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. 2017 Dec 6;208(2):705–716. doi: 10.1534/genetics.117.300538

Figure 1.

Figure 1

Drosophila Syd-1 interacts with all six Rho GTPases. Its predicted GAP domain enhances the GTPase activities of Rac1 and Cdc42 but not RhoA. (A) Western blot (probed with anti-FLAG antibodies) of protein pulled down by purified GST-Rho proteins. FLAG-tagged full-length wild-type fly Syd-1 expressed ubiquitously in adult head is pulled down by GST-tagged constitutively-active mutant forms of fly Rac1, Rac2, Mtl, RhoA, RhoL, or Cdc42, but not by GST alone. (B–D) Endpoint GTPase activity assays of wild-type fly Rac1 (B), Cdc42 (C), and RhoA (D). Each protein was assayed alone and in the presence of the negative control GST, the positive control p50 RhoGAP, the GAP domain of wild-type fly Syd-1 (Syd-1wt GAP), or the GAP domain of fly Syd-1 in which the conserved arginine of the arginine finger is replaced by alanine (Syd-1RA GAP). Activity is presented as fold-increase over the activity of each protein alone. Error bars represent SEM. n, Independent assays. As expected, GST does not increase the GTPase activity of Rac1 (1.00 ± 0.16, n = 7), Cdc42 (1.00 ± 0.09, n = 3), or RhoA (1.00 ± 0.22, n = 3), whereas p50 RhoGAP significantly increases the GTPase activity of all three (Rac1: 9.36 ± 1.16, n = 7, P = 5.9 × 10−6; Cdc42: 9.12 ± 0.22, n = 3, P = 2.2 × 10−6; RhoA: 13.32 ± 3.04, n = 3, P = 0.0077). We found that the wild-type GAP domain of Syd-1 significantly increases the GTPase activity of Rac1 (8.69 ± 1.29, n = 7, P = 3.5 × 10−5) and Cdc42 (7.83 ± 0.27, n = 3, P = 9.0 × 10−6) but not RhoA (1.48 ± 0.30, n = 3, P = 0.13). By contrast, the Syd-1RA mutant GAP domain has a severely reduced ability to increase the GTPase activity of Rac1 (1.98 ± 0.31, n = 7, P = 1.4 × 104), and Cdc42 (1.41 ± 0.08, n = 3, P = 1.1 × 10−5) and has no effect on RhoA (1.43 ± 0.18, n = 3, P = 0.10). * P < 0.05, ** P < 0.01, *** P < 0.001, based on one-tailed t-tests. (E) Like wild-type fly Syd-1 (Syd-1wt), FLAG-tagged full-length R979A mutant fly Syd-1 (Syd-1RA) expressed ubiquitously in adult head is pulled down by GST-tagged constitutively active mutant forms of Rac1, RhoA, and Cdc42 but not by GST alone.