Perspectives on Retinal Dolichol Metabolism, and Visual Deficits in Dolichol Metabolism-Associated Inherited Disorders.

De novo synthesis of dolichol (Dol) and dolichyl phosphate (Dol-P) is essential for protein glycosylation. Herein, we provide a brief overview of Dol and Dol-P synthesis and the maintenance of their cellular content. Retinal Dol metabolism and the requirement of Dol-linked oligosaccharide synthesis in the neural retina also are discussed. There are recently discovered and an emerging class of rare congenital disorders that affect Dol metabolism, involving the genes DHDDS, NUS1, SRD5A3, and DOLK. Further understanding of these congenital disorders is evolving, based upon studies utilizing yeast and murine models, as well as clinical reports of these rare disorders. We summarize the known visual deficits associated with Dol metabolism disorders, and identify the need for generation and characterization of suitable animal models of these disorders to elucidate the underlying molecular and cellular mechanisms of the associated retinopathies.

cis-prenyltransferase 3 and α/ß-hydrolase are new determinants of dolichol accumulation in Arabidopsis.

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for post-translational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this study, we have analysed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis-the AT2G17570 gene encoding a long searched for cis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/ß-hydrolase-is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans.

Inhibition of Dephosphorylation of Dolichyl Diphosphate Alters the Synthesis of Dolichol and Hinders Protein N-Glycosylation and Morphological Transitions in Candida albicans.

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.

Long-Chain Polyisoprenoids Are Synthesized by AtCPT1 in Arabidopsis thaliana.

Arabidopsis roots accumulate a complex mixture of dolichols composed of three families, (i.e., short-, medium- and long-chain dolichols), but until now none of the cis-prenyltransferases (CPTs) predicted in the Arabidopsis genome has been considered responsible for their synthesis. In this report, using homo- and heterologous (yeast and tobacco) models, we have characterized the AtCPT1 gene (At2g23410) which encodes a CPT responsible for the formation of long-chain dolichols, Dol-18 to -23, with Dol-21 dominating, in Arabidopsis. The content of these dolichols was significantly reduced in AtCPT1 T-DNA insertion mutant lines and highly increased in AtCPT1-overexpressing plants. Similar to the majority of eukaryotic CPTs, AtCPT1 is localized to the endoplasmic reticulum (ER). Functional complementation tests using yeast rer2Δ or srt1Δ mutants devoid of medium- or long-chain dolichols, respectively, confirmed that this enzyme synthesizes long-chain dolichols, although the dolichol chains thus formed are somewhat shorter than those synthesized in planta. Moreover, AtCPT1 acts as a homomeric CPT and does not need LEW1 for its activity. AtCPT1 is the first plant CPT producing long-chain polyisoprenoids that does not form a complex with the NgBR/NUS1 homologue.

Pathogenic mutations of the human mitochondrial citrate carrier SLC25A1 lead to impaired citrate export required for lipid, dolichol, ubiquinone and sterol synthesis.

Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.

Genetic defects in dolichol metabolism.

Congenital disorders of glycosylation (CDG) comprise a group of inborn errors of metabolism with abnormal glycosylation of proteins and lipids. Patients with defective protein N-glycosylation are identified in routine metabolic screening via analysis of serum transferrin glycosylation. Defects in the assembly of the dolichol linked Glc(3)Man(9)GlcNAc(2) glycan and its transfer to proteins lead to the (partial) absence of complete glycans on proteins. These defects are called CDG-I and are located in the endoplasmic reticulum (ER) or cytoplasm. Defects in the subsequent processing of protein bound glycans result in the presence of truncated glycans on proteins. These defects are called CDG-II and the enzymes involved are located mainly in the Golgi apparatus. In recent years, human defects have been identified in dolichol biosynthesis genes within the group of CDG-I patients. This has increased interest in dolichol metabolism, has resulted in specific recognizable clinical symptoms in CDG-I and has offered new mechanistic insights in dolichol biosynthesis. We here review its biosynthetic pathways, the clinical and biochemical phenotypes in dolichol-related CDG defects, up to the formation of dolichyl-P-mannose (Dol-P-Man), and discuss existing evidence of regulatory networks in dolichol metabolism to provide an outlook on therapeutic strategies.

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.

Regulation of dolichol-linked glycosylation.

