Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong Provincial Key Laboratory of Agricultural & Rural Pollution Abatement and Environmental Safety, College of Natural Resources and Environment, South China Agricultural University, Guangzhou, 510642, China.
Guangdong Provincial Key Laboratory of Microbial Signals and Disease Control, Integrate Microbiology Research Center, College of Plant Protection, South China Agricultural University, Guangzhou, 510642, China.
BMC Plant Biol. 2024 Nov 29;24(1):1148. doi: 10.1186/s12870-024-05854-3.
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.
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.
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.
自噬是真核生物中一种重要的细胞内过程，对于回收细胞质成分和维持细胞内稳态至关重要。在自噬过程中，涉及自噬相关蛋白 6（ATG6）和磷脂酰肌醇 3-激酶的蛋白质复合物的形成对于招募参与噬泡扩张的蛋白质至关重要。然而，植物中调节该蛋白质复合物的复杂分子机制仍不清楚。
在这里，我们旨在通过研究支架蛋白 14-3-3λ和 14-3-3κ在调节 ATG6 蛋白水解中的作用，揭示拟南芥自噬动力学的分子调控。表型分析表明，14-3-3λ和 14-3-3κ过表达系表现出对营养饥饿、过早叶片衰老和饥饿诱导的自噬小泡减少的敏感性增加，类似于自噬缺陷突变体的表型，表明 14-3-3 蛋白在植物中调节自噬的潜在作用。此外，我们的研究揭示了 14-3-3λ和 14-3-3κ参与 RING 指 E3 连接酶 SINAT1 介导的 ATG6 在体内的泛素化和不稳定性。我们还观察到自噬缺陷突变体中 ATG6 的周转率降低和 GFP-ATG6 易位到 mCherry-ATG8a 标记的点状结构中，这表明 ATG6 可能是自噬的靶标。此外，14-3-3λ和 14-3-3κ与肿瘤坏死因子受体相关因子 1a（TRAF1a）相互作用，以促进营养丰富条件下体内 TRAF1a 的稳定性，表明自噬存在反馈调节。这些发现表明，14-3-3λ和 14-3-3κ作为支架蛋白，通过促进 SINAT1 介导的 ATG6 蛋白水解，以直接和间接的方式，在植物中调节自噬。
14-3-3 蛋白通过直接或间接与 ATG6 和 SINAT1 结合，促进 ATG6 的泛素化和降解，从而调节自噬。14-3-3 蛋白通过促进 SINAT1 介导的 ATG6 泛素化和降解，参与调节自噬动力学。
