Neither amino nor carboxyl termini are required for function of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain.
Antibodies were raised against synthetic peptides corresponding to several regions of the rat brain gamma-aminobutyric acid (GABA) transporter. According to our model, this glycoprotein has 12 transmembrane alpha-helices with both amino and carboxyl termini located in the cytoplasm. The antibodies recognized the intact transporter on Western blots. Upon papain treatment, a reconstitutively active transporter can be isolated upon lectin chromatography (Kanner, B. I., Keynan, S., and Radian, R. (1989) Biochemistry 28, 3722-3728). The papainized transporter runs on sodium dodecyl sulfate-polyacrylamide gels as a broad band with an apparent molecular mass between about 58 and 68 kDa as compared to 80 kDa for the untreated transporter. The transporter fragment was recognized by all the antibodies, except for that raised against the amino terminus. Pronase cleaves the transporter to a relatively sharp 60-kDa band, which reacts with the antibodies against the internal loops but not with either the amino- or the carboxyl-terminal. This pronase-treated transporter, upon isolation by lectin chromatography, was reconstituted. It exhibits full GABA transport activity. This activity exhibits the same features as the intact system including an absolute dependence on sodium and chloride as well as electrogenicity. We conclude that the amino- and carboxyl-terminal parts of the transporter, possibly including transmembrane alpha-helices 1, 2, and 12, are not required for the transport function.
Mabjeesh
N J
NJ
Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Kanner
B I
BI
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D027361:Organic Anion Transporters; D011402:Pronase; D011487:Protein Conformation; D051381:Rats; D012964:Sodium / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D005680:gamma-Aminobutyric Acid; D012964:Sodium; D011402:Pronase