Identification and characterisation of the RalA-ERp57 interaction: evidence for GDI activity of ERp57.

RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro.

Protein-protein interactions identified by pull-down experiments and mass spectrometry.

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.

The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein.

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.