Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility. The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi membrane. A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner. These nucleotide sugars are competitive, alternate, noncooperative substrates. The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities. SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDPmannose. SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi. These results demonstrate that unlike most nucleotide sugar transporters, SQV-7 can transport multiple distinct nucleotide sugars. We propose that SQV-7 translocates multiple nucleotide sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.M ost cell surface and secreted proteins and some lipids undergo covalent modifications by the addition of carbohydrates (1, 2). These macromolecules play essential roles in multicellular organisms by participating in normal embryonic development and cell-cell and cell-matrix interactions. The carbohydrate-deficient glycoprotein syndromes, a group of autosomal recessive multisystemic diseases characterized by defective glycosylation of N-glycans, and studies of null mutations of N-glycan biosynthetic enzymes in mice provide strong evidence that the glycan moieties of glycoproteins play essential roles in the normal development and physiology of mammals and probably of all multicellular organisms (3-5). Disorders affecting the assembly of the glycosaminoglycan moieties of proteoglycans suggest the importance of these macromolecules in connective tissue, cartilage, and bone development (3). Heparan sulfate glycosaminoglycans (GAGs) are critical components of Wingless and fibroblast growth factor signaling in Drosophila melanogaster, and defects in heparan sulfate GAG assembly are associated with effects on Drosophila embryonic development, such as abnormalities in segment-polarity cuticle patterns and in mesodermal and tracheal cell migrations (6-9). Mutations of the EXT genes, a tumor suppressor family that includes glycosyltransferases involved in polymerization of heparan sulfates, have been associated with hereditary multiple exostoses, a skeletal dysplasia characterized by multiple cartilage-capped skeletal tumors (10).Nucleotide sugar transporters translocate nucleotide sugars from the cytosol, their site of synthesis, into the lumen of the Golgi apparatus, where they are used as sugar-donor substrates by glycosyltransferases (11). Yeast, Leishmania do...

The enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH) is responsible for the synthesis of N-glycolylneuraminic acid (Neu5Gc), a sialic acid present on the cell surface proteins of most deuterostomes. The CMAH gene is thought to be present in most deuterostomes, but it has been inactivated in a number of lineages, including humans. The inability of humans to synthesize Neu5Gc has had several evolutionary and biomedical implications. Remarkably, Neu5Gc is a xenoantigen for humans, and consumption of Neu5Gc-containing foods, such as red meats, may promote inflammation, arthritis, and cancer. Likewise, xenotransplantation of organs producing Neu5Gc can result in inflammation and organ rejection. Therefore, knowing what animal species contain a functional CMAH gene, and are thus capable of endogenous Neu5Gc synthesis, has potentially far-reaching implications. In addition to humans, other lineages are known, or suspected, to have lost CMAH; however, to date reports of absent and pseudogenic CMAH genes are restricted to a handful of species. Here, we analyze all available genomic data for nondeuterostomes, and 322 deuterostome genomes, to ascertain the phylogenetic distribution of CMAH. Among nondeuterostomes, we found CMAH homologs in two green algae and a few prokaryotes. Within deuterostomes, putatively functional CMAH homologs are present in 184 of the studied genomes, and a total of 31 independent gene losses/pseudogenization events were inferred. Our work produces a list of animals inferred to be free from endogenous Neu5Gc based on the absence of CMAH homologs and are thus potential candidates for human consumption, xenotransplantation research, and model organisms for investigation of human diseases.

No abstract

During the establishment of a bacterial infection, the surface molecules of the host organism are of particular importance, since they mediate the first contact with the pathogen. In Caenorhabditis elegans, mutations in the srf-3 locus confer resistance to infection by Microbacterium nematophilum, and they also prevent biofilm formation by Yersinia pseudotuberculosis, a close relative of the bubonic plague agent Yersinia pestis. We cloned srf-3 and found that it encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi apparatus membrane. srf-3 is exclusively expressed in secretory cells, consistent with its proposed function in cuticle/surface modification. We demonstrate that SRF-3 can function as a nucleotide sugar transporter in heterologous in vitro and in vivo systems. UDP-galactose and UDP-N-acetylglucosamine are substrates for SRF-3. We propose that the inability of Yersinia biofilms and M. nematophilum to adhere to the nematode cuticle is due to an altered glycoconjugate surface composition of the srf-3 mutant.

