Tepsin binds LC3B to promote ATG9A export and delivery at the cell periphery.
Tepsin binds LC3B to promote ATG9A export and delivery at the cell periphery.
复制标题
Tepsin 结合 LC3B 以促进 ATG9A 在细胞外周的输出和递送。
DOI：
10.1101/2023.07.18.549521
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Jackson,LaurenP
中科院分区：
文献类型：
--
作者：
Wallace,NatalieS;Gadbery,JohnE;Cohen,CameronI;Kendall,AmyK;Jackson,LaurenP
Tepsin是在适配器蛋白4(AP-4)包被的囊泡中发现的一种已确定的辅助蛋白，但Tepsin的生物学作用尚不清楚。AP-4囊泡起源于T-高尔基网络(TGN)，以ATG9A为靶点，ATG9A是自噬小体生物发生所需的一种扰乱酶，它被运送到细胞外周。利用电子计算机方法，我们在tepsin中发现了一个已知的Lc3相互作用区(LIR)基序。使用纯化的重组蛋白进行的生化实验表明，tepsin直接与哺乳动物ATG8家族的LC3B结合，但不与其他成员结合。量热和结构建模数据表明，使用已建立的LC3B LIR对接位置，这种相互作用与微摩尔亲和力发生。培养细胞中Tepsin的丢失会扰乱ATG9A从TGN的输出以及ATG9A在细胞外围的分布。在使用siRNA敲除的MRFP-GFP-LC3B HeLa报告细胞系中，Tepsin的缺失增加了自噬小体的体积和数量，但似乎不影响通过自噬途径的通量。野生型tepsin的重新引入部分挽救了ATG9A货物运输的缺陷。相反，重新引入带有突变的LIR基序或缺失N末端的Tepsin并不能完全挽救改变的ATG9A亚细胞分布。综上所述，这些数据表明tepsin在TGN的货物出口、细胞外围ATG9A阳性小泡的运输以及自噬小体结构的整体维持中发挥了作用。
Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at thetrans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Usingin silicomethods, we identified a putativeLC3-InteractingRegion (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B, but not other members, of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Re-introduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, re-introducing tepsin with a mutated LIR motif or missing N-terminus does not fully rescue altered ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; delivery of ATG9A-positive vesicles at the cell periphery; and in overall maintenance of autophagosome structure.