FIGURE 4: Carbon source dependent down regulation of Rnr1 is dependent on oxidative phosphorylation and the conserved ATP/dATP binding site. (A) WT cells transformed with a GFP-ATG8 plasmid was grown to mid log phase in leucine drop out medium supplemented with 2% glucose (SD-LEU; “D”). The cells were collected and transferred to leucine drop out medium supplemented with 2% glycerol (SGLEU; “G”) or SD-LUE2 supplemented with rapamycin (200 ng/ml; “Rap”). WCE samples were prepared from the glycerol culture at 24- and 48-hours after the medium switch or 2 hours in rapamycin, and subjected Western blot analysis using α-Rnr1 and α-GFP antibodies. Tubulin was used as a loading control. (B, C) An atg1Δ – and atg16Δ– strains transformed with the GFP-ATG8 plasmid were subjected to the analysis described in panel (A). (D) Strains of the indicated genotypes (Table S1) were grown to mid-log phase in YPD (“D”) or YPG (“G”). WCE samples were prepared and subjected to Western blot analysis using α-Rnr1 antibodies. Tubulin was used as a loading control. (E) WT and rnr1-D57N cells were grown to mid log phase in YPD (“D”). Cells were collected and released into YPG (“G”) for further incubation. Left hand panel: Western blot analysis using α-Rnr1 antibodies. Tubulin was used as a loading control. Right hand side: The Rnr1 signal in each lane was normalized to the tubulin signal in the same lane. The value obtained at each time point was expressed as a fraction of the value at t=0. (F) WT, gut2Δ, and ndi1Δ strains were cultured in YPD (“D”), YPG (“G”), and YPD supplemented with HU (10 mM) or MMS (0.01%). WCE samples in YPD were from cells in mid log phase. The YPG samples were prepared at 20 and 48 hours after medium switch to YPG. The HU and MMS samples were prepared following 2 hour exposure to the respective chemical. WCE samples were subjected to Western blot analysis using α-GFP antibodies. Tubulin was used as a loading control.