Abstract
Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an EXXK motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein.
Highlights
Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans
A bioinformatics analysis revealed that an EXXK motif in transmembrane helix 2 (TMH2) is highly conserved across solute carrier 35 family (SLC35) A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein
Mouse Slc35a3, a protein consisting of 326 amino acids, shares between 95 and 96% sequence identity with four mammalian SLC35A3 variants, which previously have been experimentally characterized as Golgi-localized UDP-GlcNAc transporters [31, 37, 45, 46]
Summary
Mouse Slc35a3 (mSLC35A3), a protein consisting of 326 amino acids, shares between 95 and 96% sequence identity with four mammalian SLC35A3 variants, which previously have been experimentally characterized as Golgi-localized UDP-GlcNAc transporters [31, 37, 45, 46]. The suspension of Kl3 cells transformed with pE4mSLC35A3 showed fluorescence levels similar to that of WT Kl8 cells, carrying the functional UDP-GlcNAc transporter, indicating that they recovered the ability to bind the lectin GSII–FITC (Fig. 1A, dark gray bars). Incubation with GSII–FITC showed that most of the cells expressing cysteine-unique mutants bound the lectin, with fluorescence intensity values higher than 40% of the WT, consistent with a restoration of the UDP-GlcNAc import into the lumen of the Golgi apparatus (Fig. 3). Kl3 cells transformed with the mutant alleles A42V, A42G, V44A, V44I, or V44L fused to GFP showed fluorescence levels similar to those observed for well-expressed proteins (white bars, Fig. S3) and values of GSII–FITC binding between 50 and 140% compared with Kl3 cells expressing the WT transporter (Fig. 5). Kinetic parameters of UDP-GlcNAc transport mediated by mSlc35a3 and selected mutants
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