Saetre Frank, Hagen Linda Korseberg, Engedal Nikolai, Seglen Per O
Cell Biology Section, Institute for Cancer Research, University of Oslo and Oslo University Hospital, Norway.
Prostate Cancer Research Group, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Norway.
FEBS J. 2015 Jun;282(11):2202-14. doi: 10.1111/febs.13268. Epub 2015 Apr 8.
Autophagy is the process by which portions of cytoplasm are enclosed by membranous organelles, phagophores, which deliver the sequestered cytoplasm to degradative autophagic vacuoles. Genes and proteins involved in phagophore manufacture have been extensively studied, but little is known about how mature phagophores proceed through the subsequent steps of expansion, closure and fusion. Here we have addressed these issues by combining our unique autophagic cargo sequestration assay (using the cytosolic enzyme lactate dehydrogenase as a cargo marker) with quantitative measurements of the lipidation-dependent anchorage and turnover of the phagophore-associated protein LC3. In isolated rat hepatocytes, amino acid starved to induce maximal autophagic activity, the two unrelated reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) both blocked cargo sequestration completely. However, whereas 3MA inhibited LC3 lipidation, TG did not, thus apparently acting at a post-lipidation step to prevent phagophore closure. Intriguingly, the resumption of cargo sequestration seen upon release from a reversible TG block was completely suppressed by 3MA, revealing that 3MA not only inhibits LC3 lipidation but also (like TG) blocks phagophore closure at a post-lipidation step. 3MA did not, however, prevent the resumption of lysosomal LC3 degradation, indicating that phagophores could fuse directly with degradative autophagic vacuoles without carrying cytosolic cargo. This fusion step was clearly blocked by TG. Furthermore, density gradient centrifugation revealed that a fraction of the LC3-marked phagophores retained by TG could be density-shifted by the acidotropic drug propylamine along with the lysosomal marker cathepsin B, suggesting physical association of some phagophores with lysosomes prior to cargo sequestration.
自噬是指细胞质的某些部分被膜性细胞器即吞噬泡包裹的过程，吞噬泡将隔离的细胞质输送到具有降解作用的自噬泡中。参与吞噬泡形成的基因和蛋白质已得到广泛研究，但对于成熟的吞噬泡如何经历随后的扩张、闭合和融合步骤却知之甚少。在此，我们通过将独特的自噬货物隔离检测方法（使用胞质酶乳酸脱氢酶作为货物标记物）与对吞噬泡相关蛋白LC3的脂化依赖性锚定和周转进行定量测量相结合，来解决这些问题。在分离的大鼠肝细胞中，通过氨基酸饥饿诱导最大自噬活性，两种不相关的可逆自噬抑制剂3-甲基腺嘌呤（3MA）和毒胡萝卜素（TG）均完全阻断了货物隔离。然而，3MA抑制LC3脂化，而TG则不抑制，因此TG显然作用于脂化后的步骤以阻止吞噬泡闭合。有趣的是，从可逆性TG阻断释放后观察到的货物隔离恢复被3MA完全抑制，这表明3MA不仅抑制LC3脂化，而且（与TG一样）在脂化后的步骤阻断吞噬泡闭合。然而，3MA并未阻止溶酶体中LC3降解的恢复，这表明吞噬泡可以直接与具有降解作用的自噬泡融合而不携带胞质货物。这一融合步骤显然被TG阻断。此外，密度梯度离心显示，TG保留的一部分LC3标记的吞噬泡可被亲酸性药物丙胺与溶酶体标记物组织蛋白酶B一起进行密度转移，这表明一些吞噬泡在货物隔离之前就与溶酶体存在物理关联。