In the majority of congenital disorders of glycosylation, the assembly of the glycan precursor GlcNAc(2)Man(9)Glc(3) on the polyprenol carrier dolichyl-pyrophosphate is compromised. Because N-linked glycosylation is essential to life, most types of congenital disorders of glycosylation represent partial losses of enzymatic activity. Consequently, increased availability of substrates along the glycosylation pathway can be beneficial to increase product formation by the compromised enzymes. Recently, we showed that increased dolichol availability and improved N-linked glycosylation can be achieved by inhibition of squalene biosynthesis. This review summarizes the current knowledge on the biosynthesis of dolichol-linked glycans with respect to deficiencies in N-linked glycosylation. Additionally, perspectives on therapeutic treatments targeting dolichol and dolichol-linked glycan biosynthesis are examined.

Approaches to homozygosity mapping and exome sequencing for the identification of novel types of CDG.

In the past decade, the identification of most genes involved in Congenital Disorders of Glycosylation (CDG) (type I) was achieved by a combination of biochemical, cell biological and glycobiological investigations. This has been truly successful for CDG-I, because the candidate genes could be selected on the basis of the homology of the synthetic pathway of the dolichol linked oligosaccharide in human and yeast. On the contrary, only a few CDG-II defects were elucidated, be it that some of the discoveries represent wonderful breakthroughs, like e.g, the identification of the COG defects. In general, many rare genetic defects have been identified by positional cloning. However, only a few types of CDG have effectively been elucidated by linkage analysis and so-called reverse genetics. The reason is that the families were relatively small and could-except for CDG-PMM2-not be pooled for analysis. Hence, a large number of CDG cases has long remained unsolved because the search for the culprit gene was very laborious, due to the heterogeneous phenotype and the myriad of candidate defects. This has changed when homozygosity mapping came of age, because it could be applied to small (consanguineous) families. Many novel CDG genes have been discovered in this way. But the best has yet to come: what we are currently witnessing, is an explosion of novel CDG defects, thanks to exome sequencing: seven novel types were published over a period of only two years. It is expected that exome sequencing will soon become a diagnostic tool, that will continuously uncover new facets of this fascinating group of diseases.

Influence of liver-X-receptor on tissue cholesterol, coenzyme Q and dolichol content.

The organ content of the mevalonate pathway lipids was investigated in liver-X-receptor (LXR) α, ß and double knock-out mice. An extensive or moderate increase of total cholesterol in the double KO mice was found in all organs elicited by the increase of the esterified form. In LXRα and double KO mice, coenzyme Q (CoQ) was decreased in liver and increased in spleen, thymus and lung, while dolichol was increased in all organs investigated. This effect was confirmed using LXR- agonist GW 3965. Analysis of CoQ distribution in organelles showed that the modifications are present in all cellular compartments and that the increase of the lipid in mitochondria was the result of a net increase of CoQ without changing the number of mitochondria. It appears that LXR influences not only cellular cholesterol homeostasis but also the metabolism of CoQ and dolichol, in an indirect manner.

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid.

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.

Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol-oligosaccharides might alter the degree of beta-1,6 branching in N-linked carbohydrates are discussed.

Long-Chain Polyprenols Promote Spore Wall Formation in Saccharomyces cerevisiae.

Dolichols are isoprenoid lipids of varying length that act as sugar carriers in glycosylation reactions in the endoplasmic reticulum. In Saccharomyces cerevisiae, there are two cis-prenyltransferases that synthesize polyprenol-an essential precursor to dolichol. These enzymes are heterodimers composed of Nus1 and either Rer2 or Srt1. Rer2-Nus1 and Srt1-Nus1 can both generate dolichol in vegetative cells, but srt1∆ cells grow normally while rer2∆ grows very slowly, indicating that Rer2-Nus1 is the primary enzyme used in mitotically dividing cells. In contrast, SRT1 performs an important function in sporulating cells, where the haploid genomes created by meiosis are packaged into spores. The spore wall is a multilaminar structure and SRT1 is required for the generation of the outer chitosan and dityrosine layers of the spore wall. Srt1 specifically localizes to lipid droplets associated with spore walls, and, during sporulation there is an SRT1-dependent increase in long-chain polyprenols and dolichols in these lipid droplets. Synthesis of chitin by Chs3, the chitin synthase responsible for chitosan layer formation, is dependent on the cis-prenyltransferase activity of Srt1, indicating that polyprenols are necessary to coordinate assembly of the spore wall layers. This work shows that a developmentally regulated cis-prenyltransferase can produce polyprenols that function in cellular processes besides protein glycosylation.

cis-Prenyltransferase and Polymer Analysis from a Natural Rubber Perspective.

