Nucleotide-Sugar-Transporter
Contact: Hans Bakker
mh-hannover.de
Molecular Cloning of two Arabidopsis UDP-galactose Transporters by Complementation of a Deficient Chinese Hamster Ovary Cell Line
Hans Bakker, Françoise Routier, Stefan Oelmann, Wilco Jordi, Arjen Lommen, Rita Gerardy-Schahn, Dirk Bosch
Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the ER and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterised. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning, using a Chinese hamster ovary cell line (CHO-Lec8) that is deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in-vitro nucleotide-sugar transport assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate and xylulose 5-phosphate. Also the latter proteins are members of the same family, which therefore has been named the Nucleotide-Sugar Transporter/Triose-Phosphate Transporter (NST-TPT) family
ER retention of the large splice variant of the UDP-galactose transporter is caused by a di-lysine motif
Roland Kabuß, Angel Ashikov, Stefan Oelmann, Rita Gerardy-Schahn and Hans Bakker
Nucleotide-sugar transporters supply mainly the Golgi glycosyltransferases with substrates. Some glycosyltransferases in the ER, however, also use these activated sugars. Recent studies have demonstrated that UDP-galactose (UDP-Gal) is the substrate for the ER resident ceramide-galactosyltransferase (cer-GalT) and cells expressing cer-GalT are able to retain the UDP-Gal transporter by physical contacts formed between the two proteins. Here, we describe a second active mechanism for ER localization of the UDP-Gal transporter. The UDP-Gal transporter is produced in two splice forms UGT1 and UGT2. The proteins vary only at their extreme C-termini but show strikingly different intracellular distribution. While N-terminally epitope tagged forms of UGT1 localize exclusively to the Golgi, similar constructs of UGT2 show both ER and Golgi localization. The di-lysine motif KVKGS contained in UGT2 can be demonstrated to be responsible for the dual localization because: (i) disturbance of the signal via site specific mutation or C-terminal extension completely shifts the transporter to the Golgi, (ii) transfer of the di-lysine motif is sufficient to redistribute the Golgi CMP-sialic acid transporter to the ER and, (iii) replacement of KVKGS by the strong ER retention signal KKNT is sufficient to completely retain UGT2 in the ER.