GORASP2 promotes phagophore closure and autophagosome maturation into autolysosomes

As the central hub of the secretory pathway, the Golgi apparatus plays a crucial role in maintaining cellular homeostasis in response to stresses. Recent studies have revealed the involvement of the Golgi tether, GORASP2, in facilitating autophagosome-lysosome fusion by connecting LC3-II and LAMP2 during nutrient starvation. However, the precise mechanism remains elusive. In this study, utilizing super-resolution microscopy, we observed GORASP2 localization on the surface of autophagosomes during glucose starvation. Depletion of GORASP2 hindered phagophore closure by regulating the association between VPS4A and the ESCRT-III component, CHMP2A. Furthermore, we found that GORASP2 controls RAB7A activity by modulating its GEF complex, MON1A-CCZ1, thereby impacting RAB7A’s interaction with the HOPS complex. The assembly of both STX17-SNAP29-VAMP8 and YKT6-SNAP29-STX7 SNARE complexes was also attenuated without GORASP2. These findings suggest that GORASP2 helps seal autophagosomes and activate the RAB7A-HOPS-SNAREs membrane fusion machinery for autophagosome maturation, highlighting its membrane tethering function in response to stresses.<b>Abbreviations</b>: BafA1: bafilomycin A₁; ESCRT: endosomal sorting complex required for transport; FPP: fluorescence protease protection; GEF: guanine nucleotide exchange factor; GFP: green fluorescent protein; GORASP2: golgi reassembly stacking protein 2; GSB: glucose starvation along with bafilomycin A₁; HOPS: homotypic fusion and protein sorting; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PK: proteinase K; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SIM: structured illumination microscopy; UVRAG: UV radiation resistance associated.作为分泌通路的核心枢纽，高尔基体（Golgi apparatus）在应对应激以维持细胞稳态中发挥关键作用。近期研究表明，高尔基体拴连蛋白GORASP2（高尔基体重排堆叠蛋白2，golgi reassembly stacking protein 2）可在营养匮乏条件下，通过连接LC3-II与LAMP2，促进自噬体与溶酶体的融合。然而，其具体分子机制仍有待阐明。本研究借助超分辨率显微镜技术，观察到葡萄糖匮乏时GORASP2定位于自噬体表面。敲除GORASP2会通过调控VPS4A与内体分选转运复合体（endosomal sorting complex required for transport）-III组分CHMP2A的结合，阻碍自噬噬泡的闭合。进一步研究发现，GORASP2可通过调控其鸟苷酸交换因子（guanine nucleotide exchange factor）复合物MON1A-CCZ1，控制RAB7A的活性，进而影响RAB7A与同型融合与蛋白分选复合体（homotypic fusion and protein sorting）的相互作用。此外，在缺失GORASP2的情况下，STX17-SNAP29-VAMP8与YKT6-SNAP29-STX7这两种可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（soluble N-ethylmaleimide-sensitive factor attachment protein receptor）复合物的组装均会受到削弱。上述研究结果提示，GORASP2可协助自噬噬泡闭合，并激活RAB7A-HOPS-SNARE膜融合机器以完成自噬体成熟，凸显了其在应激应答过程中的膜拴连功能。 缩写：BafA1：巴弗洛霉素A₁；ESCRT：内体分选转运复合体；FPP：荧光蛋白酶保护试验；GEF：鸟苷酸交换因子；GFP：绿色荧光蛋白；GORASP2：高尔基体重排堆叠蛋白2；GSB：葡萄糖匮乏联合巴弗洛霉素A₁；HOPS：同型融合与蛋白分选复合体；LAMP2：溶酶体相关膜蛋白2；MAP1LC3B：微管相关蛋白1轻链3β；PBS：磷酸盐缓冲液；PtdIns3K：磷脂酰肌醇3-激酶；PtdIns3P：磷脂酰肌醇-3-磷酸；PK：蛋白酶K；SNARE：可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体；SIM：结构照明显微镜；UVRAG：紫外线辐射抗性相关蛋白。