FIGURE 6.

The effect of amino acid withdrawal and insulin on the [³²P]guanyl nucleotide content of stably expressed RagC^WT and RagC^S75L heterodimeric complexes in ³²P_i-labeled HeLa cells. A, B, and C, replicate plates of HeLa cells stably expressing GFP-streptag, GFP-streptag-RagC^WT, or GFP-streptag-RagC^S75L were incubated in P_i-free DMEM containing ³²P_i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P_i-free medium (AA+) or P_i-free medium lacking amino acids (AA-), each containing ³²P_i (0.2 mCi/ml), for another 2 h. In A and B, insulin (1.0 μm) was added to some of the cells in DMEM (AA+/I) 30 min before harvest. The nucleotides bound to Strep-Tactin (strept) pull-downs were extracted and separated by TLC on PEI cellulose. One set of ³²P -labeled HeLa cells expressing FP-streptag, treated as described above for AA-, AA+, and AA+/I, were rinsed, extracted directly into acetonitrile, and then the solubilized total nucleotides were separated by TLC. The ³²P comigrating with GDP and GTP was quantitated by phosphorimaging. After subtraction of the averaged values found in the GFP-streptag lanes, the percentage of [³²P]GTP was calculated as [[³²P]GTP / (1.5 × [³²P]GDP + [³²P]GTP)], and the averaged values are shown. In A, the top and center panels on the right show GFP immunoblot analyses of the cell lysates and representative Strep-Tactin pull-downs (corresponding to lanes 1, 4, and 7 of each set). The bottom panel shows an immunoblot analysis of S6K(T389P) corresponding to lanes 1, 4, and 7 of each set. In B, the top panel on the right shows a GFP immunoblot analysis of the lysates, whereas the center and bottom panels show lysate immunoblot analyses for 4E-BP(T37P/T46P) and PRAS40(S246P), respectively. In C, one set of ³²P-labeled HeLa cells expressing GFP-streptag, treated as in Fig. 6, were rinsed, extracted directly into HClO₄ (0.3 m, 0 °C, HClO₄ extract). The HClO₄ supernatants were neutralized with KHCO₃, and the [³²P]guanyl nucleotides were quantified as above. In addition, the nucleotides in the lysate after pull-down of the Strep-Tactin beads were also analyzed. Immunoblot analyses of the lysates and representative Strep-Tactin pull-downs are shown in the right panel. Ori, origin; std, guanyl nucleotide standards; Ins, insulin; ACN, acetonitrile; Ct, C-terminal.