Transmembrane topology of vesicular glutamate transporter 2.
Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling in neurons and some peripheral tissues through vesicular storage of L-glutamate in secretory vesicles. To investigate the topology of VGLUT in membranes, we prepared site-directed antibodies against the amino-terminal (anti-N), 1st putative loop (anti-L), and carboxyl terminal (anti-C) regions. None of the antibodies reacted with VGLUT2 expressed in COS cells because they could not gain access to the antigen. However, both the anti-N and anti-C antibodies recognized VGLUT2 when the cells were permeabilized with digitonin, while the anti-L antibodies did not. Immunological reactivity to anti-L-antibodies appeared when the cells were permeabilized with Triton X-100. These results suggest that both the amino-terminal and carboxyl-terminal regions of VGLUT2 in membranes face the cytoplasm while the 1st loop faces the lumen.
Jung
Sun-Kyung
SK
Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan.
Morimoto
Riyo
R
Otsuka
Masato
M
Omote
Hiroshi
H
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000276:immunology; D018698:Glutamic Acid / Q000378:metabolism; D007150:Immunohistochemistry; D008566:Membranes / Q000378:metabolism; D050599:Vesicular Glutamate Transport Protein 2 / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D050599:Vesicular Glutamate Transport Protein 2; D018698:Glutamic Acid; D004247:DNA