Abstract

The research described in this proposal is designed to elucidate the mechanisms by which phospholipids are assembled into mitochondrial and plasma membranes; and to determine the role that specific phospholipids play in the structure and function of these membranes. These goals will be accomplished by establishing mutants of Chinese hamster ovary (CHO) cells that are deficient in the synthesis of phosphatidylethanolamine and sphingomyelin. Under conditions of ethanolamine starvation the synthesis of phosphatidylethanolamine in CHO cells is dependent upon the intracellular transport of phosphatidylserine from the endoplasmic reticulum to the mitochondrion. Thus, by selecting for ethanolamine auxotrophs in CHO cells it will be possible to identify a subclass of mutants that are incapable of the intracellular transport of phosphatidylserine to the mitochondria. By using the techniques of in vitro complementation between mutant and parental cells we will identify the factors responsible for this transport process, and determine whether they are related to the phospholipid exchange/transfer proteins. Ethanolamine auxotrophs will also be used to examine the effects of phosphatidylethanolamine depletion upon membrane structure and function. Sphingomyelin synthesis is dependent upon the intracellular transport of phosphatidylcholine and ceramide from the endoplasmic reticulum to the plasma membrane. By selecting mutants of CHO cells that either require sphingomyelin for growth, or are unable to express sphingomyelin as a surface antigen, it will be possible to identify mutants that are deficient in the intracellular transport of ceramide. These mutants will be used to critically evaluate the role of phospholipid exchange/transfer proteins in the assembly of plasma membranes. These mutants will also be used to determine the effects of sphingomyelin depletion upon plasma membrane structure and function. Full biochemical characterization of these mutants will provide important insights into the mechanisms of membrane biogenesis in mammalian cells. The regulation of these mechanisms must ultimately be related to control of cell growth and differentiation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032453-03
Application #
3281301
Study Section
Metabolism Study Section (MET)
Project Start
1983-07-01
Project End
1986-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
National Jewish Health
Department
Type
DUNS #
City
Denver
State
CO
Country
United States
Zip Code
80206

  Publications

Riekhof, Wayne R; Wu, Wen-I; Jones, Jennifer L et al. (2014) An assembly of proteins and lipid domains regulates transport of phosphatidylserine to phosphatidylserine decarboxylase 2 in Saccharomyces cerevisiae. J Biol Chem 289:5809-19
Gupta, Nishith; Hartmann, Anne; Lucius, Richard et al. (2012) The obligate intracellular parasite Toxoplasma gondii secretes a soluble phosphatidylserine decarboxylase. J Biol Chem 287:22938-47
Steinhauer, Josefa; Gijón, Miguel A; Riekhof, Wayne R et al. (2009) Drosophila lysophospholipid acyltransferases are specifically required for germ cell development. Mol Biol Cell 20:5224-35
Nishikawa, Yoshifumi; Quittnat, Friederike; Stedman, Timothy T et al. (2005) Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma. Cell Microbiol 7:849-67
Quittnat, Friederike; Nishikawa, Yoshifumi; Stedman, Timothy T et al. (2004) On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme. Mol Biochem Parasitol 138:107-22
Voelker, Dennis R (2003) New perspectives on the regulation of intermembrane glycerophospholipid traffic. J Lipid Res 44:441-9
Rontein, Denis; Wu, Wen-I; Voelker, Dennis R et al. (2003) Mitochondrial phosphatidylserine decarboxylase from higher plants. Functional complementation in yeast, localization in plants, and overexpression in Arabidopsis. Plant Physiol 132:1678-87
Schumacher, Marc M; Choi, Jae-Yeon; Voelker, Dennis R (2002) Phosphatidylserine transport to the mitochondria is regulated by ubiquitination. J Biol Chem 277:51033-42
Wu, Wen I; Voelker, Dennis R (2002) Biochemistry and genetics of interorganelle aminoglycerophospholipid transport. Semin Cell Dev Biol 13:185-95
Kitamura, Hidemitsu; Wu, Wen-I; Voelker, Dennis R (2002) The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function. J Biol Chem 277:33720-6

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