ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs

Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg²⁺) 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. Mg²⁺ uptake experiments with a stable isotope demonstrate that there is a significant increase of ²⁵Mg²⁺ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg²⁺ transport by promoting the complex N-glycosylation of CNNMs.