Department of Molecular, Cellular and Developmental Biology, University of Michigan , Ann Arbor , MI , USA.
Department of Neurology, University of Michigan School of Medicine , Ann Arbor , MI , USA.
Autophagy. 2019 Oct;15(10):1787-1800. doi: 10.1080/15548627.2019.1596480. Epub 2019 Apr 2.
It has been indicated that the Golgi apparatus contributes to autophagy, but how it is involved in autophagosome formation and maturation is not well understood. Here we show that amino acid starvation causes -Golgi derived membrane fragments to colocalize with autophagosomes. Depletion of the Golgi stacking protein GORASP2/GRASP55, but not GORASP1/GRASP65, increases both MAP1LC3 (LC3)-II and SQSTM1/p62 levels. We demonstrate that GORASP2 facilitates autophagosome-lysosome fusion by physically linking autophagosomes and lysosomes through the interactions with LC3 on autophagosomes and LAMP2 on late endosomes/lysosomes. Furthermore, we provide evidence that GORASP2 interacts with BECN1 to facilitate the assembly and membrane association of the phosphatidylinositol 3-kinase (PtdIns3K) UVRAG complex. These findings indicate that GORASP2 plays an important role in autophagosome maturation during amino acid starvation. : ATG14: autophagy related 14; BafA1: bafilomycin A; BSA: bovine serum albumin; CQ: chloroquine; EBSS: earle's balanced salt solution; EM: electron microscopy; EEA1: early endosome antigen 1; GFP: green fluorescent protein; GORASP1/GRASP65: golgi reassembly stacking protein 1; GORASP2/GRASP55: golgi reassembly stacking protein 2; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PK: protease K; PNS: post-nuclear supernatant; RFP: red fluorescent protein; SD: standard deviation; TGN: trans-Golgi network; UVRAG: UV radiation resistance associated.
已经表明，高尔基体有助于自噬，但它如何参与自噬体的形成和成熟尚不清楚。在这里，我们表明，氨基酸饥饿导致-Golgi 衍生的膜片段与自噬体共定位。耗尽高尔基体堆积蛋白 GORASP2/GRASP55，但不是 GORASP1/GRASP65，会增加 MAP1LC3（LC3）-II 和 SQSTM1/p62 的水平。我们证明，GORASP2 通过与自噬体上的 LC3 和晚期内体/溶酶体上的 LAMP2 相互作用，物理上连接自噬体和溶酶体，促进自噬体-溶酶体融合。此外，我们提供的证据表明，GORASP2 与 BECN1 相互作用，促进磷脂酰肌醇 3-激酶（PtdIns3K）UVRAG 复合物的组装和膜结合。这些发现表明，在氨基酸饥饿期间，GORASP2 在自噬体成熟中发挥重要作用。：ATG14：自噬相关 14；BafA1：巴弗霉素 A；BSA：牛血清白蛋白；CQ：氯喹；EBSS：Earle 的平衡盐溶液；EM：电子显微镜；EEA1：早期内体抗原 1；GFP：绿色荧光蛋白；GORASP1/GRASP65：高尔基体再组装堆积蛋白 1；GORASP2/GRASP55：高尔基体再组装堆积蛋白 2；LAMP1：溶酶体相关膜蛋白 1；LAMP2：溶酶体相关膜蛋白 2；MAP1LC3：微管相关蛋白 1 轻链 3；MTOR：雷帕霉素激酶的机制靶标；PBS：磷酸盐缓冲盐水；PtdIns3K：磷脂酰肌醇 3-激酶；PtdIns3P：磷脂酰肌醇 3-磷酸；PK：蛋白酶 K；PNS：核后上清液；RFP：红色荧光蛋白；SD：标准差；TGN：跨高尔基网络；UVRAG：紫外线辐射抗性相关。
