a Department of Biosciences , Rice University , Houston , TX , USA.
b Department of Biology , University of Mary Hardin-Baylor , Belton , TX , USA.
Autophagy. 2019 Jun;15(6):941-959. doi: 10.1080/15548627.2019.1569915. Epub 2019 Feb 8.
Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2 mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790 allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation. Abbreviations: ATG: autophagy-related; ATI: ATG8-interacting protein; Col-0: Columbia-0; DSK2: dominant suppressor of KAR2; EMS: ethyl methanesulfonate; GFP: green fluorescent protein; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; ICL: isocitrate lyase; MLS: malate synthase; NBR1: Next to BRCA1 gene 1; PEX: peroxin; PMDH: peroxisomal malate dehydrogenase; PTS: peroxisomal targeting signal; thiolase: 3-ketoacyl-CoA thiolase; UBA: ubiquitin-associated; WT: wild type.
自噬是一种细胞降解包括细胞器、蛋白聚集体和入侵病原体在内的大底物的过程。通过酵母的正向遗传学筛选，已经鉴定出超过 40 个自噬相关（ATG）基因。尽管基于同源性的分析已经在植物中鉴定出保守的 ATG 基因，但在拟南芥中，只有少数 atg 突变体是通过正向遗传学筛选得到的。我们开发了一种筛选方法，该方法利用 Lon2/At5g47040 缺陷的突变体，该突变体是一种参与过氧化物酶体质量控制的蛋白酶，一致恢复了拟南芥 atg 突变。Lon2 突变体表现出对过氧化物酶体代谢的生长素前体吲哚-3-丁酸（IBA）的反应性降低，几种过氧化物酶体基质蛋白的降解增加，以及含有 N 末端过氧化物酶体靶向信号的蛋白质的加工受损；这些缺陷通过阻止自噬得到改善。我们优化了 lon2 抑制子筛选，以加快恢复其他 atg 突变体。在筛选诱变 lon2-2 幼苗中恢复 IBA 反应性后，我们评估了过氧化物酶体蛋白的稳定性和加工、几种 ATG 蛋白的水平以及选择性自噬受体 NBR1/At4g24690 的水平，当自噬受损时，NBR1/At4g24690 会积累。我们恢复了 21 个突变体，突变了 6 个 ATG 基因：ATG2/At3g19190、ATG3/At5g61500、ATG5/At5g17290、ATG7/At5g45900、ATG16/At5g50230 和 ATG18a/At3g62770。其中 20 个是新的，3 个突变基因在公开的存储库中没有 T-DNA 插入等位基因。我们还证明了一个插入的 atg11/At4g30790 等位基因不能完全抑制 lon2 缺陷。最后，我们表明 NBR1 不是 lon2 过氧化物酶体自噬所必需的，NBR1 的过表达不足以触发幼苗过氧化物酶体的自噬，这表明拟南芥可以使用 NBR1 独立的机制将过氧化物酶体靶向自噬降解。缩写：ATG：自噬相关；ATI：ATG8 相互作用蛋白；Col-0：哥伦比亚-0；DSK2：显性抑制 KAR2；EMS：乙基甲磺酸；GFP：绿色荧光蛋白；IAA：吲哚-3-乙酸；IBA：吲哚-3-丁酸；ICL：异柠檬酸裂解酶；MLS：苹果酸合酶；NBR1：BRCA1 基因旁边 1 号；PEX：过氧化物酶体；PMSDH：过氧化物酶体苹果酸脱氢酶；PTS：过氧化物酶体靶向信号；硫酯酶：3-酮酰基辅酶 A 硫酯酶；UBA：泛素相关；WT：野生型。
