Structure-function studies on nucleotide-sugar transporter

D. ROMERO; RODOLFO M GONZÁLEZ LEBRERO; LM BREDESTON

Congreso; XLI Reunión anual de la Sociedad Argentina de Biofísica; 2012

Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of Golgi apparatus where glyconjugate biosynthesis take place by specific transferases. To these transport process an antiport mechanism, nucleotide-sugar/nucleoside monophosphate, have been proposed (Hirschberg at al, 1998). However the mechanism at the molecular level and the aminoacid residues involved in the binding and transport of substrates, 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 8-10 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 and localization 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 with the aim to obtained a collection of cysteine unique mutant.Initials studies were focused on putative TM2 where two conserved and charged aminoacids, E47 and K50, are present. Preliminary results shown: i) mutation E47C impairs transport activity; ii) more conservative substitutions E47D or K50R not recover the wild type full activity; iii) 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.