Membrane topology and glycosylation of the human multidrug resistance-associated protein.
The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.
Bakos E
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.
Hegedüs
T
T
Holló
Z
Z
Welker
E
E
Tusnády
G E
GE
Zaman
G J
GJ
Flens
M J
MJ
Váradi
A
A
Sarkadi
B
B
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D006031:Glycosylation; D018922:HL-60 Cells; D006801:Humans; D011487:Protein Conformation; D018411:Spodoptera
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004317:Doxorubicin