Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast.

To further evaluate the role of Rft1 in the transbilayer movement of Man(5)GlcNAc(2)-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc(3)Man(9)GlcNAc(2)-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[(3)H]mannose or UDP-[(3)H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [(3)H]Man(9)GlcNAc(2)-P-P-dolichol or Man(9)[(3)H]GlcNAc(2)-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc(2)-P-P-Dol(15), a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [(3)H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [(3)H]GlcNAc(2)-P-P-Dol(15) in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.

Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis.

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.

Oligosaccharyl diphosphodolichols in the ceroid-lipofuscinoses.

Autopsy brain samples from patients with late-infantile, juvenile and adult forms of ceroid-lipofuscinosis (CL) and cultured skin fibroblasts from juvenile CL were analyzed for the content of phosphodolichol (P-Dol) related compounds. The levels of P-Dol obtained on treatment with hot dilute acid of the chloroform-methanol (CM 2:1) extract and the chloroform-methanol-water (CMW 1:1:3) extract of the residue were estimated by high performance liquid chromatography. Compared to age-matched control individuals, the levels of P-Dol obtained in both the extracts were increased more than 6.6 times in all the patient samples. Further analysis of the CMW extract indicates that the increased P-Dol is primarily due to oligosaccharyl diphosphodolichol. Cultured skin fibroblasts from the juvenile form of CL show normal level of free dolichol and elevated level of phosphorylated dolichols. Glycoprotein synthesis measured by incorporation of labeled glucosamine show no deficit in the transfer of oligosaccharides from lipids to proteins. A hypothesis is presented to explain the accumulation of oligosaccharyl diphosphodolichol and deficiency of lysosomal proteases in ceroid-lipofuscinosis.

Transmembrane organization of protein glycosylation. Mature oligosaccharide-lipid is located on the luminal side of microsomes from Chinese hamster ovary cells.

The transmembrane orientation of Glc3Man9GlcNAc2-pyrophosphoryl-dolichol, which is the oligosaccharide donor in the glycosylation of asparagine residues of eukaryotic glycoproteins has been investigated. The lectin concanavalin A was used as a nonpenetrating probe to study the location of this oligosaccharide-lipid in microsomal vesicles prepared from cultured fibroblasts. Lectin treatment of intact vesicles, and vesicles made leaky with low concentrations of detergent showed that this oligosaccharide-lipid is on the luminal side of membrane. The oligosaccharide-lipid was bound by lectin only if the permeability barrier of the membrane had been destroyed by detergent; very little binding was seen in intact vesicles. This result suggests that glycosylation of nascent secretory and membrane glycoproteins occurs on the luminal side of the membrane. It also implies that sugar residues derived from cytoplasmic sugar nucleotides must be transported across the membrane at some point during the synthesis and accumulation of mature, luminal oligosaccharide-lipid, although the identity of the transported species remains unknown.

The topological orientation of N,N'-diacetylchitobiosylpyrophosphoryldolichol in artificial and natural membranes.

Purified N,N'-diacetylchitobiosylpyrophosphoryldolichol (chitobiosyl-lipid) in the presence of detergent was shown to act as a substrate for the soluble enzyme galactosyltransferase. The nature of this transfer reaction and the galactose-containing trisaccharide-lipid product have been partially characterized. Using galactosyltransferase as a probe, the topolical arrangement of chitobiosyl-lipid in both artificial and natural membranes has been examined. When incorporated into unilamellar liposomes made from phosphatidylcholine, the disaccharide residue of chitogiosyl-lipid adopts a random transbilayer orientation. Furthermore, no significant mobility in the transverse plane of the membrane (i.e. flip-flop) is detectable. Using both sealed total microsomes or isolated rough microsomes from hen oviduct, the topology of chitobiosyl-lipid after its biosynthesis in the membrane has been determined. The results suggest that, once formed, chitobiosyl-lipid is a relatively static component of the membrane and is oriented with its disaccharide residue facing the lumen of the endoplasmic reticulum.