Cystein scanning mutagenesis and precrystallization studies of the UDP-GlcNac transporter SLC35A3

FAVAROLO MB; TOSCANINI MA; LEVALLE A; TABOADA G; BREDESTON LM

Buenos Aires

Simposio; 1er simposio argentino de glicobiologia; 2014

Fundacion Instituto Leloir

Cystein scanning mutagenesis and precrystallization studies of the UDP-GlcNac transporter SLC35A3 Favarolo B, Toscanini MA, Levalle A, Taboada G, Bredeston LM IQUIFIB (CONICET-UBA), Argentina. bredes@qb.ffyb.uba.ar Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of GA and ER where glyconjugate biosynthesis take place. To these transport process an antiport mechanism, nucleotide-sugar/nucleoside monophosphate, have been proposed. However the molecular mechanism is unknown. The goals of the project are the identification of aminoacids essentials for the transport process and the production of large quantities of NST to initiate precrystallization studies. To study the Golgi SLC35A3 from mouse (specific for UDP-GlcNac) we: a) engineered a C-terminal GFP tagged SLC35A3 to detect expression in whole yeast, b) used a complementation assay in a yeast mutant to test activity and c) constructed an active mutant devoid of cysteins (mSLC35A3CL). The cystein scanning mutagenesis from Leu38 to Lys60, including the TM2 helix residues of mSLC35A3CL, was performed. All the mutants were expressed at similar levels of mSLC35A3CL-GFP. Meanwhile only two mutations, E47C and K50C, impairs the transport activity, suggesting that are important for the transport mechanism. For structural studies screening of expression and purification conditions of mSLC35A3 in S. cerevisiae were optimizing using GFP fusion technology. The expression levels of mSLC35A3-GFP were > 2mg/L culture. The efficiency of the complete NTA-Ni purification procedure monitoring by GFP fluorescence was 65% and the yield 1.3 mg of purified mSLC35A3-GFP/ L culture