TY - JOUR
T1 - Time of day-dependent sorting of the vesicular glutamate transporter to the plasma membrane
AU - Darna, Mahesh
AU - Schmutz, Isabelle
AU - Richter, Karin
AU - Yelamanchili, Sowmya V.
AU - Pendyala, Gurudutt
AU - Höltje, Markus
AU - Albrecht, Urs
AU - Ahnert-Hilger, Gudrun
PY - 2009/2/13
Y1 - 2009/2/13
N2 - Neurotransmitters are concentrated into synaptic vesicles by VGLUT (vesicular glutamate transporter) or VGAT (vesicular GABA transporter). The number of VGLUTs per vesicle determines the amount of stored neurotransmitter, thereby influencing postsynaptic response. Recently, we described a strong diurnal cycling of the amount of VGLUT1 on synaptic vesicles prepared from whole mouse brain at different times of the day (Yelamanchili, S. V., Pendyala, G., Brunk, I., Darna, M., Albrecht, U., and Ahnert-Hilger, G. (2006) J. Biol. Chem. 281, 15671-15679). To analyze whether and how much VGLUT resides in cellular versus vesicular membranes, we developed a Pronase assay. We found that VGLUT and synaptotagmin are highly accessible to proteolytic cleavage in rat and mouse synaptosomal preparations, indicating considerable amounts of these vesicular proteins at the plasma membrane, whereas only minor amounts of synaptophysin and Rab3 are digested. Sucrose stimulation increases digestion of VGLUT, synaptotagmin, and synaptophysin due to membrane fusion that exposes the lumenfacing peptides to the extracellular space. Digestion of mouse synaptosomes prepared at different times of the day revealed a diurnal cycling of VGLUT to the plasma membrane. More VGLUT is digested at noon (Zeitgeber time 6) compared with the start of the light period (Zeitgeber time 0), whereas digestion of synaptophysin and synaptotagmin is independent of diurnal cycling. In contrast to VGLUT, the amount of VGAT appears not to vary diurnally but is decreased in membrane preparations from animals kept under constant darkness. We conclude that VGLUTs are sorted diurnally to the plasma membrane to modulate glutamate transmission during a day/ night cycle, whereas VGAT expression is not oscillating but is increased in the presence of a light/dark cycle.
AB - Neurotransmitters are concentrated into synaptic vesicles by VGLUT (vesicular glutamate transporter) or VGAT (vesicular GABA transporter). The number of VGLUTs per vesicle determines the amount of stored neurotransmitter, thereby influencing postsynaptic response. Recently, we described a strong diurnal cycling of the amount of VGLUT1 on synaptic vesicles prepared from whole mouse brain at different times of the day (Yelamanchili, S. V., Pendyala, G., Brunk, I., Darna, M., Albrecht, U., and Ahnert-Hilger, G. (2006) J. Biol. Chem. 281, 15671-15679). To analyze whether and how much VGLUT resides in cellular versus vesicular membranes, we developed a Pronase assay. We found that VGLUT and synaptotagmin are highly accessible to proteolytic cleavage in rat and mouse synaptosomal preparations, indicating considerable amounts of these vesicular proteins at the plasma membrane, whereas only minor amounts of synaptophysin and Rab3 are digested. Sucrose stimulation increases digestion of VGLUT, synaptotagmin, and synaptophysin due to membrane fusion that exposes the lumenfacing peptides to the extracellular space. Digestion of mouse synaptosomes prepared at different times of the day revealed a diurnal cycling of VGLUT to the plasma membrane. More VGLUT is digested at noon (Zeitgeber time 6) compared with the start of the light period (Zeitgeber time 0), whereas digestion of synaptophysin and synaptotagmin is independent of diurnal cycling. In contrast to VGLUT, the amount of VGAT appears not to vary diurnally but is decreased in membrane preparations from animals kept under constant darkness. We conclude that VGLUTs are sorted diurnally to the plasma membrane to modulate glutamate transmission during a day/ night cycle, whereas VGAT expression is not oscillating but is increased in the presence of a light/dark cycle.
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U2 - 10.1074/jbc.M805480200
DO - 10.1074/jbc.M805480200
M3 - Article
C2 - 19103593
AN - SCOPUS:63249113373
VL - 284
SP - 4300
EP - 4307
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 7
ER -