Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains.
Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.
Wang
Pijun
P
Department of Medicine, Division of Hepatology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Hata
Soichiro
S
Xiao
Yansen
Y
Murray
John W
JW
Wolkoff
Allan W
AW
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006367:HeLa Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D029362:Organic Anion Transporters, Sodium-Independent / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D013448:Sulfobromophthalein / Q000378:metabolism; D013462:Sulfur Radioisotopes / Q000378:metabolism
D029362:Organic Anion Transporters, Sodium-Independent; D020033:Protein Isoforms; C421102:Slco1a1 protein, rat; D013462:Sulfur Radioisotopes; D013448:Sulfobromophthalein; D001216:Asparagine