The first evidence that NSTs are sufficient to generate transporting units without accessory proteins was obtained by expression of the active murine CMP-SA transporter in Saccharomyces cerevisiae (Berninsone et al., 1997). S. cerevisiae does not have genes involved in biosynthesis and utilization of CMP-SA and therefore, should not express specific proteins important for activity of the CMP-SA transporter. However, presence of general NST accessory proteins cannot be excluded. Conformations of the hypothesis that NSTs act as a single protein were obtained with reconstitution in proteoliposomes of purified GDP-Man and CMP-SA transporters (Segawa et al., 2005;Tiralongo et al., 2006). Both transporters were active in this background free system.
Biochemical characterizations of isolated Golgi vesicles and enriched or purified NSTs reconstituted in proteoliposomes have suggested that these transporters act as antiporters (figure 5). First evidence for the antiport mechanism has been obtained in the case of GDP-fucose transport, where an equimolar exchange of [14C]GDP-fucose and [3H]GMP was measured in rat liver microsomes (Capasso and Hirschberg, 1984). Preloading with UMP of proteoliposomes isolated from rat liver exhibited 5-fold higher transport of UDP-GlcNAc (Waldman and Rudnick, 1990). Further experiments with ER and Golgi vesicles from rat liver solubilized and reconstituted in proteoliposomes demonstrated, that preloading of vesicles with UMP significantly increased the transport of UDP-Gal, UDP-Xyl and UDP-GlcA (Milla et al., 1992) and the GMP preloading, stimulated the transport of GDP-Fuc (Puglielli and Hirschberg, 1999).
Experiments with the purified Leishmania GDP-Man transporter reconstituted into membranes, demonstrated higher transport of GDP-Man, GDP-Ara and GDP-Fuc in preloaded vesicles with GMP (Segawa et al., 2005). A purified CMP-SA transporter was stimulated by CMP inside the vesicles (Tiralongo et al., 2006). Based of these results a general antiporter mechanism was
Introduction
suggested. UDP-sugars and GDP-sugars are internalized into the ER and Golgi lumen and they are used by glycosyltransferases. The by-products of glycosylation, UDP and GDP, are dephosphorylated to form UMP and GMP, which are exported from the lumen by the same transporters that import the corresponding nucleotide sugars. Higher concentration of nucleoside monophosphate into the ER and Golgi was suggested to be the driving force of nucleotide sugar transport (figure 5).
ER or Golgi
Figure 5: Mechanism of nucleotide sugar transport. Nucleotide sugars are imported into the ER or Golgi via specific transporters. Subsequently, they are used as a substrate for glycosyltransferases. Resulting nucleoside diphosphates are dephosphorylated and nucleoside monophosphates are exported from the ER and Golgi lumen. The higher concentration of nucleoside monophosphates was suggested to be the driving force of the transport.
Only in the case of CMP-sialic acid, the nucleoside monophosphate is a direct product of the glycosyltransferase reaction. All other sugars are activated as nucleoside diphosphates, and these diphospho-nucleosides are the products released by glycosyltransferases. Therefore, Golgi resident dephosphatases were hypothesized and soon thereafter the Guanosine dephosphatese from Saccharomyces cerevisiae has been characterized and the gene (gda1) has been identified (Abeijon et al., 1993;Berninsone et al., 1994). A second nucleoside diphosphatase (ynd1) with a broader substrate specificity has been cloned from the same species. (Gao et al., 1999). Recently,
CMP
CMP-SA
CMP-SA CMP
UDP-Sugar
UDP-Sugar
UMP
UMP
Gycosyltransferases
GDP-Sugar GMP
Phosphatases
UDP GDP
GDP-Sugar GMP
single and double mutant of gda1 and/or ynd1 were analyzed and the surprising conclusion was drawn that absence of NDP-ase activity do not affect the entrance of UDP-Glcinto the ER lumen and does not change the UDP-Glc transporter dependent glycosylation in yeast. On the other hand, mannosylation is reduced in this mutant, but not absent (D'Alessio et al., 2005). Therefore, the presence of UMP and GMP in the lumen is enhancing nucleotide sugar transport but is not an absolute requirement, a conclusion also drawn from the in-vitro experiments described above.
Due to the lack of structural information of NSTs the recognition of substrates were hypothesized based on investigation of transporter chimeras, point mutation analyses, and comparison of phylogenetic relations of proteins with structures of the nucleotide sugar that is transported. The human transporters for UDP-Gal and CMP-SA are 43% identical at primary sequence level. In experiments set up to define the regions involved in recognition and transport of these structural distinct substrates, chimeras between these two transporters were generated and analyzed for the ability to complement Lec2 and Lec8 cells. Results obtained by these experiments demonstrated that TMDs 1 and 8 in the UDP-Gal-T were essential for UDP-Gal recognition and/or binding, and TMDs 2, 3 and 7 in the CMP-SA transporter are responsible for CMP-SA binding. Moreover, a bi-functional NST has been constructed by replacing TMDs 2, 3, and 7 of the UDP-Gal-T with the respective domains of the CMP-SA. The chimera complements Lec2 and Lec8 cells and, in-vitro, specifically transports UDP-Gal and CMP-Sia but no other nucleotide sugars. Substitution of helix 7 of CMP-SA into the corresponding part of UDP-Gal transporter was sufficient for CMP-SA transport activity and additional replacement of helix 2 or 3 of the UDP-Gal transporter with the corresponding sequence from the CMP-SA transporter lead to increased efficiency of CMP-SA transport. For the UDP-Gal transporter it has been shown that helices 9 and 10 or helices 2, 3, and 7 are required for the transport of UDP-Gal (Aoki et al., 1999;Aoki et al., 2001;Aoki et al., 2003).
Analyses of mutations causing the Lec2 and Lec8 phenotypes highlights conserved amino acids in the CMP-SA (Eckhardt et al., 1998) and UDP-Gal transporters respectively (Ishida et al., 1999b). Introducing the same mutations found in UDP-Gal transporter mutants into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter, indicating that these amino acids are of general importance for structure or transport activity and not for specificity (Oelmann et al., 2001). The Saccharomyces cerevisiae GDP-Man transporter (vrg4 gene) is essential for the live cycle of yeast. However, several viable mutants
Introduction
have been isolated which exhibited reduced uptake of GDP-Man. Analyses of primary amino acids sequences in GDP-Man transporters from different species, highlighted conserved domain in the region AA280-291 of vrg4. Studies demonstrated that mutations in the highly conserved region lead to lethality. Mutations did not interfere with Vrg4 protein stability, localization, or dimer formation. Alterations in this region were reduced in binding to guanosine 5'-[gamma-(32)P]triphosphate gamma-azidoanilide, a photo-affinity substrate analogue whose binding to Vrg4-HAp was specifically inhibited by GDP-mannose. Investigation of mutants suggested that AA280-291 region of the yeast GDP-Man transporter is involved in substrate recognition (Gao et al., 2001). In attempts to explain the substrate specificity present in different NSTs, phylogenetic analyses were combined with comparison of the structure of substrates that are recognized.
Eighty seven NSTs were analyzed divided in three groups including 13 subfamilies. For each subfamily conserved amino acids were identified. Based of the orientation of OH-groups in the sugar and type of the nucleotide part a mechanism of substrate recognition in different subfamilies was hypothesized. It has highlighted the importance of the orientation of the OH-group on position C4 in the sugar part as well the nucleotide part in discrimination of GDP-sugars from UDP-GDP-sugars (Martinez-Duncker et al., 2003). However, no direct proofs of these hypotheses, based of interaction nucleotide sugars-transporter were obtained so far.