Dataset Information

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.

SUBMITTER: Khoder-Agha F

PROVIDER: S-EPMC6453868 | biostudies-other | 2019 May

REPOSITORIES: biostudies-other

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Publications

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

Khoder-Agha Fawzi F Sosicka Paulina P Escriva Conde Maria M Hassinen Antti A Glumoff Tuomo T Olczak Mariusz M Kellokumpu Sakari S

Cellular and molecular life sciences : CMLS 20190208 9

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 th ...[more]

PMID: 30737517

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Project description:Filamentous fungi like Neurospora crassa are able to take up and metabolize important sugars present, for example, in agricultural and human food wastes. However, only a fraction of all putative sugar transporters in filamentous fungi has been characterized to date, and for many sugar substrates, the corresponding transporters are unknown. In N. crassa, only 14 out of the 42 putative major facilitator superfamily (MFS)-type sugar transporters have been characterized so far. To uncover this hidden potential for biotechnology, it is therefore necessary to find new strategies. By correlation of the uptake profile of sugars of interest after different induction conditions with the expression profiles of all 44 genes encoding predicted sugar transporters in N. crassa, together with an exhaustive phylogenetic analysis using sequences of characterized fungal sugar transporters, we aimed to identify transporter candidates for the tested sugars. Following this approach, we found a high correlation of uptake rates and expression strengths for many sugars with dedicated transporters, like galacturonic acid and arabinose, while the correlation is loose for sugars that are transported by several transporters due to functional redundancy. Nevertheless, this combinatorial approach allowed us to elucidate the uptake system for the disaccharide lactose, a by-product of the dairy industry, which consists of the two main cellodextrin transporters CDT-1 and CDT-2 with a minor contribution of the related transporter NCU00809. Moreover, a non-MFS transporter involved in glycerol transport was also identified. Deorphanization of sugar transporters or identification of transporters for orphan sugar substrates by correlation of uptake kinetics with transporter expression and phylogenetic information can thus provide a way to optimize the reuse of food industry by-products and agricultural wastes by filamentous fungi in order to create economic value and reduce their environmental impact. KEY POINTS: • The Neurospora crassa genome contains 30 uncharacterized putative sugar transporter genes. • Correlation of transporter expression and sugar uptake profiles can help to identify transporters for orphan sugar substrates. • CDT-1, CDT-2, and NCU00809 are key players in the transport of the dairy by-product lactose in N. crassa.

Dataset Information

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

Publications

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

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