RETREG1/FAM134B mediated autophagosomal degradation of AMFR/GP78 and OPA1 —a dual organellar turnover mechanism

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-<i>a</i>ssociated <i>d</i>egradation (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. <b>Abbreviations:</b> 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.

包括内质网（endoplasmic reticulum, ER）与线粒体在内的细胞器周转过程，由一套高效的细胞监视系统协同调控。我们已鉴定出应激状态下对ER与线粒体的双重调控机制。已知AMFR作为一种ER E3泛素连接酶及内质网相关降解（endoplasmic-reticulum-associated protein degradation, ERAD）调节因子，可通过蛋白酶体降解线粒体外膜（outer mitochondrial membrane, OMM）蛋白线粒体融合蛋白（mitofusins, MFNs）并触发线粒体自噬。我们的研究发现，去稳定化的线粒体几乎完全缺失外膜，进而产生"线粒体基质体（mitoplasts）"。这使得线粒体内膜（inner mitochondrial membrane, IMM）与ER邻近。当AMFR水平较高且线粒体处于应激状态时，网状自噬调节蛋白RETREG1通过与OPA1相互作用，参与线粒体自噬前体的形成。值得注意的是，与多数线粒体外膜蛋白不同，OPA1及其他线粒体内膜蛋白与AMFR一样，呈现出依赖RETREG1的自噬体降解途径。所产生的"线粒体基质体"通过网状-线粒体自噬（reticulo-mito-phagy）被降解，这一过程同时影响双细胞器的周转。
**缩写说明：**
AMFR/GP78：自分泌运动因子受体（autocrine motility factor receptor）；
BAPTA：1,2-双(2-氨基苯氧基)乙烷-N,N,N′,N′-四乙酸（1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid）；
BFP：蓝色荧光蛋白（blue fluorescent protein）；
CCCP：羰基氰化物间氯苯腙（carbonyl cyanide m-chlorophenyl hydrazone）；
CNBr：溴化氰（cyanogen bromide）；
ER：内质网（endoplasmic reticulum）；
ERAD：内质网相关蛋白降解（endoplasmic-reticulum-associated protein degradation）；
FL：荧光（fluorescence）；
GFP：绿色荧光蛋白（green fluorescent protein）；
HA：血凝素（hemagglutinin）；
HEPES：4-(2-羟乙基)-1-哌嗪乙磺酸（4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid）；
IMM：线粒体内膜（inner mitochondrial membrane）；
LIR：LC3相互作用区域（LC3-interacting region）；
MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；
MFN：线粒体融合蛋白（mitofusin）；
MGRN1： mahogunin环指蛋白1（mahogunin ring finger 1）；
NA：数值孔径（numerical aperture）；
OMM：线粒体外膜（outer mitochondrial membrane）；
OPA1：OPA1线粒体动力素样GTP酶（OPA1 mitochondrial dynamin like GTPase）；
PRNP/PrP：朊蛋白（prion protein）；
RER：粗面内质网（rough endoplasmic reticulum）；
RETREG1/FAM134B：网状自噬调节因子1（reticulophagy regulator 1）；
RFP：红色荧光蛋白（red fluorescent protein）；
RING：真核生物环指结构域（really interesting new gene）；
ROI：感兴趣区域（region of interest）；
RTN：网状蛋白（reticulon）；
SEM：均值标准误（standard error of the mean）；
SER：滑面内质网（smooth endoplasmic reticulum）；
SIM：结构光照明显微镜（structured illumination microscopy）；
SQSTM1/p62：自噬衔接蛋白p62（sequestosome 1）；
STED：受激发射损耗显微镜（stimulated emission depletion）；
STOML2： stomatin样蛋白2（stomatin like 2）；
TOMM20：线粒体外膜转位酶20（translocase of outer mitochondrial membrane 20）；
UPR：未折叠蛋白反应（unfolded protein response）。