N‑acetylglucosaminyltransferases and nucleotide sugar transporters form multi‑enzyme–multi‑transporter assemblies in golgi membranes in vivo

Session: 
S2.2 Protein N-glycosylation
Code: 
OL2.2.1
Location (hall): 
Mannose
Start/end time: 
Monday, July 1, 2019 - 14:45 to 15:00
Fawzi
Khoder-Agha

Fawzi Khoder-Agha1, Paulina Sosicka2, Maria Escriva Conde3, Antti Hassinen4, Tuomo Glumoff1, Mariusz  Olczak5, Sakari Kellokumpu1

1Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland, 2Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA, 3Faculty of Biology, University of Barcelona, Barcelona, Spain, 4Institute of Molecular Medicine, Helsinki, Finland, 5Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland

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.

A schematic model of the multi-enzyme/multi-transporter assemblies in the Golgi membranes of live cells. The complex is shown to import UDP-N-acetylglucosamine (UDP-GlcNAc) into the Golgi lumen, where after MGAT1 is adding the sugar to acceptor (not shown) and freeing UDP. UDP is then transformed to UMP by the nucleotide diphosphatase (shown as part of the same assembly). UMP then drives the import of a new UDP-GlcNAc residue for further catalysis.

Sponsors

Sponsors