Biomedical subjects

T C Huffaker

Publications and source records attributed to T C Huffaker.

22 records · Page 2Linked to original sources

PubMed1984

Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway.

Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.

Acetylglucosaminidase↗

PubMed1983

Yeast mutants deficient in protein glycosylation.

The synthesis of asparagine-linked oligosaccharides involves the formation of a lipid-linked precursor oligosaccharide that has the composition Glc3Man9GlcNAc2. We have used a [3H]mannose suicide selection to obtain mutants in yeast that are blocked in the synthesis of this precursor oligosaccharide. The alg1 mutant accumulated lipid-linked GlcNAc2, alg2 mutants accumulated Man1-2GlcNAc2, alg3 mutants accumulated Man5GlcNAc2, alg4 mutants accumulated Man1-8GlcNAc2, and alg5 and alg6 mutants accumulated Man9GlcNAc2. Some of these mutants appeared to transfer oligosaccharides other than Glc3Man9GlcNAc2 from the lipid carrier to invertase. These aberrant protein-linked oligosaccharides were processed by the addition of outer chain residues in the alg3, alg5, and alg6 mutants. There was virtually no outer chain addition in the alg2 and alg4 mutants. alg4 was the only mutant that failed to secrete invertase.

Alleles↗

PubMed1982

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly.

The formation of N-glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.

Asparagine↗

PubMed1982

Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation.

A [3H]mannose suicide selection has been used to isolate mutants in yeast which contain temperature-sensitive defects in asparagine-linked glycosylation. The surviving cells were screened at the nonpermissive temperature for a decreased ability to incorporate [3H]mannose and for defects in glycosylation of the secreted protein invertase. One of these mutants (alg1-1) has been characterized and found to be blocked in the assembly of the lipid-linked oligosaccharide precursor. The alg1-1 cells synthesize mannosyl compounds at 60% of the wild type level at the nonpermissive temperature and 105% of the wild type level at the permissive temperature. In vivo labeling experiments have demonstrated that alg1-1 cells are able to synthesize GlcNAc2-lipid but are unable to synthesize any mannose-containing oligosaccharide-lipids. This result was confirmed by in vitro labeling of yeast membranes. When incubated with UDP-[3H]GlcNAc, alg1-1 membranes synthesized GlcNAc2-lipid but failed to elongate it when GDP-Man was added. The alg1-1 membranes also failed to elongate exogenous GlcNAc2-lipid but were able to convert Man1GlcNAc2-lipid to Man5-Glc-NAc2-lipid in the presence of GDP-Man. These results indicate that the alg1-1 mutant is blocked specifically in the addition of the first mannose residue to the lipid-linked oligosaccharide precursor.

Asparagine↗