Subcellular localization of the neuronal glycine transporter GLYT2 in brainstem.
The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.
Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa" (UAM-CSIC). Universidad Autónoma de Madrid, Spain.
Enrique
E
Núñez
Gonzalo
G
Pérez-Siles
Lara
L
Rodenstein
Pablo
P
Alonso-Torres
Francisco
F
Zafra
Esperanza
E
Jiménez
Carmen
C
Aragón
Beatriz
B
López-Corcuera
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001933:Brain Stem / Q000166:cytology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D015415:Biomarkers; D005998:Glycine; D050487:Glycine Plasma Membrane Transport Proteins; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C493269:Slc6a5 protein, rat; C530052:Rab8a protein, rat