Edited by Gerald W. HartO-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.

We have functionally expressed the murine Golgi putative CMP-sialic acid transporter in Saccharomyces cerevisiae. Using a galactose-inducible expression system, S. cerevisiae vesicles were able to transport CMPsialic acid. Transport was dependent on galactose induction and was temperature-dependent and saturable with an apparent K m of 2.9 M. Transport was inhibited by CMP, and upon vesicle disruption with Triton X-100 parameters were very similar to the previously described CMP-sialic acid transport characteristics observed with mammalian Golgi vesicles. CMP-sialic acid transport induction was specific as no transport of UDPgalactose was observed even though the latter putative transporter has a high degree of amino acid sequence identity with the CMP-sialic acid transporter. Together, the above results demonstrate that the previously described cDNA encoding the putative CMP-sialic acid transporter encodes the transporter protein per se and suggests that this heterologous expression system may be used for further structural and functional studies of other Golgi membrane transporter proteins.Transporters for nucleotide sugars, nucleotide sulfate, and ATP of the Golgi apparatus membrane are required for the translocation of these solutes from the cytosol into the lumen of this organelle (1, 2). Within this compartment these nucleotide derivatives and ATP are substrates for glycosylation, sulfation, and phosphorylation of secretory and membrane-bound proteins as well as lipids (1, 2). Studies in vitro and in vivo have shown these transporters to be antiporters with the corresponding nucleoside monophosphates (1, 2). These transport activities have been detected in Golgi vesicles from mammals (1, 2), yeast (1, 2), protozoa (3), and plants (4). Mutants defective in transport activities of CMP-sialic acid (1, 2), UDPgalactose (1, 2), UDP-N-acetylglucosamine (1, 2), and GDPmannose (3, 5) have been described in these organisms and have a block of glycosylation of proteins and lipids in vivo. These phenotypes have been used as selection for expression cloning of nucleic acids, which encode proteins that correct the phenotype of the yeast UDP-GlcNAc transporter (6), the murine CMP-sialic acid transporter (7), the Leishmania donovani GDP-mannose transporter (5), and the human UDP-galactose transporter (8). So far in every instance, a highly hydrophobic multitransmembrane spanning domain protein was found to correct the mutant phenotype, suggesting that the protein is the corresponding Golgi membrane nucleotide sugar transporter. In some studies, the protein was localized to the Golgi apparatus (3, 7), while in other cases Golgi vesicles from the transfected mutant cells were shown to have recovered the ability to transport the nucleotide sugar, which the corresponding mutant cell line vesicle lacked (3, 6).We have expressed the murine Golgi apparatus membrane putative CMP-sialic acid transporter in Saccharomyces cerevisiae for the following reasons. (a) Because yeast cells do not synthesize sialoglycoconjugates and do not...

The adhesion of growing neurites into appropriate bundles or fascicles is important for the development of correct synaptic connectivity in the nervous system. We describe fasciculation defects of animals with mutations in the C. elegans gene dig-1 and show that dig-1 encodes a giant molecule (13,100 amino acids) of the immunoglobulin superfamily. Five new alleles of dig-1 were isolated in a screen for mutations affecting the morphology or function of several classes of head sensory neurons. Mutants showed process defasciculation of several classes of neurons. Analysis of a temperature-sensitive allele revealed that dig-1 is required during embryogenesis for normal process fasciculation of one class of head sensory neuron. Partial sequencing of two alleles, RNA interference (RNAi) and rescuing experiments showed that dig-1 encodes a giant molecule of the immunoglobulin superfamily. DIG-1 protein contains many domains associated with adhesion, is likely secreted, and has some features of proteoglycans. dig-1 mutants were originally isolated due to their displaced gonads [Thomas, J.H., Stern, M.J., Horvitz, H.R., 1990. Cell interactions coordinate the development of the C. elegans egg-laying system. Cell 62, 1041-52]; thus, dig-1 alleles were also characterized for their effects on gonad placement. Mutant phenotypes suggest that DIG-1 may mediate cell movement as well as process fasciculation and that different regions of the protein may mediate these functions.

No abstract