The lumens of the endoplasmic reticulum and Golgi apparatus are the subcellular sites where glycosylation, sulfation, and phosphorylation of secretory and membrane-bound proteins, proteoglycans, and lipids occur. Nucleotide sugars, nucleotide sulfate, and ATP are substrates for these reactions. ATP is also used as an energy source in the lumen of the endoplasmic reticulum during protein folding and degradation. The above nucleotide derivatives and ATP must first be translocated across the membrane of the endoplasmic reticulum and/or Golgi apparatus before they can serve as substrates in the above lumenal reactions. Translocation of the above solutes is mediated for highly specific transporters, which are antiporters with the corresponding nucleoside monophosphates as shown by biochemical and genetic approaches. Mutants in mammals, yeast, and protozoa showed that a defect in a specific translocator activity results in selective impairments of the above posttranslational modifications, including loss of virulence of pathogenic protozoa. Several of these transporters have been purified and cloned. Experiments with yeast and mammalian cells demonstrate that these transporters play a regulatory role in the above reactions. Future studies will address the structure of the above proteins, how they are targeted to different organelles, 49 0066-4154/98/0701-0049$08.00 Annu. Rev. Biochem. 1998.67:49-69

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP-and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wildtype organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.evolution ͉ N-glycans ͉ protist

Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHSI and CHS2 with the Candida albicans CHSI gene (UDP-N-acetyl-D-glucosamine:chitin 4-j3-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (=600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced amino acid sequences were aligned. With the exception of S. cerevisiae CHSI, the sequences fell into three distinct classes, which could represent separate functional groups. Within each class phylogenetic analysis was performed. Although not the major purpose of the investigation, this analysis tends to confirm some relationships consistent with current taxonomic groupings.Chitin, the ,/1-4-linked polymer of N-acetylglucosamine, is a fibrous cellulose-like polysaccharide that serves as the major cell wall/exoskeleton scaffolding in many species of fungi, arthropods, insects, and crustacea. In many yeasts chitin is used to maintain the structure of the mother-bud junction, whereas in filamentous fungi chitin is often the major supporting component of the cell wall.Early enzymatic studies in yeast and filamentous fungi showed that much of the chitin synthase activity was latent, requiring protease activation (1). Recent genetic and molecular studies in Saccharomyces cerevisiae suggest that this organism has at least two protease-activated chitin synthase zymogens as well as a more complex chitin synthase system that may not require protease activation (2).When the derived amino acid sequences of the two Sa. cerevisiae chitin synthase zymogens (from CHSJ and CHS2) were compared with the sequence of a closely related Candida albicans gene (for UDP-N-acetyl-D-glucosamine:chitin 4-f3-N-acetylglucosaminyltransferase, EC 2.4.1.16), it became clear that the enzymes contained a highly conserved sequence that possibly represents the catalytic region of the enzymes. We decided to use degenerate PCR primers that encoded short, completely conserved sequences within the three genes to probe genomic DNA from a variety offungi.** PCR-derived fragments were cloned into M13, and single nucleotide sequencing runs were used to classify the clones. Representative clones were then completely sequenced, and the deduced amino acid sequences were put into groups by the CLUSTAL program. The aligned DNA sequences within these groups, or classes, were analyzed further with the FITCH program.

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Abstract. Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O-and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.

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The endoplasmic reticulum (ER) and other secretory compartments of Saccharomyces cerevisiae have biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct. In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content. These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of the S. cerevisiae ER structure, both in intact cells and at the ultrastructural level. Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane. Double-labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment. This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycles.