Biochemical Characterization of Nucleotide and Protein Interactions of Human Multidrug Resistance Protein 1 (MRP1/ABCC1)
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Multidrug resistance protein 1 (MRP1) is an integral membrane protein belonging to the ATP-binding cassette (ABC) superfamily that utilizes ATP binding and hydrolysis to transport various endogenous substrates and/or xenobiotics across membranes against a concentration gradient. The overall goal of my research was to examine the nucleotide and protein interactions of MRP1 using various biochemical methods. In the first study, Cu2+(Ph)3 which promotes cross-linking of two nearby Cys residues and limited proteolysis were used to study conformational changes of MRP1 at different stages of ATP binding and hydrolysis at the nucleotide binding domains (NBDs). The limited trypsin digestion patterns indicated that some Cys residues of MRP1 could be cross-linked in the nucleotide-free state and that the Cys cross-linked MRP1 was more susceptible to trypsinolysis. Furthermore, binding of ATP, AMP-PNP, and trapping of ADP by MRP1 prevented the cross-linking events from occurring, but binding of ATPγS did not. However, the ATPγS-bound MRP1, like nucleotide-free MRP1, showed enhanced sensitivity towards trypsinolysis. These studies show that the two ATP analogs, AMP-PNP and ATPγS, interact with MRP1 in different ways. In the second study, the interaction of MRP1 with other cellular proteins was examined. An in vivo chemical cross-linking approach combined with affinity purification and MS analysis was initially used to identify protein partners directly interacting with MRP1. When this approach proved unsuccessful, a second approach involving immunoaffinity purification of MRP1-containing complexes followed by MS analysis was adopted. Six potential candidate interacting protein partners of MRP1 were identified via this approach and two of them, FUS and drebrin, were further characterized by co-immunoprecipitation and colocalization experiments. FUS seems unlikely to be an important binding partner of MRP1 since confocal and subcellular fractionation studies showed it to be exclusively localized in the nucleus. On the other hand, drebrin depletion by siRNA knock-down resulted in a moderate decrease in MRP1 overall expression levels although the membrane localization of MRP1 remained unchanged.