Dolichol and natural rubber are representative cis-polyisoprenoids in primary and secondary metabolism, respectively. Their biosynthesis is catalyzed by cis-prenyltransferase (CPT) by sequential condensations of isopentenyl diphosphates (IPPs) to a priming molecule. Although prokaryotic CPTs have been well characterized, the mechanism of eukaryotic CPTs in cis-polyisoprene biosynthesis was only recently revealed. It was shown that eukaryotes have evolved a unique protein complex, comprised of CPT and CPT-binding protein (CBP), to synthesize cis-polyisoprenoids. In the context of this new discovery, we found discrepancies in literature for CPT or CBP biochemical assays and in vivo CPT complementation using rer2 (yeast CPT) yeast mutant. Our study here shows that rer2 revertants occur at a frequency that cannot be disregarded and are likely accountable for the results that cannot be explained by the CPT/CBP heteroprotein complex model. To make a stable mutant, SRT1 gene (secondary CPT expressed at a basal level in yeast) was additionally deleted in the rer2Δ mutant background. This stable rer2Δ srt1Δ strain was then used to individually or simultaneously express Arabidopsis CPT1 (AtCPT1, At2g17570) and CBP (AtLEW1, At1G11755). We found that the simultaneous expression of Arabidopsis CPT1 and AtLEW1 effectively complements the rer2Δ srt1Δ strain, whereas the individual expression of AtCPT1 alone or AtLEW1 alone failed to rescue the yeast mutant. Microsomes from the dual expresser showed an efficient incorporation of IPPs into cis-polyisoprenoid (30% in 2h). These results showed that the CPT/CBP heteroprotein complex model is valid in Arabidopsis thaliana. Experimental details of these results are described in this methodology paper.

trappc11 is required for protein glycosylation in zebrafish and humans.

Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We term the pathological UPR that causes fatty liver disease a "stressed UPR." Here we investigate the mechanism of stressed UPR activation in zebrafish bearing a mutation in thetrappc11gene, which encodes a component of the transport protein particle (TRAPP) complex.trappc11mutants are characterized by secretory pathway defects, reflecting disruption of the TRAPP complex. In addition, we uncover a defect in protein glycosylation intrappc11mutants that is associated with reduced levels of lipid-linked oligosaccharides (LLOs) and compensatory up-regulation of genes in the terpenoid biosynthetic pathway that produces the LLO anchor dolichol. Treating wild-type larvae with terpenoid or LLO synthesis inhibitors phenocopies the stressed UPR seen intrappc11mutants and is synthetically lethal withtrappc11mutation. We propose that reduced LLO level causing hypoglycosylation is a mechanism of stressed UPR induction intrappc11mutants. Of importance, in human cells, depletion of TRAPPC11, but not other TRAPP components, causes protein hypoglycosylation, and lipid droplets accumulate in fibroblasts from patients with theTRAPPC11mutation. These data point to a previously unanticipated and conserved role for TRAPPC11 in LLO biosynthesis and protein glycosylation in addition to its established function in vesicle trafficking.

A novel family of longer chain length dolichols present in oleate-induced yeast Saccharomyces cerevisiae.

The typical size of the yeast dolichol family ranges from 14 to 19 isoprene units D((14-19)) with dolichol(16) being the dominating species. Induction of peroxisome proliferation by growing the cells in medium containing oleate as carbon source induces the synthesis of an additional family of longer dolichols D((19-24)) with D(21) being the most prominent. This phenomenon is abolished in the peroxisome biogenesis deficient strain in which the PEX1 gene (encoding Pex1p peroxin) has been disrupted. The total amount of dolichols in pex1Delta cells is lower than in the wild-type cells, as is the amount of phosphatidylcholine. Moreover, the levels of 3-hydroxy-3-methylglutaryl CoA reductase and farnesyl diphosphate synthase, two key enzymes in dolichol biosynthesis, are decreased in the absence of a functional PEX1 gene. The presence of longer dolichols in oleate-induced Saccharomyces cerevisiae cells, the absence of this additional family in peroxisome deficient cells, and a decrease of the total amount of dolichols in these cells indicate the involvement of peroxisomes in the biosynthesis of dolichols in this organism.

[Yeast dolichol-conjugated mannosyltransferases. A theoretical analysis of the primary structure and identification of the sites homologous to other enzymes of the dolichol cycle]. / Drozhzhevye dolikhol-sopriazhennye mannoziltransferazy. Teoreticheskii analiz pervichnoi struktury i identifikatsiia uchastikov, gomologichnykh drugim fermentam dolikhol'nogo tsikla.

