Baiying Li, Fangfang Niu, Yonglun Zeng, Man Kei Tse, Cesi Deng, Liu Hong, Shengyu Gao, Sze Wan Lo, Wenhan Cao, Shuxian Huang, Yasin Dagdas, and Liwen Jiang

PNAS January 25, 2023; 120 (5) e2208351120

Significance

Plants mobilize the endomembrane system for restoring homeostasis when stimulated by stresses. Though the function of ufmylation in ER stress and autophagy has been described in mammals, their detailed roles in plant development and stress responses remain elusive. In this study, we identified Ufl1 as an ATG8 interactor in Arabidopsis and demonstrated that Ufl1 maintains ER homeostasis upon salt stress via regulating ER-phagy by mechanistic connection with the autophagy-related (ATG) machinery. Our study thus sheds light on the pleiotropic functions of the ufmylation cascade in plants under abiotic stresses and underpins the cross-talk among ER stress, salt stress, and autophagy.

Abstract

In plants, the endomembrane system is tightly regulated in response to environmental stresses for maintaining cellular homeostasis. Autophagosomes, the double membrane organelles forming upon nutrient deprivation or stress induction, degrade bulky cytosolic materials for nutrient turnover. Though abiotic stresses have been reported to induce plant autophagy, few receptors or regulators for selective autophagy have been characterized for specific stresses. Here, we have applied immunoprecipitation followed by tandem mass spectrometry using the autophagosome marker protein ATG8 as bait and have identified the E3 ligase of the ufmylation system Ufl1 as a bona fide ATG8 interactor under salt stress. Notably, core components in the ufmylation cascade, Ufl1 and Ufm1, interact with the autophagy kinase complexes proteins ATG1 and ATG6. Cellular and genetic analysis showed that Ufl1 is important for endoplasmic reticulum (ER)-phagy under persisting salt stress. Loss-of-function mutants of Ufl1 display a salt stress hypersensitive phenotype and abnormal ER morphology. Prolonged ER stress responses are detected in ufl1 mutants that phenocopy the autophagy dysfunction atg5 mutants. Consistently, expression of ufmylation cascade components is up-regulated by salt stress. Taken together, our study demonstrates the role of ufmylation in regulating ER homeostasis under salt stress through ER-phagy.

See https://www.pnas.org/doi/10.1073/pnas.2208351120

Figure 1: Identification of Ufl1 as an ATG8e interactor under salt stress condition. (A) Purified recombinant GST-ATG8e was used as bait for IP from Arabidopsis PSB-D cell lysate upon salt stress treatment. Proteins pulled-out by GST-ATG8e were separated by SDS-PAGE, and silver-stained protein bands were subjected to tandem MS analysis for subsequent protein identification. The Right panel lists promising hits in the GST-ATG8 pull down group. The label of boxes in red, blue, and black on the gel represents the portion of gel cut where candidates in the lists labeled by the same color were identified. (B) Ufl1-YFP colocalized with mCherry-ATG8e in punctate upon their co-expression in Arabidopsis protoplasts. (Scale bar, 10 μm.) (C) CNX-RFP-ATG8e recruited Ufl1-YFP to the ER upon their co-expression in Arabidopsis protoplasts. (Scale bar, 10 μm.) (D) FRET analysis of ATG8e-CFP with Ufl1-YFP in Arabidopsis protoplasts. (Scale bar, 10 μm.) **P < 0.01, ***P < 0.001. (E) Co-immunoprecipitation (Co-IP) analysis of 3HA-Ufl1 with YFP-ATG8e in Arabidopsis protoplasts with or without salt stress treatment. YFP-ATG8e or free YFP was transiently co-expressed with 3HA-Ufl1, followed by cell lysis and IP using GFP-trap magnetic beads for subsequent western blot analysis. (F) Quantification analysis of the ratio of co-immunoprecipitated Ufl1 by ATG8e shown in (E). Means ± SD; n = 5 individual experiments, two-tailed unpaired t test, *P < 0.05. (G) Y2H analysis between ATG8e and Ufl1 in serial dilution. Transformants were screened on synthetic drop-out medium lacking Trp and Leu (SD-2). Positive colonies were then grown on SD medium lacking His, Trp, and Leu (SD-3) with 1 mM 3AT to inhibit self-activation.