Functional Mapping of ABC Transporters Protein Interaction Networks

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The human genome encodes for 48 ABC proteins that are classified into seven subfamilies (ABC A - G). Research from several laboratories has shown these ABC proteins to transport various endogenous substrates, including chloride ions, glutathione, cholesterol, LTC4, hemin, sterols and bile salts; however little is known about the mechanisms that regulate their functions. We believe that such regulatory mechanisms can be unmasked by identifying ABC transporters' interacting proteins. Using peptide-scanning approach to identify interacting proteins, we have shown that sequences in ABCB1 and ABCC1 (ABCB1/C1) linker domains (L1) bind soluble /-tubulin in cell extracts. More recently, in collaboration with Dr. Zhang's lab, we also demonstrated ABCC1-ATP synthase- interactions. The binding of ATP synthase- to ABCC1 linker domain was dramatically increased by L1 phosphorylation, while that to tubulin was abolished. We speculate that these interactions may modulate ABCB1/C1 subcellular localization or suggest a novel function for ABCC1 in regulating extracellular ATP level. The long-term objective of this program is to map the protein interaction networks of ABC transporters in an effort to understand the mechanisms that regulate their functions. We will begin by focusing on the identification and functional characterization of all interacting proteins to two well-characterized ABC transporters (i.e., ABCB1/C1). However, the same approach will be expanded to other ABC proteins. The specific aims of this application are to:***I. Identify all interacting proteins with ABCB1/C1 using various protein-interaction methods, beginning with peptide-scanning approach and mass-spectrometry. Confirm such interactions with pull-down assays using full-length and GST-fusion constructs. Compare ABCB1/C1 protein interactions using different tissue extracts.***II. In vivo validation of ABCB1/C1 protein interactions. Protein interactions will be validated using wild type and mutant variants of ABCB1/C1 interacting domains in vivo, using chemical/photo-crosslinking and confocal microscopy with antibodies to ABCB1/C1 and their respective interacting protein.***III. Assess the effects of protein interactions on ABCB1/C1 transport functions, subcellular localization, proteins half-lives, phosphorylation and the collateral sensitivity phenotype.***The completion of the above objectives should have an impact on diverse areas of studies given the role of ABC transporters in plant, yeast and parasite biology. We recognize that mapping the human ABC transporters interactome using non-computational approaches is an ambitious undertaking, however the use of high-throughput parallel peptide scanning synthesis with mass-spectrometry should facilitate this undertaking. In addition, knowledge gained from this program should enhance the predictive power of computational approaches.