Mutation of two charged aminoacids in transmembrane helix 2 impairs UDP-GlcNac transport activity of mouse SLC35A3.

TOSCANINI MA; LEVALLE A; BREDESTON LM

Villa Carlos Paz

Congreso; XLII Annual Meeting of Argentinean Biophysical Society; 2013

Sociedad Argentina de Biofisica

Mutation of two charged aminoacids in transmembrane helix 2 impairs UDP-GlcNac transport activity of mouse SLC35A3 Toscanini MA, Levalle A, Bredeston LM IQUIFIB (CONICET-UBA), Departamento de Química Biológica, Facultad. de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina. bredes@qb.ffyb.uba.ar Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of Golgi apparatus where glyconjugate biosynthesis take place. To these transport process an antiport mechanism, nucleotide-sugar/nucleoside monophosphate, have been proposed. However the amino acids residues involved in the binding and transport of NS, are unknown. In this work we studied the mouse SLC35A3 transporter (MmSLC35A3), specific for UDP-GlcNac, as a model of NST. MmSLC35A3 is a 326 aminoacids protein with a predicted 10 transmembrane helices. The goal of the project is the identification of aminoacids essentials for the transport process. For these porpoise we: a) engineered a C-terminal GFP tagged SLC35A3 to detect expression in yeast, b) used a complementation assay in a yeast mutant lacking the UDP-GlcNac transporter, to test activity and c) constructed an active mutant devoid of cysteins (SLC35A3-CL) with the aim to obtained a collection of cysteine unique mutant. The cystein scanning mutagenesis of 23 residues of SLC35A3, including the TM2 helix, from Leu38 (loop betweeen TM1-2) to Lys60 (loop betweeen TM2-3) was performed. Expression of each mutant in yeast, detected in whole cells, showed that all the mutants were expressed at similar levels of SLC35A3-CL. Meanwhile the activity, tested by the binding of the lectin GSII to the cell surface GlcNac residues, showed that two mutations E47C and K50C impairs the UDP-GlcNac transport activity of SLC35A3. More conservative substitutions E47D or K50R not recover the wild type full activity. Double mutant E47K/K50E showed a residual activity, indicating the residues are not interchangeable. All together these results suggest that both charged aminoacids are important for transport mechanism.