Comparative analysis of primary structure of the enzymes of dolichol cycle, yeast GDP-Man:Dol-PP-GlcNAc2 beta-mannosyltransferase (product of ALG1 gene) and yeast GDP-Man:Dol-PP-GlcNAc2Man2 alpha-1,3-mannosyltransferase (product of ALG2 gene) was conducted. In amino acid sequence of ALG2 protein extensive symmetrical segments were identified, in particular, 230-452 segment symmetrical relatively to Lys 346. Besides, repeated segments, homologous to the ones earlier identified by us in ALG1 protein were revealed in the molecule of ALG2 protein. The analysis of secondary structure of ALG2 protein allowed to reveal sites capable for forming alpha-helices with heptade periodicity. These helices can be included in formation of coifed-coifed structures. Similar structures were revealed earlier in the molecule of ALG1 protein. For sites able for formation of alpha-helices and neighbouring sites there was shown high homology of primary structure observed by us both at the comparison of ALG1 and ALG2 with each other and at the comparison with other enzymes of dolichol cycle. The data obtained point to evolutionary relationship between dolichol-coupled enzymes and in particular, between mannosyltransferases and dolicholphosphomannosylsynthase, using the same substrate GDP-mannose.

Antimalarial drug targets and drugs targeting dolichol metabolic pathway of Plasmodium falciparum.

Because of mutation and natural selection, development of drug resistance to the existing antimalarial is the major problem in malaria treatment. This problem has created an urgent need of novel antimalarial drug targets as well as lead compounds. The important characteristic of malaria is that it shows the phenomenon of balanced polymorphisms. Several traits have been selected in response to disease pressure. Therefore such factors must be explored to understand the pathogenesis of malaria infection in human host. Apicoplast, hub of metabolism is present in Plasmodium falciparum (causative agent of falciparum malaria) having similarities with plant plastid. Among several pathways in apicoplast, Dolichol metabolic pathway is one of the most important pathway and has been known to play role in parasite survival in the human host. In P.falciparum, a phosphorylated derivative of Dolichol participates in biosynthesis of glycoproteins. Several proteins of this pathway play role in post translational modifications of proteins involved in the signal transduction pathways, regulation of DNA replication and cell cycle. This pathway can be used as antimalarial drug target. This report has explored progress towards the study of proteins and inhibitors of Dolichol metabolic pathway. For more comprehensive analysis, the host genetic factors and drug-protein interaction have been covered.

Biochemical characterization and membrane topology of Alg2 from Saccharomyces cerevisiae as a bifunctional alpha1,3- and 1,6-mannosyltransferase involved in lipid-linked oligosaccharide biosynthesis.

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc(3)Man(9) GlcNAc(2)-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the alpha1,3- and the alpha1,6-mannose-linked residue from GDP-mannose to Man(1)GlcNAc(2)-PP-Dol forming the Man(3)GlcNAc(2)-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX(7)E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys(230), as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.

Proliferation-dependent differential regulation of the dolichol pathway genes in Saccharomyces cerevisiae.

The dolichol pathway serves in the synthesis of the dolichol-linked oligosaccharide precursor for protein N-glycosylation. Recently, we reported that mRNAs of genes that function at the early steps in the dolichol pathway in yeast, ALG7, ALG1 and ALG2, were co-ordinately induced following growth stimulation of G0-arrested cells in a manner similar to that of the transcripts of the early growth response genes (Kukuruzinska, M.A. and Lennon, K. Glycobiology, 4, 437-443, 1994). To determine whether the entire dolichol pathway was co-ordinately regulated with growth, we examined the expression of genes functioning late in the pathway, including two genes encoding oligosaccharyltransferase subunits, at two critical control points in the G1 phase of cell cycle: G0/G1 and START. We show that early in G1, at the G0/G1 transition point, the late ALG genes and the two oligosaccharyltransferase-encoding genes examined were regulated co-ordinately with the early ALG genes: they were downregulated upon exit from the mitotic cell cycle into G0, and they were induced following growth stimulation in the absence of de novo protein synthesis. All the dolichol pathway genes produced transcripts with short half-lives that were rapidly stabilized in the presence of cycloheximide. In contrast, cell division arrest late in G1, at START, was accompanied by a selective downregulation of only the first dolichol pathway gene, ALG7, and not of the genes functioning later in the pathway. These results indicate that, depending on their position in G1, cells either co-ordinately or differentially regulate the dolichol pathway genes.