995; 234: 492-500Crossref PubMed Scopus (52) Google Scholar)), the N-terminal region (N-MLK2, nucleotides 289–1777), the C-terminal region (C-MLK2, nucleotides 1778–3454), and the C-terminal 321 nucleotides (C100-MLK2, nucleotides 2830–3154) were cloned into the vector pEGFPC1 (CLONTECH). For production of tetracycline-inducible cell lines, GFP-MLK2 was subcloned into vector pTRE (CLONTECH) and co-transfected with a modified Tet-ON (CLONTECH) vector (pEFpuropTet-ON (36Zhu H.J. Iaria J. Sizeland A.M. J. Biol. Chem. 1999; 274: 29220-29227Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar)) with tetracycline-inducible expression driven by the EF-1 promoter and a gene for puromycin resistance. The pEFpuropTet-ON vector was constructed by Dr. G. Vairo (Walter and Eliza Hall Institute, Melbourne, Australia) and was a gift from Dr. H.-J. Zhu (Ludwig Institute, Melbourne, Australia). Kinase-negative MLK2 mutant, K125A-MLK2 (DN-MLK2) was made using the Altered Sites® II in vitro mutagenesis kit (Promega) essentially according to the manufacturer's instructions, as described previously (19Phelan D.R. Loveland K.L. Devereux L. Dorow D.S. Mol. Reprod. Dev. 1998; 52: 135-140Crossref Scopus (13) Google Scholar). The clathrin box mutant, 932LDM934 ⇒ AAA C100A3-MLK2 was made by PCR-based mutagenesis. HEK-293 cells were maintained in DMEM supplemented with 10% fetal calf serum and 20 μg/ml (20 IU) gentamycin at 37 °C in 5% CO2 in air. For transient expression, cells (0.5–1 × 106) were transfected with 2–5 μg of plasmid DNA using FuGENE 6TM transfection reagent and lysed for assay 24 h later. For GFP-stable transfectants, cells were transfected with 5 μg of plasmid DNA and grown in the presence of 400 μg/ml G418 antibiotic. Clones of resistant cells were isolated and assayed by microscopy for expression of the GFP fusion protein and by immunoblotting with anti-GFP antibodies to confirm the size of the expressed protein. Tet-ON lines were made by co-transfecting pEFpuropTet-ON vector together with a 10-fold excess of GFP-MLK2 pTRE into HEK-293 cells, and the cells were grown in the presence of 2 μg/ml puromycin. Resistant cells were cloned, and individual clones were selected for expression of correctly sized GFP fusion proteins following induction with 2 μg/ml doxycycline. GFP-MLK2 was immunoprecipitated with anti-GFP antibodies from untreated or doxycycline-treated (2 μg/ml, 24 h) GFP-MLK2-Tet-ON cells using anti-GFP antibody. Precipitated proteins were separated by SDS-PAGE on a 4–20% acrylamide gradient gel (NOVEX) and visualized by fast Coomassie Blue staining (37Wong C. Sridhara S. Bardwell J.C.A. Jakob U. BioTechniques. 2000; 28: 426-432Crossref PubMed Scopus (107) Google Scholar). Protein bands were excised and digestedin situ with 0.5 μg of trypsin as described previously (38Moritz R.L. Simpson R.J. J. Chromatogr. 1992; 599: 119-130Crossref PubMed Scopus (56) Google Scholar, 39Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). An electrospray ion trap mass spectrometer (model LCQ; Finnigan, San Jose, CA) coupled on-line with a capillary high pressure liquid chromatography was used for peptide sequencing (38Moritz R.L. Simpson R.J. J. Chromatogr. 1992; 599: 119-130Crossref PubMed Scopus (56) Google Scholar, 39Moritz R.L. Eddes J.S. Reid G.E. Simpson R.J. Electrophoresis. 1996; 17: 907-917Crossref PubMed Scopus (92) Google Scholar). Electrospray ionization parameters are as follows: spray voltage, 4.5 kV; sheath and auxiliary gas flow rates, 30 and 5 arbitrary values, respectively; capillary temperature, 150 °C; capillary voltage, 20 V; tube lens offset, 16 V. The sheath liquid was 2-methoxyethanol (99.9% high pressure liquid chromatography grade) delivered at a flow rate of 3 μl/min. The electron multiplier was set to 860 V. The ion trap automatic gain control parameter was turned on for all experiments, and this, coupled with a maximum injection time of 200 ms, ensured that the number of ions in the trap was automatically maintained at a constant preset value. All data were collected in centroid mode using a “triple play” experiment. After acquisition of a full mass spectrometry (MS) scan (350–2000 Da) in the first scan event, the most intense ion present above a preset threshold of 1 × 105 counts was isolated. In the second and third scan events, a high resolution (Zoom) scan and a collision-induced dissociation tandem mass spectrometry (MS/MS) scan of the selected ion were performed. The collision energy for the MS/MS scan events was preset at a value of 55%. The sequences of uninterpreted collision-induced dissociation spectra were identified by correlation with peptide sequences present in a non-redundant protein sequence data base (comprising ∼849,000 entries) using the SEQUEST algorithm (version 27, revision 11) incorporated into the Finnigan-BioBrowserTM software (version 2.0). The SEQUEST search results were assessed by examination of the Xcorr (cross-correlation) and Cn (normalized correlation) scores. Manual inspection of each spectrum was performed to confirm the SEQUEST result. Cells were washed in phosphate-buffered saline and lysed in ice-cold lysis buffer (50 mm HEPES, pH 7.2, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm EGTA, 1.5 mmMgCl2, 50 mm NaPO4, 5 mm dithiothreitol with the following protease inhibitors: 1 mm phenylmethylsulfonyl fluoride and 1 μg/ml each aprotinin, leupeptin, and pepstatin). Lysates were incubated on ice for 10 min and then clarified by centrifugation at 13,000 rpm, 4 °C in a microcentrifuge. Clarified lysate protein concentrations were determined using the bicinchoninic acid assay (40Smith P.K. Krohn R.L. