GORASP2/GRASP55 collaborates with the PtdIns3K UVRAG complex to facilitate autophagosome-lysosome fusion.

ABSTRACT

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.