William Dowhan, Ph. D.
Extended Research Objectives
Role of Phospholipids in Cell Function: My thoughts and approaches in this area are outlined in a recent review article . The basic approach now being applied in both E. coli and S cerevisiae is to construct null mutants in specific steps of phospholipid biosynthesis. For the most part such mutants are not viable at least under normal growth conditions. Next copies of the respective wild type genes under control of regulateable promoters are introduced into the null mutants so that individual steps of phospholipid metabolism and the level of specific phospholipids can be regulated exogenously. Such mutants of E. coli, as outlined in the above review, have established the role at the molecular level for the involvement of specific phospholipids in initiation of DNA replication, translocation of proteins across membranes, physical organization of the lipid bilayer, signal transduction pathways in response to cell stress, assisting membrane protein folding as a molecular chaperone , and initial steps of cell division . We have now generated similar mutants of S cerevisiae in which anionic phospholipid (phosphatidylglycerol and cardiolipin) content of the mitochondria can be varied and controlled (see below). Since these phospholipdis are essential for normal mitochondrial function and have been implicated in protein import, oxidative phosphorylation, membrane permeability, and ion sequestration, such mutants can now be used to extend the successful approach used in E. coli to eukaryotic systems to define the role of specific phospholipids in cell function.
Using the above mutants projects are possible to define both in vivo and in vitro the specific role of phospholipids in many cellular processes and to carry out detailed biochemical studies on each of these specific phospholipid-protein interactions. For the most part each of these interactions has proved to be unique and novel and therefore are of considerable importance and significance for further investigate.
Eukaryotic Phospholipid Metabolism: We have constructed mutants of and cloned the genes encoding several enzymes of phospholipid metabolism in S. cerevisiae and have isolated cDNA's encoding somatic cell enzymes of phospholipid metabolism . In yeast a major focus is to understand novel and understudied aspects of regulation of phospholipid metabolism. Cross pathway control by inositol has been extensively studied; therefore, we are concentrating on a new regulatory process involving the regulation of biosynthetic steps downstream of CDP-diacylglycerol formation by the cellular capacity of the cell to make this liponucleotide intermediate . This regulatory pathway may be operative in somatic cells because alteration in the biosynthetic capacity for this intermediate does affect cellular lipid-dependent signal transduction . A major goal currently is to identify the cis and trans acting elements responsible for this novel regulation. Phosphatidylglycerol and cardiolipin as essential lipids of the mitochondria and until recently have been understudied as to function and regulation of synthesis because of the lack of mutants and cloned genes. With the cloning of genes necessary for their synthesis in yeast as well as somatic cells studies are now underway to understand the regulation of their synthesis and their function (see above). The levels of these lipids and their respective biosynthetic pathways respond to cross pathway control, the level of CDP-diacylglycerol, and mitochondrial development. All of these areas of regulation are under investigation in the laboratory.
Enzymology of Phospholipid Metabolism: Critical to a complete understanding of lipid metabolism is a characterization of the biochemical and enzymological properties of phospholipid biosynthetic enzymes. My laboratory has recognized expertise in isolation and characterization of such enzymes. With the various cloned genes in hand we are in a position to overproduce and isolate almost all of these enzymes from E. coli and yeast as well as selective enzymes from somatic cells. Of immediate interest are the following: CDP-diacylglycerol synthase, one from yeast and two distinct isoforms from humans; phosphatidylglycerophosphate (PGP) synthase, one from yeast, one from E. coli, and one from CHO cells; cardiolipin synthase, one from yeast, one from E. coli, and one from humans. Of particular interest is the fact that the catalytic mechanisms for the eukaryotic versus the prokaryotic PGP and cardiolipin synthases are considerably different . Therefore, these steps in prokaryotic metabolism are potential sites for antibiotic development that will require more detailed understanding of the respective enzyme mechanisms. CDP-diacylglycerol synthase is a central intermediate in lipid metabolism as well as the precursor in somatic cells to the mitochondrial anionic phospholipids and the lipid-dependent signaling precursor phosphatidylinositol. This enzyme is important in the recycling of breakdown products from the lipid-dependent signal transduction pathways and its catalytic activity has been shown to affect these processes, yet little is known about its regulation.
1. Dowhan, W. (1997). Molecular basis for membrane phospholipid diversity: Why are there so many phospholipids. Annu. Rev. Biochem. 66: 199-232.
2. Bogdanov, M. and Dowhan, W. (1998). Phospholipid-assisted protein folding: Phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease. EMBO J. 17: 5255-5264.
