Independent and simultaneous translocation of two substrates by a nucleotide sugar transporter

Abstract

Nucleotide sugar transporters play an essential role in protein and lipid glycosylation, and mutations can result in developmental phenotypes. We have characterized a transporter of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine encoded by the Caenorhabditis elegans gene C03H5.2. Surprisingly, translocation of these substrates occurs in an independent and simultaneous manner that is neither a competitive nor a symport transport. Incubations of Golgi apparatus vesicles of Saccharomyces cerevisiae expressing C03H5.2 protein with these nucleotide sugars labeled with 3 H and 14 C in their sugars showed that both substrates enter the lumen to the same extent, whether or not they are incubated alone or in the presence of a 10-fold excess of the other nucleotide sugar. Vesicles containing a deletion mutant of the C03H5.2 protein transport UDP-N-acetylglucosamine at rates comparable with that of wild-type transporter, whereas transport of UDP-N-acetylgalactosamine was decreased by 85-90%, resulting in an asymmetrical loss of substrate transport.C. elegans ͉ glycosylation ͉ Golgi

Functional Redundancy between Two Caenorhabditis elegans Nucleotide Sugar Transporters with a Novel Transport Mechanism

“…5A). This result resembles the one previously described for transporter CO3H5.2 and suggests that transporter SRF-3, which is closely related to transporter CO3H5.2 in the phylogenetic tree, is also closely related in the manner by which it transports substrates, namely a non-competitive, independent, and simultaneous manner (11).…”

“…Very recently, a new nucleotide sugar multisubstrate transporter from C. elegans was characterized, CO3H5.2, with a novel, simultaneous, and non-competitive transport mechanism (11). We have now demonstrated that SRF-3, another C. elegans multisubstrate transporter, has a translocation mechanism that is similar to the one described for C03H5.2 and different from the one previously shown for SQV-7 (10).…”

“…In the present instance, nucleotide sugar transporters CO3H5.2 and SRF-3 share partial substrate and tissue specificity (11,12). Although srf-3 mutants have no morphological phenotype when grown on E. coli (12) and CO3H5.2 RNAi inactivation does not cause visual morphological defects in wild type animals, inactivation of CO3H5.2 in srf-3 animals leads to abnormal gonad morphology and oocyte accumulation in the proximal gonad arms, strongly suggesting genetic enhancement between these transporters.…”

“…Evolutionary Relationship among C. elegans Nucleotide Sugar Transporter Mechanisms-We have recently demonstrated that transporter CO3H5.2 translocates UDP-GlcNAc and UDP-GalNAc in an independent and simultaneous manner (11). This novel translocation mechanism is different from the competitive and non-cooperative transport of multiple substrates by SQV-7 (10) and prompted us to examine the transport mechanism of the multisubstrate transporter SRF-3 (12).…”

Cosmc is an essential chaperone for correct protein O-glycosylation

“…Because mouse ES cells are male and hemizygous for Cosmc, we could use these cells to examine the consequences of complete Cosmc loss on protein glycosylation. The complex biosynthesis of glycoproteins requires the activities of many dozens of enzymes in the ER, Golgi, and other organelles in the secretory pathway, including nucleotide sugar transporters, glycosidases, glycosyltransferases, and other chaperones involved in glycoprotein assembly and movement to the cell surface (22)(23)(24)(25)(26). We found that although mouse ES cells with a null Cosmc lack T-synthase activity and express Tn antigen, all other protein glycosylation, including total N-glycan profiles by mass spectrometry, and cell growth appeared normal in vitro.…”