Time of day-dependent sorting of the vesicular glutamate transporter to the plasma membrane

Mahesh Darna, Isabelle Schmutz, Karin Richter, Sowmya V Yelamanchili, Gurudutt N Pendyala, Markus Höltje, Urs Albrecht, Gudrun Ahnert-Hilger

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)4300-4307
Number of pages8
JournalJournal of Biological Chemistry
Volume284
Issue number7
DOIs
StatePublished - Feb 13 2009

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Vesicular Glutamate Transport Proteins
Cell membranes
Sorting
Cell Membrane
Synaptotagmins
Synaptophysin
Digestion
Synaptic Vesicles
Membranes
Neurotransmitter Agents
Pronase
Membrane Fusion
Synaptosomes
Darkness
Photoperiod
Extracellular Space
Sucrose
Rats
Glutamic Acid
Assays

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Time of day-dependent sorting of the vesicular glutamate transporter to the plasma membrane. / Darna, Mahesh; Schmutz, Isabelle; Richter, Karin; Yelamanchili, Sowmya V; Pendyala, Gurudutt N; Höltje, Markus; Albrecht, Urs; Ahnert-Hilger, Gudrun.

In: Journal of Biological Chemistry, Vol. 284, No. 7, 13.02.2009, p. 4300-4307.

Research output: Contribution to journalArticle

Darna, Mahesh ; Schmutz, Isabelle ; Richter, Karin ; Yelamanchili, Sowmya V ; Pendyala, Gurudutt N ; Höltje, Markus ; Albrecht, Urs ; Ahnert-Hilger, Gudrun. / Time of day-dependent sorting of the vesicular glutamate transporter to the plasma membrane. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 7. pp. 4300-4307.
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