Delivery of n-Rosella to the vacuole of nitrogen-starved wild type cells.

(A) Wild type (BY4741) cells expressing n-Rosella were subjected to nitrogen starvation and then sampled at selected time points (0, 6, 24, 48 and 72 hours after commencement of nitrogen starvation). Accumulation of diffuse red fluorescence in the vacuole was taken as evidence of nucleophagy. (B) The percentage of cells showing accumulation of diffuse red fluorescence in the vacuole at various time points is shown. (C) Cells expressing n-Rosella were starved in SD(-N) medium for 0, 3, 6, 9, 12, 15, 18, 21, 24, 48 and 72 hours, and the increase in processed degradation product (free GFP) levels was monitored by immunoblotting as described in Materials and Methods. Cytosolic PGK was detected as a loading control. *indicates the presumptive degradation product of n-Rosella lacking the NAB35 nuclear targeting signal. (D) Staining with Hoechst 33258 was performed to confirm the targeting of n-Rosella (red and green fluorescence) to the nucleus 24 hours after commencement of nitrogen starvation. (E) Staining with the vacuolar lumen dye, CMAC-Arg was performed to confirm the delivery of n-Rosella (diffuse red fluorescence) to the vacuole (24 hours after commencement of nitrogen starvation).