Rab7 is required for the normal progression of the autophagic pathway in mammalian cells
Rab7 is required for the normal progression of the autophagic pathway in mammalian cells
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DOI：
10.1242/jcs.01114
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发表时间：
2004-06-01
影响因子：
4
通讯作者：
Colombo, MI
中科院分区：
文献类型：
--
作者：
Gutierrez, MG;Munafó, DB;Colombo, MI
自噬是一种正常的降解途径，包括将细胞质成分和细胞器隔离在一个被称为自噬体的液泡中，最终与溶酶体融合。Rab7是Rab7家族的一员，参与转运到晚期核内体和核周溶酶体室的生物发生。为了评估Rab7在自噬中的作用，我们用野生型pEGFP-Rab7和突变体T22N （GTP型）和Q67L （GTP型）稳定转染CHO细胞。氨基酸饥饿诱导细胞自噬，自噬液泡用单甘油三酯标记。通过荧光显微镜，我们观察到Rab7wt和活性突变体Rab7Q67L与单氨尸胺标记的环状囊泡相关，表明这些Rab7wt蛋白与自噬囊泡的膜相关。正如预期的那样，在转染阴性突变体Rab7T22N的细胞中，该蛋白弥散分布在细胞质中。然而，在氨基酸饥饿或雷帕霉素处理诱导自噬后，该突变体清楚地修饰了单胺尸胺标记的囊泡。此外，通过过表达失活突变体T22N，观察到饥饿诱导的单氨尸胺标记液泡的大小显着增加。同样，LC3标记的囊泡大小增加，LC3是一种特异性定位于自噬体膜上的蛋白质。综上所述，Rab7的功能对细胞自噬的正常进行至关重要。
Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called an autophagosome that finally fuses with the lysosome. Rab7 is a member of the Rab family involved in transport to late endosomes and in the biogenesis of the perinuclear lysosome compartment. To assess the role of Rab7 in autophagy we stably transfected CHO cells with wild-type pEGFP-Rab7, and the mutants T22N (GDP form) and Q67L (GTP form). Autophagy was induced by amino acid starvation and the autophagic vacuoles were labeled with monodansylcadaverine. By fluorescence microscopy we observed that Rab7wt and the active mutant Rab7Q67L were associated with ring-shaped vesicles labeled with monodansylcadaverine indicating that these Rab proteins associate with the membrane of autophagic vesicles. As expected, in cells transfected with the negative mutant Rab7T22N the protein was diffusely distributed in the cytosol. However, upon induction of autophagy by amino acid starvation or by rapamycin treatment this mutant clearly decorated the monodansylcadaverine-labeled vesicles. Furthermore, a marked increase in the size of the monodansylcadaverine-labeled vacuoles induced by starvation was observed by overexpression of the inactive mutant T22N. Similarly, there was an increase in the size of vesicles labeled with LC3, a protein that specifically localizes on the autophagosomal membrane. Taken together the results indicate that a functional Rab7 is important for the normal progression of autophagy.