Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole

Abstract

Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1’s function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants.

The phosphatidylinositol 3-phosphate effector FYVE3 regulates FYVE2-dependent autophagy in Arabidopsis thaliana

“…The phenotypic consequence of fyve3 mutation is not evident unless it is combined with fyve2 mutation, which prevented us and others from identifying its involvement in autophagy ( Kim et al., 2022 ; Zhao et al., 2022 ). In our previous work ( Kim et al., 2022 ), we employed 48-h nitrogen starvation for autophagic flux assay, whereas 24-h starvation was used for the assay in this study.…”

“…Nevertheless, we observed a similar suppression in the autophagic flux analysis in which 21-d-old leaves of fyve2 fyve3 mutants were used ( Kim et al., 2022 ). Zhao et al. (2022) reported that the steady-state level of the autophagic cargo receptor NBR1 was comparable between fyve2 and fyve2 fyve3 , but did not directly assess the autophagic flux of fyve2 fyve3 (i.e., cfs1 cfs2 ) double mutants.…”

“…For example, SNX1 ( Pourcher et al., 2010 ) and SNX2b ( Phan et al., 2008 ) localize to endosomes, and FYVE4 ( Liu et al., 2021 ) is primarily cytosolic but can be recruited to endosomal membranes. FYVE1/FREE1 ( Gao et al., 2014 ; Gao et al., 2015 ), ATG18A ( Zhuang et al., 2017 ; Kang et al., 2018 ; Kim et al., 2022 ) and FYVE2/CFS1 (Cell death-related endosomal FYVE/SYLF protein 1) ( Sutipatanasomboon et al., 2017 ; Kim et al., 2022 ; Zhao et al., 2022 ) localize at both endosomes and autophagosomes. Genetic analyses of mutants defective in the PI3P-binding proteins indicated that these proteins are involved in endosomal sorting (e.g., SNX1, SNX2b, FYVE4), autophagic degradation (e.g., ATG18A, FYVE2/CFS1), or both (e.g., FYVE1/FREE1).…”

“…Both FYVE2/CFS1 and FYVE3/CFS2 proteins harbor the PI3P-binding FYVE domain and C-terminal SYLF domain, which assists yeast SYLF-containing proteins to bind actin filaments and phosphoinositides ( Robertson et al., 2009 ; Urbanek et al., 2015 ; Sutipatanasomboon et al., 2017 ) ( Figure S1A ). FYVE2/CFS1 facilitates the efficient progression of autophagy and interacts with a variety of proteins such as ATG18A, SAR1B, and VPS23A, which are important for autophagosome biogenesis, COPII vesicle trafficking, and membrane scission, respectively ( Sutipatanasomboon et al., 2017 ; Kim et al., 2022 ; Zhao et al., 2022 ). By contrast, no apparent defect in autophagic flux was observed in its paralogous fyve3/cfs2 single mutants ( Kim et al., 2022 ; Zhao et al., 2022 ).…”

“…FYVE2/CFS1 facilitates the efficient progression of autophagy and interacts with a variety of proteins such as ATG18A, SAR1B, and VPS23A, which are important for autophagosome biogenesis, COPII vesicle trafficking, and membrane scission, respectively ( Sutipatanasomboon et al., 2017 ; Kim et al., 2022 ; Zhao et al., 2022 ). By contrast, no apparent defect in autophagic flux was observed in its paralogous fyve3/cfs2 single mutants ( Kim et al., 2022 ; Zhao et al., 2022 ). Thus, currently no evidence supports the involvement of FYVE3/CFS2 in autophagy and other membrane trafficking routes.…”