A pull-down procedure for the identification of unknown GEFs for small GTPases

Abstract

Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.

Keywords: GEF; enrichment; in vitro transcription/translation; nucleotide exchange factor; pull-down; small GTPase; yeast two-hybrid.

Figure 1.

Scheme for the specific enrichment and identification of unknown GEFs of small GTPases. (A) General reaction scheme of guanine nucleotide exchange factors for small GTPases. GEFs operate by transiting from a low affinity ternary GTPase:GXP:GEF intermediate to a high-affinity binary GTPase:GEF complex and back. In a first step, the GEF binds the GTPase:GXP complex with low affinity. GEF-mediated release of the nucleotide in the second step leads to a high affinity GEF:GTPase complex. In the reverse reaction, a different guanine nucleotide can bind, thereby completing the exchange reaction. (B) We envision to enrich the specific GEFs from cell lysates by exploiting the high-affinity of the intermediary nucleotide-free GTPase:GEF complex. The immobilized GTPase of interest is incubated with cell lysate containing its cognate GEF (green) among several other proteins (yellow and orange). Upon formation of a binary GTPase:GEF complex, the released nucleotide is degraded by exogenously added alkaline phosphatase to further stabilize the GTPase:GEF complex. After washing and removal of unbound proteins, the GEF can be specifically eluted by addition of GDP or GTP followed by subsequent identification via mass spectrometry. Since complex mixtures such as cell lysates may give rise to false positive targets, we designed the following procedures for GEF validation: I) An in vitro translation system coupled with an activity based GEF assay and II) a Y2H experiments with specifically designed GTPase mutants favoring GEF-interaction. The use of Gateway compatible vectors for both validation procedures greatly simplifies the cloning of different target proteins into expression vectors or vectors for Y2H experiments.

Figure 2.

Proteins that were specifically enriched in the pull-down experiments. (A) and (B) show the two independent experimental replicates of the pull-down experiments with each single experiment performed in triplicate. The average values of the label free quantification (LFQ) with their standard deviations are indicated in the graphs for all proteins that were significantly enriched in at least one triplicate (red: elution with 500 µM GTP; orange: control experiment not containing immobilized Sec4 and elution with 500 µM GTP; blue: elution with 10 mM GTP; cyan: control experiment not containing immobilized Sec4 and elution with 10 mM GTP; * indicates statistically relevant enrichment comparing the pull-down and the control experiment (t-test)). The insets show the same graphs with logarithmic scale of the LFQ intensities for better visualization of the differences between the experiments and the corresponding controls.

Figure 3.

Nucleotide exchange assay using proteins from cell free expression. Sec4:mantGDP (1 µM) was incubated with 200 µM GDP (step 1) without addition of cell-free expressed protein showing the intrinsic rate of nucleotide exchange (black curve) and after addition (step 2) of 20 µl cell-free expression mixture (red curve) as control experiments. These experiments show that the cell-free expression mixture does not contain factors that accelerate nucleotide exchange. Additionally, similar experiments were repeated after expression of the putative targets (green and blue curves: 10 µl and 20 µl cell free expression mixture after expression of Sec2, respectively; cyan: 20 µl cell free expression mixture after expression of Dss4). Observed rate constants are indicated for each curve.

Figure 4.

Results of the yeast two-hybrid screens with Sec4. (A) All 10 putative targets from the pull-down experiments were cloned into bait-vectors and tested for auto-activation against empty prey-vectors. In these experiments, only Mrs6 displayed auto-activation. (B) The Y2H experiments were repeated using the prey-vectors containing Sec4_WTΔC, Sec4_S34NΔC, Sec4_Q79LΔC or Sec4_D136NΔC to test for interaction with putative binding partners. Growth on the selective medium lacking histidine (-His) indicates an interaction of both known GEFs (Sec2 and Dss4) with Sec4_S34NΔC and Sec4_D136NΔC, but not with Sec4_WTΔC and Sec4_Q79LΔC. However the interaction is probably weak (as expected for an enzymatic interaction) and turnover of the chromogenic substrate X-α-Gal could only be observed for Dss4, but not Sec2 ((+) and (−): positive (pGBKT7–53/pGADT7-T) and negative (pGBKT7-Lam/pGADT7-T) controls).

Figure 5.

Results of the yeast two-hybrid screens with known Rab:GEF pairs. (A) The GEF domains of GRAB (aa 73–154 cloned into the vector pGADT7) and (B) Rabin8 (aa 153–237 in vector pGADT7) were tested for interaction with Rab8A (aa 1–203 in vector pGBKT7) and different mutants of Rab8 (Rab8_T22N, Rab8_Q67L, Rab8_N121I, Rab8_D124N, Rab8_{N121I, D124N}). (C) In a similar experiment, the GEF domain of the Legionella pneumophila GEF DrrA (aa 340–533) was tested for interaction with Rab1B (aa 1–199) and the corresponding mutants of Rab1B (Rab1_S22N, Rab1_Q67L, Rab1_N121I, Rab1_D124N, Rab1_{N121I, D124N}). All experiments were also performed with the corresponding empty vectors (−) to exclude auto-activation. (D) Shown are the positive (pGBKT7–53/pGADT7-T) and negative (pGBKT7-Lam/pGADT7-T) controls as well as the control with empty vectors (−/−).