Abstract

Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC₃₅A₃ nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT₁-MGAT₅). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT₁, MGAT₂, MGAT₃, MGAT₄B (but not MGAT₅) and Golgi alpha-mannosidase IIX (MAN₂A₂) form several distinct molecular assemblies with each other and that the MAN₂A₂ acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC₃₅A₂, SLC₃₅A₃, and SLC₃₅A₄. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.