Time of day-dependent sorting of the vesicular glutamate transporter to the plasma membrane.
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 lumen-facing 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.
Darna
Mahesh
M
AG Functional Cell Biology, Institute for Integrative Neuroanatomy, Charité Centre 2 for Basic Medicine, 10115 Berlin, Germany.
Schmutz
Isabelle
I
Richter
Karin
K
Yelamanchili
Sowmya V
SV
Pendyala
Gurudutt
G
Höltje
Markus
M
Albrecht
Urs
U
Ahnert-Hilger
Gudrun
G
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D002940:Circadian Rhythm / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000378:metabolism*; D050857:Synaptotagmins / Q000378:metabolism; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism*; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000378:metabolism; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism
D008840:Microfilament Proteins; C493465:Slc17a7 protein, mouse; C493466:Slc17a7 protein, rat; C118623:Slc32a1 protein, rat; C503968:Synpo protein, mouse; C503969:Synpo protein, rat; D050598:Vesicular Glutamate Transport Protein 1; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; C492244:Viaat protein, mouse; D050857:Synaptotagmins; D018698:Glutamic Acid; D020692:rab3 GTP-Binding Proteins