A rat homologue of CED-6 is expressed in neurons and interacts with clathrin.
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.
Martins-Silva
Cristina
C
Departamento de Farmacologia, Universidade Federal de Minas Gerais, Brazil.
Ferreira
Lucimar Teodoro
LT
Cyr
Michel
M
Koenen
Janaina
J
Fernandes
Diane Ramires
DR
Carvalho
Natalia Rodrigues
NR
Ribeiro
Caroline Batista Franco
CB
Marion
Sébastien
S
Chavez-Olortegui
Carlos
C
Prado
Marco Antonio Maximo
MA
Prado
Vania Ferreira
VF
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
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