Figure 3.

Loss of Ncr1 does not impair protein trafficking to the yeast vacuole. (A) ALP-AP-3 pathway: wild-type (wt), ncr1∆, and yck3∆ cells carrying a plasmid expressing a GFP-tagged Nyv1-Snc1 fusion protein grown to exponential phase were incubated with 0.024 mM FM4-64 (vacuolar membrane probe) for 1 h and observed by fluorescence microscopy. (B) CPY pathway: cells were grown to exponential phase and proteins were analyzed by immunoblotting, using anti-CPY antibody. Bands corresponding to the proenzyme (pCPY) or the mature enzyme (mCPY) are indicated. Pgk1 was used as loading control. Cells lacking Pep4, which is involved in the proteolytic activation of pCPY to mCPY, were used as a negative control. As a positive control, wild-type cells were treated with rapamycin (200 ng mL⁻¹) for 4 h (wt + Rap). The original figure is shown in Supplementary Figure S4. (C) Cvt pathway: cells were grown to exponential (log) and post-diauxic shift [(PDS), 48 h after log] phases, and the processing of pApeI to mApeI was analyzed by immunoblotting, using anti-ApeI antibody. Pgk1 was used as loading control. Cells lacking Pep4, which is involved in the proteolytic activation of pApeI to mApeI, were used as control. The original figure is shown in Supplementary Figure S5. Representative images of at least three independent experiments are shown.