Genomes and Genes
LIPID CARRIERS IN MEMBRANE GLYCOPROTEIN SYNTHESIS
Principal Investigator: William Lennarz
Affiliation: State University of New York
Abstract: The goal of this research is to investigate two separate but related issues pertaining to polyprenois: 1) The topological organization in endoplasmic reticulum (ER) of glucosyl- P-dollchol (Glc-P-Dol) and mannosyl-P-dollchol (Man-P-Dol) and the enzymes that catalyze their synthesis. Two steps in the overall oligosaccharide assembly process that occurs in the ER, the synthesis of Glc-P-Dol and Man-P-Dol, will be studied in yeast. These two reactions were chosen because, although they both are chemically very similar, in rat liver ER the former is believed to involve a sugar nucleotide transporter and occurs in the lumen, whereas the latter is thought to not require a transporter and occurs at the cytoplasmic face. To study their synthesis probes will be developed for the glycose moelty of each lipid intermediate in phospholipid liposomes, and then these will be applied to topology studies in yeast ER. Impermeable probes to be used will be metaperlodate, which will oxidize the sugar residues, and enzymes that will either covalently alter or release the sugar residues. Having established the topological orientation of the glycose moelties of Glc-P- Dol and Man-P-Dol, the orientation of the synthases that catalyze their formation will be studied. To accomplish this in yeast, Glc-P-Dol synthase, which has recently been identified by photoaffinity labelling will be cloned and sequenced. The deducted sequence will be used to interpret studies on the topological orientation of the protein after its 1) in vitro synthesis and insertion in the ER and 2) reconstitution in liposomes. Similar studies will be carried out with yeast Man-P-Dol synthase which recently has been cloned and sequenced. These experiments should allow one to determine if the assembly of Glc-P-Dol and Man-P-Dol involves vectorial translocation of these lipid-linked sugars. 2) The mechanism and function of polyprenol attachment to proteins. The objective of these studies will be to elucidate the mechanism and function of the protein polyprenylation process. The two systems to be utilized are cultured insect cells and developing sea urchin embryos. Preliminary experiments indicate that in vivo incorporation of labeled polyprenyl groups (such as farnesol) into proteins using labeled mevalonic acid as precursor is readily detected in insect cells, presumably because these cells lack the enzyme system that converts farnesol to cholesterol. Pulse-chase labeling experiments will be carried out to study the kinetics of synthesis and turnover of these polyprenylated proteins. In addition, the mechanism of the polyprenylation process will be studied in vitro using apoproteins or synthetic peptides as acceptors, and by varying the structure of the polyprenol donor. Efforts will be made to use exogenous acceptor peptides as substrates so as to inhibit specifically protein polyprenylation in vivo, and thereby obtain insight into the function(s) of this process. Finally, because palmitoylation of proteins has already been demonstrated in the developing sea urchin embryo, which initially undergoes rapid cell proliferation and subsequently extensive cell differentiation, protein polyprenylation will be examined in this system. These studies should define the possible relationship between the biosynthetic pathway for polyprenylated proteins, signal transduction and cell proliferation.
Funding Period: 1989-08-01 - 1994-11-30
more information: NIH RePORT
- Misfolding of glycoproteins is a prerequisite for peptide: N-glycanase mediated deglycosylationShivanjali Joshi
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA
FEBS Lett 579:823-6. 2005..Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins...
- Pkc1p modifies CPY* degradation in the ERAD pathwayMihai Nita-Lazar
Department of Biochemistry and Cell Biology and the Institute of Cell and Developmental Biology, SUNY Stony Brook, NY, USA
Biochem Biophys Res Commun 332:357-61. 2005..In addition, we show that Cdc48p interacts in the cytosol with the deglycosylation enzyme, PNGase, only when Cdc48p is associated with a misfolded glycoprotein...
- Studies on the intracellular localization of hHR23BSamiksha Katiyar
Department of Biochemistry and Cell Biology, The Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA
Biochem Biophys Res Commun 337:1296-300. 2005..When the cell enters mitosis, hHR23B relocalizes in the cytoplasm without association with chromatin. These results indicate that the intracellular distribution hHR23B is cell cycle dependent...
- The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sitesGeng Tian
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, NY 11794, USA
Cell 124:61-73. 2006..The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds...
- The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptorGuangtao Li
Department of Biochemistry and Cell Biology, 450 Life Sciences Building, Stony Brook University, Stony Brook, NY 11794 5215, USA
Proc Natl Acad Sci U S A 103:8348-53. 2006....
- The catalytic activity of protein-disulfide isomerase requires a conformationally flexible moleculeGeng Tian
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794 5215, USA
J Biol Chem 283:33630-40. 2008....