The aim of this unit is to provide a method for the identification of new protein‐protein interactions. Pull‐down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein‐protein interactions. When coupled with mass spectrometry, pull‐downs can be considered as the protein‐based equivalent of a yeast two‐hybrid screen. To improve the isolation of specific binding partners, pull‐down methods are described involving the use of cross‐linking, large‐scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state‐dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry‐based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in‐gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.
P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.
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