GFP-LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy.
GFP-LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy.
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DOI：
10.1002/jcb.22103
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发表时间：
2009-05-01
影响因子：
4
通讯作者：
Korkhov, Vladimir M.
中科院分区：
文献类型：
--
作者：
Korkhov, Vladimir M.
自噬的破坏导致细胞内多层包涵体的积累，形态上类似于有组织的滑面内质网（OSER）膜。然而，这些膜隔室与自噬的关系尚不清楚。本研究的目的是测试OSER是否在自噬蛋白降解途径中起作用。在此，GFP-LC 3显示定位于由瞬时转染的HEK 293细胞和小鼠胚胎成纤维细胞中的钙连接蛋白表达诱导的OSER膜。与GFP-LC 3相反，在正常条件下以及细胞饥饿后，内源性LC 3被从这些膜中排除。此外，YFP-Atg 5，自噬所必需的蛋白质，已知驻留在自噬膜上，被排除在钙连接蛋白阳性包涵体结构之外。在缺乏Atg 5的细胞中，自噬所必需的蛋白质并且已知存在于自噬膜上，钙连接蛋白与GFP-LC 3在多层体内的共定位得以保留。我发现，钙连接蛋白，蛋白质丰富的OSER，是不受自噬或溶酶体降解。最后，靶向这些膜的GFP-LC 3不依赖于其加工，并且对调节自噬和溶酶体蛋白降解的药物不敏感。这些观察结果与自噬/溶酶体降解在清除包含OSER的多层体中的作用不一致。此外，广泛用作自噬囊泡和自噬前区室的标记物的融合蛋白GFP-LC 3可能被捕获在该区室中，并且如果构建体用于可视化自噬膜，则必须考虑到这种假象。J.细胞。107：86-95，2009.© 2009 Wiley-Liss公司。
Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP-LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP-LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP-Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin-positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP-LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP-LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP-LC3, a fusion protein widely used as a marker for autophagic vesicles and pre-autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley-Liss, Inc.
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DOI：
10.1083/jcb.152.4.657
发表时间：
2001-02-19
期刊：
The Journal of cell biology
影响因子：
--
作者：
Mizushima N;Yamamoto A;Hatano M;Kobayashi Y;Kabeya Y;Suzuki K;Tokuhisa T;Ohsumi Y;Yoshimori T
通讯作者：
Yoshimori T
影响因子：
13.3
作者：
Cieehomska, Iwona A.;Tolkovsky, Aviva M.
通讯作者：
Tolkovsky, Aviva M.
影响因子：
3.5
作者：
Fujita, Eriko;Kouroku, Yoriko;Momoi, Takashi
通讯作者：
Momoi, Takashi
影响因子：
5.4
作者：
Firdaus, Wance J. J.;Wyttenbach, Andreas;Arrigo, Andre-Patrick
通讯作者：
Arrigo, Andre-Patrick
影响因子：
4
作者：
Mizushima, N;Kuma, A;Yoshimori, T
通讯作者：
Yoshimori, T