ARMH3 acts as a central scaffold at the Golgi/TGN through interactions with Arl5, GBF1, and PI4KB.

The armadillo repeat protein ARMH3 regulates the activity and localization of the Golgi resident lipid kinase phosphatidylinositol 4 kinase IIIβ (PI4KB) and the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) that activates Arf1. ARMH3 localizes to the trans-Golgi network (TGN) via the GTPase Arl5. We used hydrogen deuterium exchange mass spectrometry (HDX-MS) and AI-enabled modeling to define the interfaces of ARMH3 with its binding partners Arl5, PI4KB, and GBF1. The ARMH3-Arl5 interface was determined to consist of regions near the N and C termini of ARMH3, with Arl5 binding causing allosteric conformational changes in ARMH3 located at a shared PI4KB/GBF1 interface. ARMH3 binds to PI4KB and GBF1 at this shared competitive interface, with GBF1 binding to ARMH3 through a disordered loop we have named the ARMH3-binding region (ABR). Both GBF1 and PI4KB can form ternary complexes with ARMH3-Arl5. The ARMH3 interfaces in PI4KB and GBF1 contain phosphosites, with the phosphomimetic mutation of GBF1 blocking complex formation. These findings provide new insights into the role of ARMH3 as a master coordinator of GTPase and phosphoinositide signaling at the Golgi/TGN.