Figure 4.
Mutational analysis of evolutionary conserved residues.A, alignment of hSLC35B1 and putative orthologs from evolutionary distant organism: A. thaliana (AtUTr3), S. pombe (Sphut1), and C. elegans (CeHut1). Conserved charged residues were labeled in yellow, and the C-terminal ER retention motif was labeled in red. B, proteins purified from yeast membranes expressing WT, K117A, K120A, or K277A variants were separated by SDS-PAGE and GFP fusions visualized by gel fluorescence and CB staining. C, [α32P]ATP uptake activity of purified hSLC35B1-GFP variants inserted in liposomes loaded with 10 mM of ADP, ATP, or UDP, and incubated with 200 μM [α32P]ATP for 1 min at 37 °C. The bars represent the mean values + SEM (n = 3). ER, endoplasmic reticulum; hSLC35B1, human SLC35B1 isoform.