3. Bogdanov, M. and Dowhan, W. (1999). Phospholipid-assisted refolding of an integral membrane protein. J. Biol. Chem. 274: 12339-12345.
4. Mileykovskaya, E., Sun, Q., Margolin, W. and Dowhan, W. (1998). Localization and function of early cell division proteins in filamentous Escherichia coli cells lacking phosphatidylethanolamine. J. Bacteriol. 179: 4252-4257.
5. Aitken, J. F., van Heusden, G. P. H., Temkin, M. and Dowhan, W. (1990). The gene encoding the phosphatidylinositol transfer protein is essential for cell growth. J. Biol. Chem. 265: 4711-4717.
6. Bankaitis, V. A., Aitken, J. R., Cleves, A. E. and Dowhan, W. (1990). An essential role for a phospholipid transfer protein in yeast Golgi function. Nature 347: 561-562.
7. Clancey, C. J., Chang, S. C. and Dowhan, W. (1993). Cloning of a gene (PSD1) encoding phosphatidylserine decarboxylase from Saccharomyces cerevisiae by complementation of an Escherichia coli mutant. J. Biol. Chem. 268(33): 24580-24590.
8. Shen, H., Heacock, P. N., Clancey, C. J. and Dowhan, W. (1996). The CDS1 gene encoding CDP-diacylglycerol synthase in Saccharomyces cerevisiae is essential for cell growth. J. Biol. Chem. 271: 789-795.
9. Chang, S.-C., Heacock, P. N., Clancey, C. J. and Dowhan, W. (1998). The PEL1 gene (renamed PGS1) encodes the phosphatidylglycerophosphate synthase of Saccharomyces cerevisiae. J. Biol. Chem. 273: 9829-9836.
10. Chang, S.-C., Heacock, P. N., Mileykovskaya, E., Voelker, D. R. and Dowhan, W. (1998). Isolation And Characterization of the Gene (CLS1) Encoding Cardiolipin Synthase in Saccharomyces cerevisiae. J. Biol. Chem. 273: 14933-14941.
11. Weeks, R., Dowhan, W., Shen, H., Balantac, N., Meengs, B., Nudelman, E. and Leung, D. W. (1997). Isolation and expression of an isoform of human CDP-diacylglycerol synthase cDNA. DNA Cell Biol. 16: 281-289.
12. Kawasaki, K., Kuge, O., et al. (1999). Isolation of a Chinese Hamster Ovary (CHO) cDNA Encoding Phosphatidylglycerophosphate (PGP) Synthase, Expression of Which Corrects the Mitochondrial Abnormalities of a PGP Synthase-Defective Mutant of CHO-K1 Cells. J. Biol. Chem. 274: 1828-1834.
13. Shen, H. and Dowhan, W. (1997). Regulation of phospholipid biosynthetic enzymes by the level of CDP-diacylglycerol synthase activity. J. Biol. Chem. 272: 11215-11220.
14. Shen, H. and Dowhan, W. (1998). Regulation of phosphatidylglycerophosphate synthase levels in Saccharomyces cerevisiae. J. Biol. Chem. 273: 11638-11642.
15. Bogdanov, M., Umeda, M. and Dowhan, W.: Phospholipid-assisted refolding of an integral membrane protein. J. Biol. Chem. 274: 12339-12345, 1999.
16. Rilfors, L., Niemi, A., Haraldsson, S., Edwards, K., Andersson, A.-S., and Dowhan. W.: Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles. Biochim. Biophys. Acta, 1438: 281-294, 1999.
17. Stallkamp, I., Dowhan, W., Altendorf, K., and Jung, K.: Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli. Arch. Microbiol. 172: 295-302, 1999
18. Mileykovskaya, E. and Dowhan, W.: Visualization of phospholipid domains in Escherichia coli by using cardiolipin specific fluorescent dye 10-N-nonyl-acridine orange. J. Bacteriol. 182: 1172-1175, 2000
19. Mikhaleva, N. I., Golovastov, V. V., Zolov, S. N., Bogdanov, M. V., Dowhan, W., and Nesmeyanova, M. A.: Depletion of phosphatidylethanolamine affects secretion of Escherichia coli alkaline phosphatase and its transcriptional expression. FEBS Lett. 30: 85-90, 2001
20. Ostrander, D. B., Sparagna, G. C., Amoscato, A. A., Dowhan, W., and McMillin, J. B.: Decreased Cardiolipin Synthesis Corresponds with Cytochrome c Release in Palmitate-Induced Cardiomyocyte Apoptosis. J. Biol. Chem. 276: in press, 2001.