Wilson-Zbinden Caroline, dos Santos Aline Xavier da Silveira, Stoffel-Studer Ingrid, van der Vaart Aniek, Hofmann Kay, Reggiori Fulvio, Riezman Howard, Kraft Claudine, Peter Matthias
Department of Biology, Institute of Biochemistry, ETH Zurich, 8093 Zürich, Switzerland.
Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.
Genetics. 2015 Feb;199(2):475-85. doi: 10.1534/genetics.114.169797. Epub 2014 Dec 17.
Autophagy is a highly regulated pathway that selectively degrades cellular constituents such as protein aggregates and excessive or damaged organelles. This transport route is characterized by engulfment of the targeted cargo by autophagosomes. The formation of these double-membrane vesicles requires the covalent conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE). However, the origin of PE and the regulation of lipid flux required for autophagy remain poorly understood. Using a genetic screen, we found that the temperature-sensitive growth and intracellular membrane organization defects of mcd4-174 and mcd4-P301L mutants are suppressed by deletion of essential autophagy genes such as ATG1 or ATG7. MCD4 encodes an ethanolamine phosphate transferase that uses PE as a precursor for an essential step in the synthesis of the glycosylphosphatidylinositol (GPI) anchor used to link a subset of plasma membrane proteins to lipid bilayers. Similar to the deletion of CHO2, a gene encoding the enzyme converting PE to phosphatidylcholine (PC), deletion of ATG7 was able to restore lipidation and plasma membrane localization of the GPI-anchored protein Gas1 and normal organization of intracellular membranes. Conversely, overexpression of Cho2 was lethal in mcd4-174 cells grown at restrictive temperature. Quantitative lipid analysis revealed that PE levels are substantially reduced in the mcd4-174 mutant but can be restored by deletion of ATG7 or CHO2. Taken together, these data suggest that autophagy competes for a common PE pool with major cellular PE-consuming pathways such as the GPI anchor and PC synthesis, highlighting the possible interplay between these pathways and the existence of signals that may coordinate PE flux.
自噬是一条高度受调控的途径，可选择性地降解细胞成分，如蛋白质聚集体以及过多或受损的细胞器。这条转运途径的特征是自噬体对靶向货物的吞噬。这些双膜囊泡的形成需要泛素样蛋白Atg8与磷脂酰乙醇胺（PE）共价结合。然而，PE的来源以及自噬所需脂质通量的调节仍知之甚少。通过基因筛选，我们发现mcd4 - 174和mcd4 - P301L突变体的温度敏感型生长和细胞内膜组织缺陷可通过缺失必需的自噬基因（如ATG1或ATG7）得到抑制。MCD4编码一种磷酸乙醇胺转移酶，该酶将PE用作合成糖基磷脂酰肌醇（GPI）锚定物关键步骤的前体，GPI锚定物用于将一部分质膜蛋白连接到脂质双层。与编码将PE转化为磷脂酰胆碱（PC）的酶的CHO2基因缺失类似，ATG7基因缺失能够恢复GPI锚定蛋白Gas1的脂化和质膜定位以及细胞内膜的正常组织。相反，在限制温度下生长的mcd4 - 174细胞中，Cho2的过表达是致死性的。定量脂质分析表明，mcd4 - 174突变体中的PE水平大幅降低，但可通过缺失ATG7或CHO2恢复。综上所述，这些数据表明自噬与主要的细胞PE消耗途径（如GPI锚定物合成和PC合成）竞争共同的PE库，突出了这些途径之间可能的相互作用以及可能协调PE通量的信号的存在。
