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. Author manuscript; available in PMC: 2013 Jul 31.
Published in final edited form as: Science. 2013 May 31;340(6136):1100–1106. doi: 10.1126/science.1232044

Figure 4.

Figure 4

GATOR1 is a GTPase activating protein complex for RagA and RagB. (A)–(D) GATOR1 stimulates GTP hydrolysis by RagA and RagB. RagA-RagCX, RagB-RagCX, RagC-RagBX or the control GTPase Rap2a were loaded with [α-32P] GTP and incubated with GATOR1 (20 pmols) or the control Leucyl tRNA synthetase (LRS) (20 pmols). GTP hydrolysis was determined by thin layer chromatography (see methods). Each value represents the normalized mean ± SD (n = 3). (E) GATOR1 increases GTP hydrolysis by RagB in a time dependent manner. RagB-RagCX was loaded with [γ-32P]GTP, incubated with GATOR1 or a control and hydrolysis was determined by phosphate capture (see methods) Each value represents the normalized mean ± SD (n = 3). (F) GATOR1 preferentially interacts with the dominant active Rag heterodimer. In vitro binding assay in which FLAG-GATOR1 was incubated with immobilized HA-GST-tagged RagBT54N-RagCQ120L (dominant negative), RagBQ99L-RagCS75N (dominant active) or Rap2a. HA-GST precipitates were analyzed by immunoblotting for the levels of FLAG-GATOR1.