RESUMEN
The endoplasmic reticulum (ER) is crucial for maintaining cell homeostasis because it is the primary site for synthesizing secreted and transmembrane proteins and lipids. The unfolded protein response (UPR) is activated to restore ER homeostasis under ER stress. However, the relationship between lipids and the ER stress response in plants is not well understood. Arabidopsis Golgi anti-apoptotic proteins (GAAPs) are involved in resisting ER stress. To elucidate the function of GAAPs, PASTICCINO2 (PAS2), involved in very long-chain fatty acid (VLCFA) synthesis, was found to interact with GAAPs and IRE1. Single pas2 and gaap1/gaap2pas2 double mutants exhibited increased seedling damage and impaired UPR response under chronic ER stress. Site mutation combined with genetic analysis revealed that the role of PAS2 in resisting ER stress depended on its VLCFA synthesis domain. VLCFA contents were upregulated under ER stress, which required GAAPs. Exogenous VLCFAs partially restored the defect in UPR upregulation caused by PAS2 or GAAP mutations under chronic ER stress. These findings demonstrate that the association of PAS2 with GAAPs confers plant resistance to ER stress by regulating VLCFA synthesis and the UPR. This provides a basis for further studies on the connection between lipids and cell fate decisions under stress.
RESUMEN
Maintaining proteostasis in the endoplasmic reticulum (ER) is critical for cell viability and plant survival under adverse conditions. The unfolded protein response (UPR) pathways interact with reactive oxygen species (ROS) to precisely trigger adaptive outputs or cell death under ER stress with varying degrees. However, little information is known about the relationship between UPR signalling and ROS regulation. Here, Arabidopsis GOLGI ANTI-APOPTOTIC PROTEIN1 (GAAP1)-GAAP4 were found to play redundant positive roles under ER stress. Genetic analysis showed that GAAP4 played a role in INOSITOL-REQUIRING ENZYME (IRE1)-dependent and -independent pathways. In addition, GAAPs played negative roles to activate the adaptive UPR under conditions of stress. Quantitative biochemical analysis showed that mutations in GAAP genes decreased the oxidised glutathione content and altered the pattern of ROS and glutathione in early ER stress. When plants were challenged with unmitigated ER stress, mutations in GAAP advanced ROS accumulation, which was associated with a decline in adaptive UPR. These data indicated that GAAPs resist cell death by regulating glutathione content to inhibit ROS accumulation and maintain UPR during ER stress. They provide a basis for further analysis of the regulation of cell fate decision under ER stress.
Asunto(s)
Arabidopsis , Estrés del Retículo Endoplásmico , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Arabidopsis/metabolismo , Muerte Celular , Estrés del Retículo Endoplásmico/fisiología , Glutatión/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Cell viability requires the maintenance of intracellular homeostasis through the unfolded protein response mediated by receptors localized on the endoplasmic reticulum (ER) membrane. The receptor IRE1 mediates not only various adaptive outputs but also programmed cell death (PCD) under varying stress levels. However, little is known about the mechanism by which the same receptors trigger different responses in plants. Arabidopsis Golgi anti-apoptotic protein 1 (GAAP1) and GAAP3 resist PCD upon ER stress and negatively modulate the adaptive response of the IRE1-bZIP60 pathway through IRE1 association. To elucidate the mechanism underlying the anti-PCD activity of GAAPs, we attempted to isolate interactors of GAAPs by yeast two-hybrid screening. Membrane-associated progesterone receptor 3 (MAPR3) was isolated as one of the factors interacting with GAAP. Mutations in GAAP1/GAAP3 and/or MAPR3 enhanced the sensitivity of seedlings to ER stress. Whole-transcriptome analysis combined with quantitative reverse transcription-PCR and cellular analysis showed that regulated IRE1-dependent decay (RIDD) and autophagy were impaired in mutants mapr3, gaap1mapr3, and gaap3mapr3. MAPR3, GAAP1, and GAAP3 interacted with IRE1B as determined by protein interaction assays. These data suggest that the interaction of GAAP1/GAAP3 with MAPR3 mitigates ER stress to some extent through regulating IRE10-mediated RIDD and autophagy.