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. 2013 Dec 11;289(5):2658–2674. doi: 10.1074/jbc.M113.528505

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FIGURE 3. — Rag heterodimers lack detectable GTPase activity in vitro. A, the RagB/C wild-type heterodimer does not hydrolyze [γ-³²P]GTP in vitro. GST-Ha-Ras^WT (▵), GST-Ha-Ras^G12V (▴), and GST-RagB^WT/FLAG-RagC^WT (●) were charged with [γ-³²P]GTP and examined for intrinsic GTPase activity. The amount of unhydrolysed [γ-³²P]GTP on proteins was measured at intervals thereafter by nitrocellulose filter binding assay. B, a recombinant catalytic fragment of NF1 stimulates GTP hydrolysis by Ha-Ras^WT but not Ha-Ras^G12V or RagB^WT/C^WT. GST-Ha-Ras^WT (●, dashed line), GST-Ha-Ras^G12V (○, dashed line), or GST-RagB^WT/FLAG-RagC^WT (●, solid line) were charged with [γ-³²P]GTP, and a catalytic fragment of NF1 was added. The release of ³²P_i at 30 °C was measured at intervals thereafter by the charcoal method. C, neither RagB wild-type or RagC wild-type within a heterodimer hydrolyze [α-³²P]GTP detectably in vitro. GST and various GST-RagB/FLAG-RagC heterodimers were charged with [α-³²P]GTP, and at intervals thereafter, nucleotides were extracted and separated on TLC on PEI cellulose. D, extracts from HEK293T cells do not promote the ability of RagB/C to hydrolyze [γ-³²P]GTP. HEK293T cells were lysed in the absence of detergent (solid line) or with CHAPS (0.3%, dotted line), TritonX-100 (1%, short dashes), or RIPA buffer (Pierce) (long dashes). GST-Ha-Ras^WT (triangles) and GST-RagB^WT/FLAG-RagC^WT (circles) charged with [γ-³²P]GTP were mixed with each of these extracts at 30 °C, and the amount of protein-bound [γ-³²P]GTP was measured by filtration through nitrocellulose filters. E, MonoQ anion exchange chromatography of a HEK293T cell extract contains GAP activity toward Ha-Ras wild-type, not RagB/C wild-type. HEK293T cells were extracted by freezing and thawing and separated by anion exchange chromatography. Each fraction was incubated at 30 °C with GST-Ha-Ras^WT (▵, dashed lines), GST-Ha-Ras^G12V (▴, dashed lines), and GST-RagB^WT/FLAG-RagC^WT, each charged with [γ-³²P]GTP. After 30 min, protein-bound [γ-³²P]GTP was measured as in C.