Fig. 5: CtNbr1 mediates vacuolar targeting of CtAms1 via conventional autophagy machinery in S. pombe.

a CtNbr1 interacts with SpAtg8 in Pil1 co-tethering assay. SpAtg8(1−115), which lacks the G116 residue required for lipidation, was fused to Pil1-mCherry and co-expressed with mECitrine-tagged CtNbr1 in S. pombe. Pil1-mCherry served as a negative control. Cells were observed by fluorescence microscopy. Bar, 3 μm. b CtNbr1 can enter the vacuole through autophagy in nitrogen-starved S. pombe cells. CtNbr1-mCherry was expressed in wild-type and atg5Δ cells. SpCpy1-Venus was used as a vacuole lumen marker. Cells were collected before (0 h) and 8 h after nitrogen starvation (-N) and examined by fluorescence microscopy. Bar, 3 μm. c CtNbr1 can promote the vacuolar entry of CtAms1 in nitrogen-starved S. pombe cells in an autophagy-dependent manner. C-terminally mCherry-tagged CtAms1, CtAms1-G2A, and CtAms1-G3A were individually co-expressed with untagged CtNbr1 in wild-type S. pombe cells. CtAms1-mCherry alone expressed in wild-type cells served as a negative control. CtAms1-mCherry was also co-expressed with untagged CtNbr1 in atg5Δ cells. Cells were collected before (0 h) and 3 h after nitrogen starvation and were examined by fluorescence microscopy. Bar, 3 μm. The data shown are representative of two independent experiments with similar results. Source data are provided as a Source Data file.