Figure 7.
Leep2 complex promotes GTP hydrolysis of RasB, RasD, and RasG. (A) GAPs interact preferentially with their target GTPases during the transition state of GTP hydrolysis. Top: Schematic view of GAP-stimulated GTP hydrolysis. Bottom: GAP-GTPase interaction during the transition state of GTP hydrolysis (left) or in the presence of GDP and AlFx (right). (B) Proteomic identification of the substrates of Leep2. The Leep2 complex was immunoprecipitated from cell lysates of DKO cells expressing GFP-Leep2A and RFP-Leep2B, supplemented with or without GDP and AlFx, using GFP-trap. The bound proteins were analyzed by mass spectrometry. The abundance ratio was determined by dividing the abundance from AlFx-containing samples by that from AlFx-free samples. (C) Pull-down of Leep2 complex from cell lysates using GST or GST-fused RasB, RasD, and RasG immobilized on beads. The cell lysates were obtained from WT cells expressing Flag-Leep2A and GFP-Leep2B, supplemented with or without GDP and AlFx. Samples were probed with Flag antibody. Coomassie bright blue-stained gel at the bottom shows purified GST and GST-fusion proteins. (D) Pull-down of Leep2 complex from cell lysates using GST or GST-fused Ras subfamily GTPases immobilized on beads. The cell lysates were obtained from WT cells expressing GFP-Leep2A and RFP-Leep2B, supplemented with GDP and AlFx. Samples were probed with GFP antibody. Coomassie bright blue-stained gel at the bottom shows purified GST and GST-fusion proteins. (E) Pull-down of Leep2 components from cell lysates using GST, GST-RasB, GST-RasD, or GST-RasG. The cell lysates were obtained from DKO cells coexpressing GFP-Leep2A and RFP-Leep2B or DKO cells expressing GFP-Leep2A or GFP-Leep2B, supplemented with GDP and AlFx. Samples were probed with GFP or RFP antibody. Coomassie bright blue-stained gel at the bottom shows purified GST and GST-fusion proteins. (F) Luminescence-based GAP assay: Purified recombinant GST, GST-RasB, GST-RasD, or GST-RasG immobilized on beads were incubated with immunopurified complexes of Flag-Leep2A and GFP-Leep2B, Flag-Leep2AN1474K and GFP-Leep2B, as well as Flag-Leep2A, or Flag control. The luminescence of GST incubated with the Flag control was set to 1, and other values were normalized correspondingly (means ± SD, n = 3). AU, arbitrary unit. Source data are available for this figure: SourceData F7.

Leep2 complex promotes GTP hydrolysis of RasB, RasD, and RasG. (A) GAPs interact preferentially with their target GTPases during the transition state of GTP hydrolysis. Top: Schematic view of GAP-stimulated GTP hydrolysis. Bottom: GAP-GTPase interaction during the transition state of GTP hydrolysis (left) or in the presence of GDP and AlFx (right). (B) Proteomic identification of the substrates of Leep2. The Leep2 complex was immunoprecipitated from cell lysates of DKO cells expressing GFP-Leep2A and RFP-Leep2B, supplemented with or without GDP and AlFx, using GFP-trap. The bound proteins were analyzed by mass spectrometry. The abundance ratio was determined by dividing the abundance from AlFx-containing samples by that from AlFx-free samples. (C) Pull-down of Leep2 complex from cell lysates using GST or GST-fused RasB, RasD, and RasG immobilized on beads. The cell lysates were obtained from WT cells expressing Flag-Leep2A and GFP-Leep2B, supplemented with or without GDP and AlFx. Samples were probed with Flag antibody. Coomassie bright blue-stained gel at the bottom shows purified GST and GST-fusion proteins. (D) Pull-down of Leep2 complex from cell lysates using GST or GST-fused Ras subfamily GTPases immobilized on beads. The cell lysates were obtained from WT cells expressing GFP-Leep2A and RFP-Leep2B, supplemented with GDP and AlFx. Samples were probed with GFP antibody. Coomassie bright blue-stained gel at the bottom shows purified GST and GST-fusion proteins. (E) Pull-down of Leep2 components from cell lysates using GST, GST-RasB, GST-RasD, or GST-RasG. The cell lysates were obtained from DKO cells coexpressing GFP-Leep2A and RFP-Leep2B or DKO cells expressing GFP-Leep2A or GFP-Leep2B, supplemented with GDP and AlFx. Samples were probed with GFP or RFP antibody. Coomassie bright blue-stained gel at the bottom shows purified GST and GST-fusion proteins. (F) Luminescence-based GAP assay: Purified recombinant GST, GST-RasB, GST-RasD, or GST-RasG immobilized on beads were incubated with immunopurified complexes of Flag-Leep2A and GFP-Leep2B, Flag-Leep2AN1474K and GFP-Leep2B, as well as Flag-Leep2A, or Flag control. The luminescence of GST incubated with the Flag control was set to 1, and other values were normalized correspondingly (means ± SD, n = 3). AU, arbitrary unit. Source data are available for this figure: SourceData F7.

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