The long-term goals of this research proposal are the following: (a) To understand the cellular mechanisms which regulate the glycosylation of proteins and lipids in the rough endoplasmic reticulum and Golgi apparatus and, (b) to establish the role of protein and lipid glycosylation in organelle topography and compartmentation during secretion and membrane biogenesis. We propose to pursue the following specific aims towards achieving these goals: (1) To continue with our studies on the mechanisms of glycosylation in the Golgi apparatus of mammalian cells. We will use biochemical and molecular biological approaches to purify the Golgi CMPNeuAc and UDP-Galactose transporters and to clone the respective genes. Using our recently described proteoliposome reconstitution system, we will use affinity and conventional chromatography to purify the transporters. We will also rescue plasmids, containing cDNAs of the Golgi UDP-Galactose and CMPNeuAc transporters, from transfectants of mutant Chinese hamster ovary cells, which are deficient in the above transport activities. Antibodies against the transporters will be made from the proteins or based on the cDNAs and used in immunoelectronmicroscopy studies to determine whether the transporters are polarized within the Golgi apparatus. In conjunction with proteases, the antibodies will also be used to study the arrangement of the transporter proteins in the Golgi membrane. The cDNAs will be used to study the structure of the transporter genes and how the expression of these proteins is regulated. (2) To continue with our studies on the subcellular organization and topography of glycosylation in the Golgi apparatus of yeast. We will transform, with a genomic library made from wild-type DNA, a mutant of K. lacis. recently characterized by us to be deficient in UDP-GlcNAc transport into Golgi-like vesicles in order to isolate and characterize the Golgi UDP-GlcNAc transporter gene. We will clone and disrupt the gene of the Golgi membrane GDPase, a lumenal marker enzyme from S. cerevisiae recently purified in our laboratory and hypothesized to be necessary for Golgi mannosylation. Antibodies against the GDPase and the K. lactis UDP-GlcNAc transporters (obtained via their DNA sequence) will be used to study, (a) by immunoelectronmicroscopy, their location within the cell, and (b) via sensitivity towards proteases to establish the proteins' topography in the Golgi membrane. (3) To continue with our studies on the topography of glycosylation in the rough endoplasmic reticulum. Using membrane impermeable probes, we will attempt to demonstrate, directly, translocation of dolichol-oligosaccharide derivatives from the cytosolic side of the membrane into the lumen. Reconstitution studies with endoplasmic reticulum (ER) membrane proteins and liposomes will be attempted to demonstrate the occurrence of a dolichol-oligosaccharide translocator protein in the ER membme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
7R37GM030365-24
Application #
2734451
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1987-07-01
Project End
2001-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
24
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Hirschberg, Carlos B (2018) My journey in the discovery of nucleotide sugar transporters of the Golgi apparatus. J Biol Chem 293:12653-12662
Caffaro, Carolina E; Koshy, Anita A; Liu, Li et al. (2013) A nucleotide sugar transporter involved in glycosylation of the Toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites. PLoS Pathog 9:e1003331
Liu, Li; Hirschberg, Carlos B (2013) Developmental diseases caused by impaired nucleotide sugar transporters. Glycoconj J 30:5-10
Xu, Yu-Xin; Liu, Li; Caffaro, Carolina E et al. (2010) Inhibition of Golgi apparatus glycosylation causes endoplasmic reticulum stress and decreased protein synthesis. J Biol Chem 285:24600-8
Liu, Li; Xu, Yu-Xin; Hirschberg, Carlos B (2010) The role of nucleotide sugar transporters in development of eukaryotes. Semin Cell Dev Biol 21:600-8
Uccelletti, D; Pascoli, A; Farina, F et al. (2008) APY-1, a novel Caenorhabditis elegans apyrase involved in unfolded protein response signalling and stress responses. Mol Biol Cell 19:1337-45
Caffaro, Carolina E; Luhn, Kerstin; Bakker, Hans et al. (2008) A single Caenorhabditis elegans Golgi apparatus-type transporter of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine. Biochemistry 47:4337-44
Caffaro, Carolina E; Hirschberg, Carlos B; Berninsone, Patricia M (2007) Functional redundancy between two Caenorhabditis elegans nucleotide sugar transporters with a novel transport mechanism. J Biol Chem 282:27970-5
Caffaro, Carolina E; Hirschberg, Carlos B; Berninsone, Patricia M (2006) Independent and simultaneous translocation of two substrates by a nucleotide sugar transporter. Proc Natl Acad Sci U S A 103:16176-81
Caffaro, Carolina E; Hirschberg, Carlos B (2006) Nucleotide sugar transporters of the Golgi apparatus: from basic science to diseases. Acc Chem Res 39:805-12

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