Heterologous expression and purification of RhoGAP and RhoGEF

In preparation of activity assays and to test in silico gene structure predictions by the Broad Neurospora crassa Database, cDNA covering the full-length coding regions of most of the N. crassa RhoGAP and RhoGEF genes was amplified from total RNA. Detailed results of the ensuing cDNA sequencing in comparison with the two latest annotation versions of the database, .3 and .4, are summarized in Supplementary Table 1 (p.104). With few exceptions, own findings conform to annotation version .4; deviations from the predictions are not accompanied by changes in type or order of conserved domains within the proteins encoded by the cDNAs. Full-length cDNAs were used as templates to amplify regions encoding RhoGAP or classical/non.classical RhoGEF domains (the latter sometimes in combination with PH or BAR domains) for insertion into a derivative of the pMal-c2X expression vector (New England Biolabs, USA). From the resulting vectors, RhoGAP and RhoGEF constructs (Table 4) were expressed as N-terminally maltose binding protein (MBP)-tagged fusion proteins in E. coli cells and enriched by affinity purification using amylose resin (see Supplementary Figure 1, p.103 for an overview of a representative purification procedure).

Table 4: RhoGAP and RhoGEF constructs expressed as MBP fusion proteins. For convenience, amino acid (aa) positions in the table refer to the current annotation version .4 of the Broad Neurospora crassa Database. Note that the NCU09492 non-classical GEF domain constructs are based on DHR-2 (Dock Homology Region-2) sequence alignments presented in (Côté and Vuori, 2002, 2006) rather than the less extended Ded-cyto domain predictions by InterProScan Sequence Search (see section 4.10.1).

construct (MBP-…) region (aa positions)

As shown in Figure 6, all fusion proteins can be purified in a soluble state. Only little degradation is observed, and a high degree of purity is achieved, making the proteins suitable for in vitro activity assays. Identity of selected fusion proteins with Coomassie stained bands was verified by performing parallel Western Blot experiments using MBP-antiserum (data not shown).

Figure 6: MBP fusion proteins of RhoGAP (A) and RhoGEF (B) constructs are soluble and can be enriched to a high degree of purity by affinity purification. Coomassie stained SDS polyacrylamide gels loaded with equal volumes of eluate fractions of the indicated constructs (two each in case of the RhoGAP constructs) are shown. Predicted fusion protein molecular weights (MW) are given below the corresponding lanes.

A

B

60 5. Results 5.1.3 In vitro GEF activity assays

All RhoGEF constructs purified as described in the preceding section were tested for their ability to stimulate Rho GTPase nucleotide exchange activity in vitro. For this, the six N. crassa Rho GTPases were likewise purified as MBP fusion proteins (Figure 7 A), and their nucleotide exchange activity in the absence or presence of RhoGEF constructs (Figure 7 B) was assessed fluorospectrometrically employing mant-GDP (2'-(3')- O- (N'- methylanthraniloyl)-GDP). Presumably due to relief of quenching interactions existing in solution between the mant moiety and the nucleotide base, emission intensity of this fluorescent nucleotide analogue is enhanced two- to threefold upon binding to a protein, in this case the Rho GTPases (Scheidig et al., 1995; Jameson and Eccleston, 1997).

Fluorescence emission was monitored over time, and the mean linear slopes of the kinetic curves calculated to serve as a measure for intrinsic and GEF-stimulated nucleotide exchange activity.

Figure 7: (A) Result of a representative purification of MBP-Rho GTPase fusion proteins used in in vitro GEF activity assays. A Coomassie stained SDS polyacrylamide gel loaded with equal volumes of eluate fractions of the indicated constructs is shown. Predicted fusion protein molecular weights (MW) are given below the corresponding lanes. (B) Schematic representation of RhoGEF constructs tested as MBP fusion proteins in in vitro activity assays. Numbers indicate amino acid positions referring to the corresponding full-length protein (annotation version .4 of Broad Neurospora crassa Database).

Conserved domains predicted by InterProScan Sequence Search (see section 4.10.1) are depicted as boxes of varying shades of grey.

B

A

In the assays, all Rho GTPases exhibited intrinsic nucleotide exchange activity, although its extent differed considerably, with RHO1 and RHO2 displaying the lowest and RHO3 the highest exchange rates (cp. Supplementary Figures 2 to 5, p.106-109). While MBP alone had no effect on the exchange activity of any of the Rho GTPases (data not shown), several MBP-GEF fusion constructs clearly showed GEF activity towards specific Rho GTPases.

