Division of Membrane Dynamics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.
J Biochem. 2022 Sep 30;172(4):245-257. doi: 10.1093/jb/mvac060.
Endoplasmic reticulum (ER) tubules are interconnected by three-way junctions, resulting in the formation of a tubular ER network. Lunapark (Lnp) localizes to and stabilizes the three-way junctions. The N-terminal cytoplasmic domain in Lnp has a ubiquitin ligase activity. However, the molecular mechanism of how the ubiquitin ligase activity of Lnp is involved in the formation of the tubular ER network remains unknown. In this study, we examined whether the ER membrane proteins responsible for the formation of the tubular ER network are ubiquitinated by Lnp. We found that atlastin-2 (ATL2), an isoform of the ATL family mediating the generation of the three-way junctions by connecting the ER tubules, is a novel substrate for ubiquitination by Lnp. The localization of Lnp at the three-way junctions is important for ubiquitination of ATL2. Lysine 56, 57, 282 and 302 are the potential ubiquitination sites by Lnp. Silencing ATL2 decreased the number of the three-way junctions, and the expression of the ATL2 mutant in which the lysine residues are substituted with arginine failed to rescue the decrease of the three-way junctions in the ATL2 knocked-down cells. These results suggest that Lnp ubiquitinates ATL2 at the three-way junctions for the proper tubular ER network formation.
内质网（ER）小管通过三向连接相互连接，形成管状 ER 网络。Lunapark（Lnp）定位于三向连接并稳定三向连接。Lnp 的 N 端细胞质结构域具有泛素连接酶活性。然而，Lnp 的泛素连接酶活性如何参与管状 ER 网络的形成的分子机制尚不清楚。在这项研究中，我们检查了负责管状 ER 网络形成的 ER 膜蛋白是否被 Lnp 泛素化。我们发现，atlastin-2（ATL2），一种 ATL 家族的同工酶，通过连接 ER 小管介导三向连接的产生，是 Lnp 泛素化的新底物。Lnp 在三向连接处的定位对于 ATL2 的泛素化很重要。赖氨酸 56、57、282 和 302 是 Lnp 泛素化的潜在位点。沉默 ATL2 减少了三向连接的数量，并且表达赖氨酸残基被精氨酸取代的 ATL2 突变体不能挽救 ATL2 敲低细胞中三向连接的减少。这些结果表明，Lnp 在三向连接处泛素化 ATL2 以形成适当的管状 ER 网络。
