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

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. Electronic supplementary material The online version of this article (10.1007/s00018-019-03032-5) contains supplementary material, which is available to authorized users.

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

“…Interestingly, we found that both SLC35A2 and SLC35A3 interact with mannoside N-acetylglucosaminyltransferases (Mgats) including Mgat5 as demonstrated by the FLIM-FRET and in situ PLA analyses (5). Recently, several ternary complexes formed with the involvement of some Mgats and the selected SLC35A subfamily members have been demonstrated (7). According to this study, Mgat5 was the only Mgat not being capable of interacting with other Mgats.…”

“…Importantly, we demonstrated that biological function of UDP-GlcNAc and UDP-Gal transporters in glycosylation of proteins may be coupled. This hypothesis was based on: i) the observation that overexpression of UDP-GlcNAc transporter in mutant cells defective in UDP-Gal transporter partially restores galactosylation of N-glycans (37), ii) chimeric proteins composed of regions derived from both transporters restore galactosylation in cells defective in UDP-Gal transporter (38,39), iii) both overexpressed and native transporters form complexes detected not only by Fluorescence Lifetime Imaging Microscopy -Förster Resonance Energy Transfer (FLIM-FRET) or in situ Proximity Ligation Assay (PLA) analyses, but importantly also by coimmunoprecipitation assay, suggesting by guest on October 30, 2020 http://www.jbc.org/ Downloaded from not only their close localization but also strong interaction (3,7,40).…”

“…NSTs also transport the nucleotide monophosphate byproduct of the glycosyltransferase reaction back to the cytoplasm. The ability of NSTs to form dimers or higher order oligomers (2) allows for multiple and simultaneous interactions between several NSTs as well as between NSTs and glycosyltransferases (3)(4)(5)(6)(7). Initially, NSTs were demonstrated to be specific for the translocation of a single nucleotide sugar (8), but later multisubstrate transporters were reported (9)(10)(11).…”

N-glycosylation of the human β1,4-galactosyltransferase 4 is crucial for its activity and Golgi localization

“…Since recently it was revealed that some transferases can form multi-enzyme complexes with nucleotide sugar transporters, we decided to explore B4GalT4 interactions with the Golgi-residing UDP-Gal transporter (UGT1) and how it can be affected by glycan removal on transferase [34]. Interestingly, observed interactions between transferase and transporter shown by the split luciferase assay were abolished in the case of B4GalT4 delocalized versions (MT3 and MT23) while removal of the N-glycosylation site essential for enzymatic activity (MT2) showed an increase of them.…”

Translation of genome to glycome: role of the Golgi apparatus

“…The specificity determinants of this complex formation have been identified in some cases. For example, B4GALT1 and ST6GAL1 interact through their CTS region , while medial Golgi localized N‐glycosylation or O‐glycosylation enzymes interact through their luminal catalytic and/or stem domains (, see also ). Further appropriate glycosylation in the luminal domain of ST8SIA1 (GD3 synthase) was shown to be required for its complex formation with B4GALNT1 (GM2/GD2 synthase) .…”