Diagrams in Figures 8 to 11 below summarize the results of the in vitro experiments for NCU00668, CDC24, BUD3 and NCU10282, respectively. Accompanying representative plots of fluorescence intensity over time as a measure for mant-GDP incorporation and thus nucleotide exchange activity for individual reaction samples can be found in Supplementary Figures 2 to 5 (p.106-109).

While NCU00668 functions as a RHO1-specific GEF (Figure 8), CDC24 stimulates nucleotide exchange in both RAC and CDC42 to a similar extent (Figure 9). RHO4 is the exclusive target GTPase of BUD3 (Figure 10), while NCU10282 strongly enhances nucleotide exchange in CDC42 (Figure 11 B). Note, however, that NCU10282 also increases the rate of nucleotide exchange in RAC, albeit more gradually, as evident in the curve progression when fluorescence emission intensity is plotted over time (Figure 11 A). Indeed, when calculated for a longer period of time (ca. 24 min), relative exchange activity of RAC in the presence of the GEF construct is 165±37 (mean±SD, [%]), as compared to an intrinsic activity of 100±14.

Although two different constructs each were tested (see Figure 7 B), no in vitro GEF activity was observed for NCU02764 and NCU09492 (data not shown). As GEF activity of RGF3 towards RHO4 was determined in parallel to this work (Justa-Schuch et al., 2010), the complete set of putative N. crassa RhoGEFs has now been analyzed with regard to in vitro specificity (see Figure 12 for a summary).

Figure 8: NCU00668 is a RHO1-specific GEF in vitro. Nucleotide exchange activity calculated as mean linear slope of fluorescence intensity over a period of ca. 24 minutes [arbitrary units/second] is displayed normalized to the intrinsic exchange activity (“no GEF”) of each Rho GTPase, which was set to 100% . n gives the number of independent experimental replicates, each of which was performed in technical duplicates. Error bars indicate standard deviation. See section 4.7.7 for details on data evaluation.

62 5. Results

Figure 9: CDC24 specifically stimulates nucleotide exchange activity of both RAC and CDC42 in vitro.

Nucleotide exchange activity calculated as mean linear slope of fluorescence intensity over a period of ca. 24 minutes [arbitrary units/second] is displayed normalized to the intrinsic exchange activity (“no GEF”) of each Rho GTPase, which was set to 100%. n gives the number of independent experimental replicates, each of which was performed at least in technical duplicates. Error bars indicate standard deviation. See section 4.7.7 for details on data evaluation.

Figure 10: BUD3 is a RHO4-specific GEF in vitro. Nucleotide exchange activity calculated as mean linear slope of fluorescence intensity over a period of ca. 8 minutes [arbitrary units/second] is displayed normalized to the intrinsic exchange activity (“no GEF”) of each Rho GTPase, which was set to 100%.

n gives the number of independent replicates, each of which was performed in technical duplicates. Error bars indicate standard deviation. See section 4.7.7 for details on data evaluation.

Figure 11: NCU10282 is a RhoGEF acting on CDC42 and, to a lesser extent, on RAC in vitro. (A) Curves of fluorescence emission intensity [a.u. =arbitrary units] plotted over time [min] for representative individual experiments testing the nucleotide exchange activity of RAC and CDC42 in the absence (“no GEF”) or presence (“+10282”) of MBP-10282GEFBAR. (B) Nucleotide exchange activity calculated as mean linear slope of fluorescence intensity over a period of ca. 8 minutes [arbitrary units/second] is displayed normalized to the intrinsic exchange activity (“no GEF”) of each Rho GTPase, which was set to 100%.

n gives the number of independent replicates, each of which was performed in technical duplicates. Error bars indicate standard deviation. See section 4.7.7 for details on data evaluation.

A

B

64 5. Results

Figure 12: Summary of RhoGEF specificities in S. cerevisiae (S.c.), S. pombe (S.p.) and N. crassa (N.c.).

Target GTPases newly determined in this study are highlighted by frames. Question marks denote N. crassa RhoGEFs showing no in vitro stimulatory activity under the assay conditions. See legend of Figure 5 for details on phylogenetic tree construction and references.