Cloning and functional characterization of human nucleotide-sugar transporters

The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells, showed specific uptake for a total of eight nucleotide sugars, for activated sulfate (Adenosine 3'-phospho 5'-phosphosulfate, PAPS) and ATP. By phenotypic correction of biochemically characterized mutants several transporters have been identified and revealed that NSTs are type III membrane proteins with eight to ten trans-membrane domains (TMD). When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatics methods were used to display putative NST-sequences in the human genome. Twenty eight human and eight yeast open reading frames were selected as putative NSTs. Nine proteins were hetero- or homologeously expressed in yeast and transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells, demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the ER and Golgi lumen, our data suggest that two ways exist to deliver UDP-Xyl to the Golgi apparatus. While these studies identified the first UDP-Xyl transporter a, potential UDP-Glc transporter could not be found due to the high endogenous transport background present in S. cerevisiae. To identify this remaining activity and to search for a specific ATP transporter six yeast strains with defects in the SLC35 gene family were used as expression systems of candidate genes. Testing of prepared Golgi fractions demonstrated, however, that redundancy exists in the transport of these essential solute compounds and a single gene deletion is not enough to reduce the endogenous transport. Importantly, observations made in these assays are in excellent agreement with the lately identified multi-substrate specificity of many members in the SLC35 family, which clearly contrasts earlier biochemical data describing NSTs as mono-substrate specific. Low (better zero) -background systems are, therefore, needed to further investigate SLC35 family members. Towards this goal my subsequent studies were concentrated at generating and evaluating such systems. With SLC35B4 as model system reconstitution of recombinantly expressed protein in artificial proteoliposomes was tested. Because expression of the SLC35B4 protein in E. coli was not possible, purification of this recombinant protein after expression in S. cerevisiae was used but resulted in copurification of endogenous Golgi proteins and thus hampered the reconstitution experiments. Consequently, efforts were focused at cell free synthesis of SLC35B4. Using this method homogenous protein was obtained in sufficient concentration for reconstitution in true zero background systems. The reconstituted SLC35B4 confirmed specific transport for UDP-Xyl. However, the second transport activity for UDP-GlcNAc could not be detected in this system. Together, the results clearly demonstrate that the SLC35B4 protein is a human UDP-Xyl transporter. The second activity determined in the yeast system is, by all likelihood, formed in the cell by involvement of endogenous factors. The method newly developed in this study provides a crucial step forward should help to reevaluate transport specificities of know proteins and to characterize unknown members in the SLC35 family.