Current research in this laboratory focuses on the way in which membrane glycoproteins are expressed at their site of synthesis in the rough endoplasmic reticulum and what particular properties of the protein sequence and carbohydrate microstructure are responsible for the sorting of the membrane glycoproteins to their final membrane compartment. We are studying particular membrane glycoproteins in the rough endoplasmic reticulum by using membrane maturing viruses as a probe. Rotavirus SA11 matures by budding into the RER of infected cells. The membrane, initially acquired from the host cell (RER) membrane, is later lost giving rise to the double protein shelled virus typical of the Reoviridae. The structural glycoprotein VP7, also the neutralizing antigen, and the nonstructural glycoprotein, NCVP5, have been located to the RER by immunoelectronmicroscopy and by immunofluorescence. These integral membrane glycoproteins are not found in the Golgi apparatus or the plasma membrane using the cloned genes. We are performing in vitro site-specific mutagenesis in order to identify regions in the gene products which are important in the RER location of the molecules. We have shown that disruption of the second of 2 hydrophobic domains at the N-ter of VP7 causes it to be secreted with terminal glycosylation, consistent with passage through the Golgi-Hybrid molecules of functional sequences from rotavirus genes. Other molecules are being constructed to demonstrate the molecular basis for such translocation. The oligosaccharide portion of the rotavirus glycoproteins undergo a set of specific processing steps by enzymes located in the ER and the Golgi appartus. The form of the oligosaccharide therefore provides an indication of where the glycoproteins are located in the cell and what compartments they have already migrated through. Comparative studies of the processing of VP7 carbohydrate, which is retained in the ER, and of vesicular stomatitis virus G protein, which is exported, indicate a subcompartment of the ER containing VP7. It is being studied further. We are using high field (500 MHz) spectroscopy in combination with conventional complex carbohydrate chemistry to elucidate the molecular structure of N-linked glycoprotein oligosaccharides. We plan to seek alpha 1,2-mannosidases which may be growth-dependent and also located in the rough endoplasmic reticulum. (A)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA013402-14
Application #
3163758
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1979-01-01
Project End
1986-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
14
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Maass, D R; Atkinson, P H (1994) Retention by the endoplasmic reticulum of rotavirus VP7 is controlled by three adjacent amino-terminal residues. J Virol 68:366-78
Verostek, M F; Atkinson, P H; Trimble, R B (1993) Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum. J Biol Chem 268:12095-103
Verostek, M F; Atkinson, P H; Trimble, R B (1993) Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. II. Structure of novel Man6-10GlcNAc2 processing intermediates on secreted invertase. J Biol Chem 268:12104-15
Trimble, R B; Atkinson, P H (1992) Structural heterogeneity in the Man8-13GlcNAc oligosaccharides from log-phase Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic study. Glycobiology 2:57-75
Poruchynsky, M S; Maass, D R; Atkinson, P H (1991) Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER. J Cell Biol 114:651-6
Trimble, R B; Atkinson, P H; Tschopp, J F et al. (1991) Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris. J Biol Chem 266:22807-17
Poruchynsky, M S; Atkinson, P H (1991) Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles. J Virol 65:4720-7
Verostek, M F; Atkinson, P H; Trimble, R B (1991) Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides. J Biol Chem 266:5547-51
Maass, D R; Atkinson, P H (1990) Rotavirus proteins VP7, NS28, and VP4 form oligomeric structures. J Virol 64:2632-41
Bergmann, C C; Maass, D; Poruchynsky, M S et al. (1989) Topology of the non-structural rotavirus receptor glycoprotein NS28 in the rough endoplasmic reticulum. EMBO J 8:1695-703

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