Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region.
The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.
Westlake
Christopher J
CJ
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Payen
Lea
L
Gao
Mian
M
Cole
Susan P C
SP
Deeley
Roger G
RG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003062:Codon; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004742:Enhancer Elements, Genetic; D006868:Hydrolysis; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D017997:Leukotriene C4 / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013997:Time Factors; D014638:Vanadates / Q000737:chemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003062:Codon; D017997:Leukotriene C4; D014638:Vanadates; D000255:Adenosine Triphosphate