MUTANTS OF THE CMP-SIALIC ACID TRANSPORTER CAUSING THE LEC2 PHENOTYPE

Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementat ion group are unable to translocate CMP-sialic acid to the lumen of th e Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter f rom mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid t ransport, To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyz ed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutatio n. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural cha nges on the expression and subcellular targeting of the transporter pr oteins. Mutants derived from deletions showed reduced protein expressi on and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation prod uct derived from the point mutated cDNA (G189E) was expressed at the l evel of the wild-type transporter and co-localized with the Golgi mark er cu-mannosidase ll. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. Whi le the G189A mutant was able to complement CMP-sialic acid transport-d eficient Chinese hamster ovary mutants, the exchange of glycine 189 in to glutamine or isoleucine dramatically affected the transport activit y of the CMP-sialic acid transporter.