Identification of essential amino acid residues in the hydrophilic loop regions of the CMP-sialic acid transporter and UDP-galactose transporter.

The Golgi CMP-sialic acid transporter (CST) is a type III transmembrane protein with 10 transmembrane domains that are linked by eight hydrophilic loops. To investigate the function of these hydrophilic loops, the green fluorescent protein (GFP) was inserted into each loop of the transporter. Expression and localization of the resulting CST-GFP fusion proteins were confirmed by analyzing the fluorescence of GFP. The transport activity of the CST-GFP proteins was analyzed by a previously described erythropoietin/isoelectric focusing assay in CST-deficient MAR-11 cells. Interruption of the second and fourth lumenal loops and the fourth cytosolic loop of CST with GFP resulted in complete or partial loss of transport activity. Regions in these loops that play crucial roles in CST's activity were identified by Gly substitutions. Single amino acid substitution experiments revealed that Lys(272) of the fourth loop on the cytosolic side of CST is essential for transport activity. Mutation of the conserved Lys residue (Lys(297)) in the UDP-galactose transporter (UGT) also resulted in a complete loss of its activity. Point mutations of highly conserved amino acid residues in the loop regions identified Leu(136) of CST as essential for its activity. However, mutation of the conserved Leu residue in UGT (Leu(160)) did not affect the transport activity of UGT. Finally, mutation of Leu(224) in UGT completely inactivated the activity of UGT, although mutation of its conserved counterpart in CST, Leu(199), did not have any effect on CST. This study provides a structure-function analysis of the loop regions in CST and UGT.