Stable polarized expression of hCAT-1 in an epithelial cell line.
Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-[3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-[3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization.
Cariappa
R
R
Department of Pediatrics, Washington University School of Medicine, and St. Louis Children's Hospital, One Children's Place, St. Louis, MO 63110, USA.
Heath-Monnig
E
E
Furesz
T C
TC
Kamath
S G
SG
Smith
C H
CH
D000818:Animals; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D017136:Ion Transport / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D018613:Microscopy, Confocal; D009865:Oocytes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism*; D014162:Transfection; D014981:Xenopus
D027201:Cationic Amino Acid Transporter 1; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C420825:SLC7A1 protein, human; D049452:Green Fluorescent Proteins; D008239:Lysine