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.
Highlights
Glycosylation is the most common modification of proteins and lipids, and crucial for multicellular life given its roles in a plethora of basic cellular functions
We found that MGAT1 interacted with MGAT2 and with MGAT4B, but not with MGAT3, MGAT5 or MAN2A2 (Fig. 1a)
We found that the MGAT1–MGAT2 interaction (Fig. 2a) was significantly inhibited by MGAT1, and by MGAT3, while MGAT4B, MGAT5 or MAN2A2 had no effect
Summary
Glycosylation is the most common modification of proteins and lipids, and crucial for multicellular life given its roles in a plethora of basic cellular functions. Glycosylation helps proteins to fold correctly, ensures their optimal transport to the cell surface, and mediates a variety of interactions between cells, cells and the extracellular matrix and certain pathogens that use glycans as specific recognition and attachment sites. Processing of high mannose N-glycans to complex N-glycans commences in the cis/medial-Golgi. It relies on the activity of several distinct N-acetylglucosaminyltransferases termed MGAT1, MGAT2, MGAT3, MGAT4A,4B, MGAT5A,5B and possibly MGAT6 [37] that each add N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to a specific mannose in the core of a N-glycan. MGAT1 adds the first GlcNAc residue to the C-2 of the
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