Rosalind and Morris Goodman Cancer Research Centre, McGill University, 1160 Pine Avenue W, Montréal, QC, H3A 1A3, Canada.
Department of Biochemistry, McGill University, Montréal, QC, H3G 1Y6, Canada.
Cell Mol Life Sci. 2021 Jul;78(13):5427-5445. doi: 10.1007/s00018-021-03832-8. Epub 2021 Jun 5.
Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-β-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg uptake experiments with a stable isotope demonstrate that there is a significant increase of Mg uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg transport by promoting the complex N-glycosylation of CNNMs.
周期素 M (CNNM1-4) 蛋白维持细胞和体内镁 (Mg) 稳态。我们使用各种生化方法鉴定出 CNNM 家族的成员是 ADP-核糖基化因子样 GTP 酶 15 (ARL15) 的直接相互作用伙伴，ARL15 是一种小 GTP 结合蛋白。ARL15 与其羧基末端保守半胱氨酸-β-合成酶 (CBS) 结构域的 CNNM 相互作用。对 CNNM2 和 ARL15 之间相互作用的计算机建模支持小 GTP 酶特异性结合 CBS1 和 CNBH 结构域。免疫细胞化学实验表明 CNNM2 和 ARL15 在肾脏中共定位，这两种蛋白质在细胞质内质网、高尔基体和质膜中均有亚细胞定位。最重要的是，我们发现 ARL15 是形成 CNNM 复杂 N-糖基化所必需的。ARL15 的过表达促进了 CNNM3 的复杂 N-糖基化。用稳定同位素进行的 Mg 摄取实验表明，在多种肾癌细胞系中敲低 ARL15 后，Mg 摄取显著增加。总之，我们的结果确立了 ARL15 作为一种新型的 Mg 转运负调节剂，通过促进 CNNM 的复杂 N-糖基化来发挥作用。
