title: A rat homologue of CED-6 is expressed in neurons and interacts with clathrin.
abstract: We isolated from a brain library a cDNA encoding an isoform of rat CED-6 that has not been previously described. This transcript results from alternative splicing of the ced-6 gene present on chromosome 9. We expressed this isoform as his-tagged protein in E. coli and used the purified protein to raise antibodies to investigate the expression of CED-6 in rat brain. Immunoblot analysis showed the presence of CED-6 as a doublet of approximately 34 and 33 kDa in cortex, hippocampus and cerebellum, indicating that the protein was present in different regions of the brain. Subcellular fractionation experiments showed that CED-6 immunoreactivity did not concentrate in GFAP-containing glial vesicles, whereas it showed a distribution similar to the synaptotagmin in synaptosomes-enriched fractions, suggesting that CED-6 is present in neurons. CED-6 immunoreactivity was also investigated using immunohistochemistry analysis and it was found in several brain regions, being particularly strong in the cell body of some groups of neurons such as Purkinje cell layer of cerebellum, and pyramidal cells of the hippocampal formation and also in epithelial cells from the choroid plexus. Importantly, CED-6 immunoreactivity colocalized with a neuronal marker but not with a glial marker. Considering that several PTB-containing proteins bind clathrin, we investigated whether rat CED-6 would also have this property. Yeast two-hybrid and GST pull-down analysis indicated that ratCED-6 interacts with clathrin and in cultured cells we detected colocalization between CED-6 and clathrin-coated vesicles. The present findings suggest that CED-6 may have a role in endocytic trafficking or signaling in neurons.
authors.affiliation: Departamento de Farmacologia, Universidade Federal de Minas Gerais, Brazil.
authors.forename: Cristina
authors.initials: C
authors.lastname: Martins-Silva
authors.forename: Lucimar Teodoro
authors.initials: LT
authors.lastname: Ferreira
authors.forename: Michel
authors.initials: M
authors.lastname: Cyr
authors.forename: Janaina
authors.initials: J
authors.lastname: Koenen
authors.forename: Diane Ramires
authors.initials: DR
authors.lastname: Fernandes
authors.forename: Natalia Rodrigues
authors.initials: NR
authors.lastname: Carvalho
authors.forename: Caroline Batista Franco
authors.initials: CB
authors.lastname: Ribeiro
authors.forename: Sébastien
authors.initials: S
authors.lastname: Marion
authors.forename: Carlos
authors.initials: C
authors.lastname: Chavez-Olortegui
authors.forename: Marco Antonio Maximo
authors.initials: MA
authors.lastname: Prado
authors.forename: Vania Ferreira
authors.initials: VF
authors.lastname: Prado
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D002831:Choroid Plexus / Q000378:metabolism / Q000648:ultrastructure; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles; D004705:Endocytosis / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D021381:Protein Transport / Q000502:physiology; D011817:Rabbits; D051381:Rats; D009435:Synaptic Transmission / Q000502:physiology; D013574:Synaptosomes / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D051017:Apoptosis Regulatory Proteins; C112888:CED-6 protein, C elegans; D029742:Caenorhabditis elegans Proteins; D002966:Clathrin; C516481:Gulp1 protein, rat; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins