Figure 4.

mTOR-raptor complex formation during G₁ progression. (A) Western blot analysis of G₁ cells sorted by mitochondrial membrane potential demonstrates no significant change in absolute levels of mTOR, raptor, or the activation by phosphorylation of S6K. (B) Formation of mTOR-raptor complexes increases from early to late G₁. Equal amounts of protein lysate were immunoprecipitated (IP) for mTOR and the amount of coprecipitated raptor assessed by Western blotting (WB). Quantifica- ion is from three separate experiments. (C) Determination of oxygen consumption in cells isolated so as to be in early G₁ (ΔΨ_mL). Oxygen consumption was measured for cells that were maintained in serum free conditions or for an equal number of cells stimulated for 18 hours with serum in the presence or absence of rapamycin. (D) Mitochondrial membrane potential determines the efficacy of rapamycin-induced G₁ delay. Cell cycle analysis of G₁ cells sorted for either high or low membrane potential. Cells still remaining in G₁ were determined as percentage of BrdU-negative cells 18 hours after serum stimulation in the presence or absence of rapamycin (mean ± SD, n = 3). (E) Model of the G₁ phase. The concerted rise of mitochondrial oxidative capacity and formation of mTOR-raptor complex during G₁ progression may play a role for the passage of a putative metabolic checkpoint between early and late G₁.