MOLECULAR-CLONING OF THE HAMSTER CMP-SIALIC ACID TRANSPORTER

Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 compleme ntation group are unable to express sialylated glycoproteins and glyco lipids due to a defect in the Golgi CMP-sialic acid transporter (CMP-S ia-Tr). Using an expression cloning strategy, we isolated a cDNA encod ing the hamster CMP-Sia-Tr which complements the Lec2 phenotype. The d educed amino acid sequence of the cloned cDNA shows 95% identity to th e recently cloned murine CMP-Sia-Tr. The expression of a hamster CMP-S ia-Tr fusion protein with an N-terminal MDYKDDDDK (FLAG) sequence reve aled Golgi localisation of the transporter Amino acid sequence compari son revealed strong similarity (44.6% identity and 19.3% similarity) o f CMP-Sia-Tr to the recently cloned human UDP-galactose transporter (U DP-Gal-Tr). In contrast, sequence similarities to the yeast UDP-N-acet ylglucosamine transporter (UDP-GlcNAc-Tr) and the GDP-mannose transpor ter (GDP-Man-Tr) of Leishmania donovani are restricted to a region enc oding the two most C-terminally located transmembrane helices. A compu ter-based structural analysis of CMP-Sia-Tr proposes an eight transmem brane helix model with the N- and C-termini located on the cytosolic s ide of the Golgi membrane.