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. 2016 Jul 6;7:12119. doi: 10.1038/ncomms12119

Figure 3. LC-MS/MS analysis of nucleotide sugar transport activities for GFT1 and other GONST members.

Figure 3

(a) Immunoblot analysis of empty vector, GFT1, GONST1 and GONST2 expression in proteo-liposomes (2.5 μg protein per lane). (b) Separation of a 20 nucleotide/nucleotide sugar mix (1, CMP; 2, UMP; 3, UDP-GalA; 4, UDP-GlcA; 5, CMP-Sia; 6, UDP-Arap; 7, UDP-Rha; 8, UDP-Gal; 9, UDP-Glc; 10, UDP-Xyl; 11, UDP-GlcNAc/GalNAc; 12, UDP-Araf; 13, PAPS; 14, GMP; 15, AMP; 16, GDP-Man; 17, GDP-Gal; 18, GDP-Glc, 19, GDP-Fuc; 20, ADP-Glc). LC-MS/MS analysis of proteo-liposomes derived from (c) yeast transformed with the empty vector (control) or (d) yeast expressing GFT1 after simultaneous incubation with 16 nucleotide sugar substrates. (e) Quantification of nucleotide sugar transport into proteo-liposomes pre-loaded with 10 mM GMP. (f and g) Exchange substrate specificities of GFT1 and GONST1. Proteo-liposomes were loaded with 1 mM GDP-Fuc (f) or 1 mM GDP-Man (g) and incubated with a mix of UMP, GMP, CMP, AMP at a concentration of 200 μM. Data are mean and s.d. of n=4 assays.