engleski

A pipeline for testing interaction partners of small GTPases in Dictyostelium

Small GTPases from the Ras superfamily are monomeric GTP-hydrolyzing proteins which act as molecular switches, regulating a variety of cellular processes such as cell motility, proliferation, endocytosis, cytokinesis, membrane trafficking etc., by alternating between active (GTP-bound) and inactive (GDP- bound) forms. Since many of them have mutually related domain architecture, regulation and function, it is important to identify specific interactions between a given GTPase and its regulating and effector proteins reliably. In recent years, our group investigated the roles of Rac and Ras proteins and their regulators and effectors in amoeba Dictyostelium discoideum. When analyzing interactions and functions of a GTPase, it is crucial to distinguish between its active and inactive forms, since usually only the active form is conveying biological activity of the protein. We therefore introduced a pipeline of assays to determine specific interactions between candidate proteins or protein domains and a panel of small GTPases. Initially, we test potential interactions using yeast two-hybrid (Y2H) screen using constitutively active and inactive GTPase mutants as preys. Next, positive interactions are further tested biochemically using GST pull-down assay, where potential interactions are verified in relation to the activation status of a GTPase. As a final step, the apparent interactions are examined in vivo, using fluorescence-based proximity assays which enable discrimination between direct and indirect interactions, such as Fluorescence Resonance Energy Transfer (FRET) measurements realized by sensitized emission of the acceptor or Fluorescence Lifetime Imaging Microscopy (FLIM), and Bimolecular Fluorescence Complementation (BiFC). We will present the results provided by application of this pipeline on two examples: development of probes for tracking the intracellular dynamics of active Rac1 proteins, and discovery of a specific interaction between IQGAP-related protein IqgC active RasG. We will provide a comparison of results obtained by equivalent methods in these studies and especially point out certain subtle disparities between the results of different assays. Taken together, results obtained by using this set of methods provide robust data for evaluating the interactions, and their nature, between small GTPases and their potential interaction partners.

small GTPases ; protein interaction ; Dictyostelium

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