Localization, topology, and substrate specificity of the human ATP -binding cassette half-transporter ABCG2

Ndeye Khady Diop, Purdue University

Abstract

The human ATP-binding cassette half-transporter ABCG2 is a 72 kDa glycoprotein that confers multidrug resistance to cells in culture when overexpressed. Our lab has investigated the localization, topology, and substrate specificity of ABCG2. While in most tumors, ABCG2 is located in the membrane, additional cytoplasmic staining is also observed. To determine the exact subcellular localization of ABCG2, we fused a green fluorescent protein (GFP) on either terminus of ABCG2 and monitored expression in vivo using our vaccinia virus transient system. Confocal microscopy analysis of the GFP fusion proteins indicates expression in the cytoplasm and the plasma membrane. Hydropathy analysis of the amino acid sequence of ABCG2 reveals three potential N-linked glycosylation sites at amino acids 418, 557 and 596. Site-directed mutagenesis experiments, in which each Asn was changed to Gin independently, revealed that only asparagine 596 is glycosylated. We compared expression levels, catalytic and drug transport properties of the glycosylation deficient protein to its glycosylated counterpart and concluded that glycosylation is not essential for the expression, trafficking to the plasma membrane, nor the overall function of ABCG2. Using the glycosylation deficient mutant as a template, we introduced fourteen glycosylation consensus sites into the predicted intracellular and extracellular loops of ABCG2 and tested their ability to undergo glycosylation. Our data provide evidence that G577N, F586S, and A606T are located in the extracellular space. The distance constraint between oligosaccharyl transferase and the glycosylation site allowed us to map residues 565 through 620 to the extracellular space. Furthermore, we introduced FLAG tags at either terminus of ABCG2 and found these sequences to be cytosolic. We now have a clearer picture of ABCG2 in the membrane. ABCG2 variants, cloned from several drug resistant cells, differ in their substrate transport phenotype due to a mutation at position 482. To study the function of residue 482, we changed the wild type arginine 482 to the nineteen other standard amino acids. Although the mutants differ in transport and ATPase activities, they specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but not in substrate binding.

Degree

Ph.D.

Advisors

Hrycyna, Purdue University.

Subject Area

Biochemistry

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