Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.
Mutations in either ATP-binding cassette (ABC) G5 or ABCG8 cause sitosterolemia, an autosomal recessive disorder of sterol trafficking. To determine the site of action of ABCG5 and ABCG8, we expressed recombinant, epitope-tagged mouse ABCG5 and ABCG8 in cultured cells. Both ABCG5 and ABCG8 underwent N-linked glycosylation. When either protein was expressed individually in cells, the N-linked sugars remained sensitive to Endoglycosidase H (Endo H). When ABCG5 and ABCG8 were coexpressed, the attached sugars were Endo H-resistant and neuraminidase-sensitive, indicating that the proteins were transported to the trans-Golgi complex. The mature, glycosylated forms of ABCG5 and ABCG8 coimmunoprecipitated, consistent with heterodimerization of these two proteins. The Endo H-sensitive forms of ABCG5 and ABCG8 were confined to the endoplasmic reticulum (ER), whereas the mature forms were present in non-ER fractions in cultured hepatocytes. Immunoelectron microscopy revealed ABCG5 and ABCG8 on the plasma membrane of these cells. In polarized WIF-B cells, recombinant ABCG5 localized to the apical (canalicular) membrane when coexpressed with ABCG8, but not when expressed alone. To our knowledge this is the first direct demonstration that trafficking of an ABC half-transporter to the cell surface requires the presence of its dimerization partner.
McDermott Center for Human Growth and Development, Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9046, USA.
Gregory A
GA
Graf
Wei-Ping
WP
Li
Robert D
RD
Gerard
Ingrid
I
Gelissen
Ann
A
White
Jonathan C
JC
Cohen
Helen H
HH
Hobbs
D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D000939:Epitopes; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D022781:Hepatocytes / Q000378:metabolism; D008074:Lipoproteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
C462600:ABCG5 protein, mouse; C462601:ABCG8 protein, mouse; D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters; D000939:Epitopes; D008074:Lipoproteins; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D006026:Glycoside Hydrolases