Identification of endophilins 1 and 3 as selective binding partners for VGLUT1 and their co-localization in neocortical glutamatergic synapses: implications for vesicular glutamate transporter trafficking and excitatory vesicle formation.
1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.
De Gois
Stephanie
S
Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Jeanclos
Elisabeth
E
Morris
Marie
M
Grewal
Sukhjeevan
S
Varoqui
Helene
H
Erickson
Jeffrey D
JD
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018698:Glutamic Acid / Q000378:metabolism; D008954:Models, Biological; D019579:Neocortex / Q000201:enzymology* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000201:enzymology* / Q000378:metabolism; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D050598:Vesicular Glutamate Transport Protein 1; C519036:endophilin 3, rat; D018698:Glutamic Acid; D000217:Acyltransferases; C018764:2-acylglycerophosphate acyltransferase