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fugimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18645) Google Scholar), and protein concentrations were equalized for immune precipitation or immunoblotting. For immune precipitation, primary antibodies were added 1 h prior to addition of protein A-Sepharose (or a mixture of protein A-Sepharose and protein G-Sepharose in the case of anti-clathrin immune precipitation), and the mixture was incubated overnight at 4 °C on a rotating wheel. Sepharose beads were washed three times in lysis buffer and resuspended in SDS-PAGE sample buffer. Immune precipitates or aliquots of cellular lysate containing 30–50 μg of protein were subjected to SDS-PAGE and transferred to nitrocellulose for immunoblotting. CCVs were isolated from cultured cells as described by Metzler et al.(67Metzler M. Legendre-Guillemin V. Gan L. Chopra V. Kwok A. McPherson P.S. Hayden M.R. J. Biol. Chem. 2001; 276: 39271-39276Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Briefly, 8 × 107 cells were homogenized in 3 ml of ice-cold buffer A (0.1 m MES, pH 6.5, 1 mm EGTA, 0.5 mm MgCl2 with protease inhibitors, as above) on ice using a glass Dounce homogenizer (10 strokes; fraction H). All subsequent steps were performed on ice or at 4 °C. The homogenate was clarified by centrifugation at 17,800 × g for 20 min, and the supernatant was collected and centrifuged at 56,100 × g for 1 h. The pellet from the second centrifugation was resuspended in 500 μl of buffer A, loaded onto buffer A containing 8% sucrose, and centrifuged (115,800 × g, 2 h). The supernatant was removed, and the final pellet containing CCVs was resuspended in 30 μl of buffer A (fraction SGP). Equal amounts of protein from SGP fractions were analyzed by SDS-PAGE. CCVs were isolated from mouse brain essentially following the procedure of Maycox et al. (41Maycox P.R. Link E. Reetz A. Morris S.A. Jahn R. J. Cell Biol. 1992; 118: 1379-1388Crossref PubMed Scopus (246) Google Scholar), with minor modifications. Briefly, 10 mouse brains were homogenized in 20 ml of cold buffer A using a glass homogenizer (10 strokes; fraction H) with subsequent steps on ice or at 4 °C. The homogenate was centrifuged at 20,000 × gfor 20 min. The pellet was resuspended in 5 ml of buffer A (fraction P1), and the supernatant (fraction S1) was centrifuged at 55,000 × g for 1 h. The supernatant was drained, and the pellet was resuspended in 1.2 ml of buffer A and homogenized as above (three strokes) followed by dispersion through a 26-gauge needle. The suspension (fraction P2) was then mixed with 1.2 ml of buffer A containing 12.5% Ficoll and 12.5% sucrose and centrifuged (40,000 × g, 40 min). The supernatant was diluted to 3.9 ml in buffer A and centrifuged (100,000 × g, 1 h) to pellet the vesicles. The supernatant was drained, and the pellet was resuspended in 1.0 ml of buffer A and dispersed through a 26-gauge needle. This suspension was cleared by centrifugation (20,000 × g, 20 min), and the pellet was resuspended in 100 μl of buffer A (fraction P4). The supernatant was layered on top of buffer A containing 8% sucrose and centrifuged for 2 h at 115,800 × g. The supernatant (fraction S4) was drained, and the final pellet was resuspended in 70 μl of buffer A (fraction SGP). Equal amounts of protein from fractions were analyzed by immunoblotting with anti-CHC, anti-MLK2, or anti-Myc tag (nonspecific) antibodies. Cells were plated onto poly-l-lysine-coated glass coverslips at a density of 1.5 × 106 cells/90-mm tissue culture plate, transfected as described above, and serum-starved overnight. Cells were then incubated with 25 μg/ml Texas Red-conjugated human transferrin in DMEM for 20 min, washed briefly in phosphate-buffered saline, and fixed for 10 min in 4% paraformaldehyde in PME buffer (100 mm Pipes, pH 6.9, 5 mm MgSO4, 10 mm EGTA, and 2 mm dithiothreitol). Cells with significant internal clustering of transferrin in recycling endosomes were counted using a Zeiss Axioskop 2 fluorescence microscope as follows: each coverslip was divided into quadrants, and 100 transfected cells (25 per quadrant) were evaluated for extent of transferrin clustering. Cells were scored as positive if significant clustering of transferrin was present and negative if only punctate distribution of red fluorescence was observed. Untransfected or transfected cells were counted on 6 independent coverslips (600 cells total) per expression construct. Although we (21Rasmussen R.K., Ji, H. Eddes J.S. Moritz R.L. Reid G.E. Simpson R.J. Dorow D.S. 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