RESUMO
Reactive oxygen species (ROS)-induced oxidative stress has been associated with diseases such as amyotrophic lateral sclerosis, stroke, and cancer. While the effect of ROS on mitochondria and endoplasmic reticulum (ER) has been well documented, its consequence on the Golgi apparatus is less well understood. In this study, we characterized the Golgi structure and function in HeLa cells after exposure to hydrogen peroxide (H2O2), a reagent commonly used to introduce ROS to cells. Treatment of cells with 1 mM H2O2 for 10 min resulted in the degradation of Arl1 and dissociation of GRIP domain-containing proteins Golgin-97 and Golgin-245 from the trans-Golgi. This effect could be rescued by treatment of cells with a ROS scavenger N-acetyl cysteine or protease inhibitors. Structurally, H2O2 treatment reduced the number of cisternal membranes per Golgi stack, suggesting a loss of trans-Golgi cisternae. Functionally, H2O2 treatment inhibited both anterograde and retrograde protein transport, consistent with the loss of membrane tethers on the trans-Golgi cisternae. This study revealed membrane tethers at the trans-Golgi as novel specific targets of ROS in cells.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo/fisiologia , Fatores de Ribosilação do ADP/efeitos dos fármacos , Autoantígenos/metabolismo , Transporte Biológico/fisiologia , Complexo de Golgi/fisiologia , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Membranas Intracelulares/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Transporte Proteico/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Rede trans-Golgi/metabolismoRESUMO
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 trans-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. Abbreviations: ATG14: autophagy related 14; BafA1: bafilomycin A1; 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.
Assuntos
Autofagossomos/fisiologia , Proteínas da Matriz do Complexo de Golgi/fisiologia , Lisossomos/fisiologia , Fusão de Membrana/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/genética , Células Cultivadas , Proteínas da Matriz do Complexo de Golgi/genética , Células HeLa , Humanos , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Ratos , Proteínas Supressoras de Tumor/fisiologiaRESUMO
In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.
