SHISA5/SCOTIN restrains spontaneous autophagy induction by blocking contact between the ERES and phagophores

The phagophore expands into autophagosomes in close proximity to endoplasmic reticulum (ER) exit sites (ERESs). Here, we propose that a single-pass ER transmembrane protein, SHISA5/SCOTIN, acts as an autophagy suppressor under basal condition by blocking the contact between the phagophore and ERES. HeLa cells lacking SHISA5 displayed higher levels of macroautophagy/autophagy. The enhanced autophagy in SHISA5 KO cells requires class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) activity and functional assembly of ERES, but not ULK1 activity. A proximity ligation assay (PLA) of SEC16A (Sec16 homolog A, endoplasmic reticulum export factor)-WIPI2 (WD repeat domain, phosphoinositide interacting 2) and SEC31A (Sec31 homolog A, COPII coat complex component)-MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) demonstrated that contact between the ERES and phagophore increased in SHISA5 KO cells, and the cytosolic domain of SHISA5 was sufficient to rescue this phenotype. Close proximity between ERES and phagophore in SHISA5 KO cells was also visualized by performing an ultrastructure correlative image analysis of SEC31A associated with LC3-positive membranes. Furthermore, we observed that SHISA5 was located near ERES under basal conditions, but displaced away from ERES under autophagy-inducing conditions. These data suggest that SHISA5 functions to block spontaneous contact between ERES and phagophore, and the blockage effect of SHISA5 should be relieved for the proper induction of autophagy. ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle’s balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type

吞噬泡（phagophore）会在内质网（endoplasmic reticulum, ER）出口位点（ER exit sites, ERESs）附近延伸形成自噬体。本研究提出，单次跨膜内质网蛋白SHISA5/SCOTIN可在基础状态下作为自噬（autophagy）抑制因子，通过阻断吞噬泡与ERES的接触发挥作用。缺失SHISA5的HeLa细胞中，巨自噬（macroautophagy）水平更高。SHISA5 KO细胞中增强的自噬依赖于III型磷脂酰肌醇3-激酶复合物I（class III phosphatidylinositol 3-kinase complex I, PtdIns3K-C1）的活性以及ERES的功能性组装，而非UNC-51样自噬激活激酶1（unc-51-like autophagy activating kinase 1, ULK1）的活性。通过对SEC16A（SEC16同源物A，内质网输出因子）-WIPI2（WD重复结构域磷酸肌醇相互作用蛋白2）以及SEC31A（SEC31同源物A，COPII包被复合物组分）-MAP1LC3B/LC3B（微管相关蛋白1轻链3β）开展邻近连接试验（proximity ligation assay, PLA），结果显示SHISA5 KO细胞中ERES与吞噬泡的接触增多，且SHISA5的胞质结构域足以挽救该表型。通过对结合LC3阳性膜的SEC31A开展超微结构相关成像分析，同样可观察到SHISA5 KO细胞中ERES与吞噬泡存在紧密邻近关系。此外，本研究观察到，基础状态下SHISA5定位于ERES附近，但在自噬诱导条件下会从ERES处移位。上述数据表明，SHISA5的功能是阻断ERES与吞噬泡之间的自发性接触，而要正确诱导自噬，需解除SHISA5的阻断作用。

以下为相关术语缩写说明：
ATG2：自噬相关蛋白2（autophagy-related 2）
BECN1：Beclin 1（beclin 1）
COPII：包被蛋白II（coat protein II, COPII）
DMSO：二甲基亚砜（dimethyl sulfoxide, DMSO）
EBSS：伊格尔平衡盐溶液（Earle’s balanced salt solution, EBSS）
EGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）
ER：内质网（endoplasmic reticulum, ER）
ERES：内质网出口位点（ER exit site(s), ERES）
GFP：绿色荧光蛋白（green fluorescent protein, GFP）
H89：H-89二盐酸盐水合物（H-89 dihydrochloride hydrate, H89）
LAMP1：溶酶体相关膜蛋白1（lysosomal-associated membrane protein 1, LAMP1）
MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3, MAP1LC3/LC3）
MTORC1：哺乳动物雷帕霉素靶蛋白复合物1（mechanistic target of rapamycin kinase complex 1, MTORC1）
NS5A：非结构蛋白5A（nonstructural protein 5A, NS5A）
PIK3C3/VPS34：磷脂酰肌醇3-激酶催化亚基3型（phosphatidylinositol 3-kinase catalytic subunit type 3, PIK3C3/VPS34）
PLA：邻近连接试验（proximity ligation assay, PLA）
PtdIns3P：磷脂酰肌醇-3-磷酸（phosphatidylinositol-3-phosphate, PtdIns3P）
RB1CC1/FIP200：RB1诱导性卷曲螺旋1（RB1 inducible coiled-coil 1, RB1CC1/FIP200）
RFP：红色荧光蛋白（red fluorescent protein, RFP）
RPS6KB1/S6K：核糖体蛋白S6激酶B1（ribosomal protein S6 kinase B1, RPS6KB1/S6K）
SBP：链霉亲和素结合蛋白（streptavidin binding protein, SBP）
SEC16A：SEC16同源物A，内质网输出因子（SEC16 homolog A, endoplasmic reticulum export factor, SEC16A）
SEC31A：SEC31同源物A，COPII包被复合物组分（SEC31 homolog A, COPII coat complex component, SEC31A）
siRNA：小干扰RNA（small interfering RNA, siRNA）
Str：链霉亲和素（streptavidin, Str）
ULK1：UNC-51样自噬激活激酶1（unc-51-like autophagy activating kinase 1, ULK1）
VSVG：水疱性口炎病毒糖蛋白（vesicular stomatitis virus glycoprotein, VSVG）
WIPI2：WD重复结构域磷酸肌醇相互作用蛋白2（WD repeat domain, phosphoinositide interacting 2, WIPI2）
WT：野生型（wild type, WT）