ABSTRACT

Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate “mitoplasts”. This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The “mitoplasts” generated are degraded by reticulo-mito-phagy – simultaneously affecting dual organelle turnover.

Abbreviations: AMFR/GP78: autocrine motility factor receptor; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BFP: blue fluorescent protein; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; CNBr: cyanogen bromide; ER: endoplasmic reticulum; ERAD: endoplasmic-reticulum-associated protein degradation; FL: fluorescence, GFP: green fluorescent protein; HA: hemagglutinin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN: mitofusin, MGRN1: mahogunin ring finger 1; NA: numerical aperature; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; PRNP/PrP: prion protein; RER: rough endoplasmic reticulum; RETREG1/FAM134B: reticulophagy regulator 1; RFP: red fluorescent protein; RING: really interesting new gene; ROI: region of interest; RTN: reticulon; SEM: standard error of the mean; SER: smooth endoplasmic reticulum; SIM: structured illumination microscopy; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STOML2: stomatin like 2; TOMM20: translocase of outer mitochondrial membrane 20; UPR: unfolded protein response.

摘要

细胞器的周转，包括内质网 (ER) 和线粒体，由有效的细胞监测系统协调。我们已经确定了在压力下对 ER 和线粒体进行双重调节的机制。已知 AMFR、ER E3 连接酶和 ER - a相关d降解（ERAD）调节剂，通过蛋白酶体降解线粒体外膜（OMM）蛋白，MFN（mitofusins）并触发线粒体自噬。我们表明，不稳定的线粒体几乎没有 OMM 并产生“线粒体”。这将线粒体内膜 (IMM) 带到了 ER 附近。当 AMFR 水平高且线粒体受到压力时，网状吞噬调节蛋白 RETREG1 通过与 OPA1 相互作用参与线粒体的形成。有趣的是，与大多数 OMM 蛋白不同，OPA1 和其他 IMM 蛋白表现出与 AMFR 相似的 RETREG1 依赖性自噬体降解。产生的“线粒体”被网状线粒体自噬降解——同时影响双细胞器周转。

缩写：AMFR/GP78：自分泌运动因子受体；BAPTA：1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸；BFP：蓝色荧光蛋白；CCCP：羰基氰间氯苯腙；CNBr：溴化氰；ER：内质网；ERAD：内质网相关蛋白降解；FL：荧光，GFP：绿色荧光蛋白；HA：血凝素；HEPES：4-(2-羟乙基)-1-哌嗪乙磺酸；IMM：线粒体内膜；LIR：LC3相互作用区；MAP1LC3/LC3：微管相关蛋白1轻链3；MFN：mitofusin，MGRN1：mahogunin 无名指 1；NA：数值孔径；OMM：线粒体外膜；OPA1：OPA1 线粒体动力蛋白，如 GTPase；PRNP/PrP：朊病毒蛋白；RER：粗面内质网；RETREG1/FAM134B：网状吞噬调节剂 1；RFP：红色荧光蛋白；戒指：非常有趣的新基因；ROI：感兴趣区域；RTN：网状；SEM：均值的标准误；SER：平滑内质网；SIM：结构照明显微镜；SQSTM1/p62：隔离体 1；STED：受激发射损耗；STOML2：像 2 一样的生长素；TOMM20：线粒体外膜转位酶 20；UPR：未折叠蛋白反应。