Transport of endo-β-mannosidase AtEBM from the cytoplasm to the vacuole through the autophagy pathway in <i>Arabidopsis</i>

Macroautophagy/autophagy is a conserved eukaryotic intracellular degradation pathway, with specialized forms such as the biosynthetic cytoplasm-to-vacuole targeting (Cvt) pathway in yeast, which selectively delivers several resident hydrolase precursors to the vacuole rather than for turnover. Although the core autophagy machinery is highly conserved between yeast and plants, whether autophagy plays a biosynthetic role in transporting vacuolar hydrolases in plants has remained unclear. Here, we show that Arabidopsis thaliana AtEBM (endo-β-mannosidase), a glycosidase involved in the degradation of vacuolar N-glycans, is selectively transported to the vacuole via autophagy. Further analyses revealed that AtEBM directly interacts with the core autophagy protein ATG8 through an ATG8-family interacting motif (AIM), and this interaction is essential for sequestering AtEBM into phagophores. Notably, the AIM is conserved among EBM orthologs across the green plant lineage. Phenotypic analyses of CRISPR-Cas9-generated atebm mutants revealed no defects in canonical autophagy markers (such as early senescence and nutrient starvation hypersensitivity); instead, the mutants exhibit reduced fecundity. Collectively, our findings demonstrate that AtEBM is targeted to the vacuole through a selective biosynthetic autophagy, which is functionally similar to, but molecularly distinct from the yeast Cvt pathway.Abbreviations: AIM: ATG8-family interacting motif; Ams1: α-mannosidase; Ape1: aminopeptidase I; ATG: autophagy related; Co-IP: co-immunoprecipitation; ConA: concanamycin A; Prc1/CPY: proteinase C; Cvt cytoplasm‑to‑vacuole targeting; DMSO: dimethyl sulfoxide; EE: early endosome; EBM: endo-β-mannosidase; ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; FUC1: α1,3-fucosidase; GFP: green fluorescent protein; GST: glutathione S-transferase; LCI: luciferase complementation imaging; LDS: LIR-docking site; LIR: LC3-interacting region; LUC: luciferase; mAIM: mutated AIM; MS: Murashige and Skoog; MVB: multivesicular body; NBR1: next to BRCA1 gene 1; NVT: NBR1-mediated vacuolar targeting pathway; RFP: red fluorescent protein; TGN: trans-Golgi network; VPE: vacuolar processing enzyme; WT: wild-type; Y2H: yeast two-hybrid;