Conserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3

• Published in: The Journal of biological chemistry Vol. 294; no. 26; pp. 10042 - 10054
• Main Authors: Toscanini, M. Agustina, Favarolo, M. Belén, Gonzalez Flecha, F. Luis, Ebert, Berit, Rautengarten, Carsten, Bredeston, Luis M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28-06-2019; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; antiporter; congenital glycosylation disorder; Glutamic Acid - metabolism; glycoprotein biosynthesis; Golgi; Golgi Apparatus - metabolism; Ion Transport; Lysine - metabolism; Membrane Biology; membrane protein; membrane transport; Mice; Models, Molecular; Protein Conformation; secretory pathway; Sequence Homology; Sodium-Phosphate Cotransporter Proteins, Type IIc - chemistry; Sodium-Phosphate Cotransporter Proteins, Type IIc - genetics; Sodium-Phosphate Cotransporter Proteins, Type IIc - metabolism; solute carrier family 35 member A3 (SLC35A3); structural model; UDP-GlcNAc metabolism; Uridine Diphosphate N-Acetylglucosamine - genetics; Uridine Diphosphate N-Acetylglucosamine - metabolism; Uridine Monophosphate - metabolism; antiporter; glycoprotein biosynthesis; congenital glycosylation disorder; UDP-GlcNAc metabolism; membrane transport; membrane protein; secretory pathway; solute carrier family 35 member A3 (SLC35A3); Golgi; structural model
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.008827

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.