14-3-3 proteins inhibit autophagy by regulating SINAT-mediated proteolysis of ATG6 in Arabidopsis

Background: Autophagy is a conserved cellular process crucial for recycling cytoplasmic components and maintaining cellular homeostasis in eukaryotes. During Autophagy, the formation of a protein complex involving AUTOPHAGY-RELATED PROTEIN 6 (ATG6) and phosphatidylinositol 3-kinase is pivotal for recruiting proteins involved in phagophore expansion. However, the intricate molecular mechanism regulating this protein complex in Plants remains elusive.

Results: Here, we aimed to unravel the molecular regulation of Autophagy dynamics in Arabidopsis thaliana by investigating the involvement of the scaffold proteins 14-3-3λ and 14-3-3κ in regulating the proteolysis of ATG6. Phenotypic analyses revealed that 14-3-3λ and 14-3-3κ overexpression lines exhibited increased sensitivity to nutrient starvation, premature leaf senescence, and a decrease in starvation-induced autophagic vesicles, resembling the phenotypes of autophagy-defective mutants, suggesting the potential roles of 14-3-3 proteins in regulating Autophagy in Plants. Furthermore, our investigation unveiled the involvement of 14-3-3λ and 14-3-3κ in the RING finger E3 Ligase SINAT1-mediated ubiquitination and destabilization of ATG6 in vivo. We also observed repressed turnover of ATG6 and translocation of GFP-ATG6 to mCherry-ATG8a-labelled punctate structures in the autophagy-defective mutant, which suggesting that ATG6 is probably a target of Autophagy. Additionally, 14-3-3λ and 14-3-3κ interacted with Tumor necrosis factor Receptor Associated Factor 1a (TRAF1a) to promote the stability of TRAF1a in vivo under nutrient-rich conditions, suggesting a feedback regulation of Autophagy. These findings demonstrate that 14-3-3λ and 14-3-3κ serve as scaffold proteins to regulate Autophagy by facilitating the SINAT1-mediated proteolysis of ATG6, involving both direct and indirect mechanisms, in Plants.

Conclusions: 14-3-3 proteins regulate Autophagy by directly or indirectly binding to ATG6 and SINAT1 to promote ubiquitination and degradation of ATG6. 14-3-3 proteins are involved in modulating Autophagy dynamics by facilitating SINAT1-mediated ubiquitination and degradation of ATG6.