Isolation and characterization of rat liver amphisomes - Evidence for fusion of autophagosomes with both early and late endosomes
Isolation and characterization of rat liver amphisomes - Evidence for fusion of autophagosomes with both early and late endosomes
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DOI：
10.1074/jbc.273.34.21883
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发表时间：
1998-08-21
影响因子：
4.8
通讯作者：
Seglen, PO
中科院分区：
文献类型：
--
作者：
Berg, TO;Fengsrud, M;Seglen, PO
两性体是自噬体和核内体融合后形成的自噬液泡，迄今为止仅从间接的功能方面对其进行了表征。为了区分自噬体和两体，在与AOM-gold-loaded核内体（核内体被asialoorosomucide缀合的金颗粒致密化，在亚细胞分离前被分离的大鼠肝细胞内吞）融合后，后者在蔗糖梯度中选择性地密度转移。根据这一标准，在对照肝细胞中，两性体只占AV的一小部分，而用胰肽（一种溶酶体蛋白降解抑制剂）处理的细胞导致了大约一半AV群体中两性体的积累。定量电镜研究证实，白细胞介素诱导肝细胞两性小体数量增加数倍（通过其金颗粒含量来识别；否则，它们的超微结构与自噬小体非常相似），此外，白细胞介素导致选择性保留内吞的aom -金在两性小体中，以牺牲溶酶体为代价，与抑制两性小体-溶酶体融合一致。电镜显示，自噬体可以经历多次独立的融合，多泡（晚期）内体形成两性体，小溶酶体形成大的自噬酶体。通过一种新方法纯化的自噬体和两性体之间的生化比较表明，两性体在早期核内体标记物（asialal糖蛋白受体和早期核内体相关蛋白I）和晚期核内体标记物（不依赖阳离子的甘露糖6-磷酸受体）中富集。因此，两性体似乎能够同时接受来自早期和晚期核内体的输入。
Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prier to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphi somes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes), Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein I) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.