Membrane topology of the mammalian CMP-sialic acid transporter

Nucleotide sugar transporters form a family of distantly related membrane p roteins of the Gels apparatus and the endoplasmic reticulum, The first tran sporter sequences have been identified within the last 2 years. However, in formation about the secondary and tertiary structure for these molecules ha s been limited to theoretical considerations. In the present study, an epit ope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out t o analyze the orientation of the introduced epitopes in semipermeabilized c ells. Both an amino-terminally introduced FLAG sequence and a carboxyl-term inal hemagglutinin tag were found to be oriented toward the cytosol, Result s obtained with CMP sialic acid transporter variants that contained the hem agglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions, This building co ncept seems to be preserved also in other mammalian and nonmammalian nucleo tide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loo p structures have no detectable effect on the activity. This study points t he way toward understanding structure-function relationships of nucleotide sugar transporters.