DAVID R. NELSON
ASSISTANT PROFESSOR


EDUCATION:

B.S. 1977 University of Washington
Ph.D. 1985 University of Texas Health Sciences Center at San Antonio
Postdoctoral 1985-88 University of Texas Health Sciences Center at Houston
Postdoctoral 1988-1993 University of North Carolina at Chapel Hill


RESEARCH INTERESTS: From yeast to whales, most eucaryotes are inextricably linked to their mitochondria. At the most primitive level, there are several groups of eucaryotes that do not have mitochondria and these are considered to be the most similar extant organisms to the original eucaryote host organism. These include the diplomonads like Giardia, the trichomonads and the microsporidians. However, the vast majority of eucaryotes do have mitochondria. The mitochondrial inner membrane is impermeant to small molecules and ions, even protons, yet there is an urgent need for these organelles. How is this possible? The answer is transport, a fundamental biological process. The mitochondrial inner membrane contains a family of related proteins called carriers that move small molecules across this membrane. These carriers are essential for the TCA cycle, the urea cycle and the synthesis and reexport of ATP. Of the dozen or more mitochondrial carriers whose functions are known, two key players are the phosphate carrier and the ADP/ATP carrier. These proteins supply the subtrates for ATP synthesis in the mitochondria and the means to export ATP back to the cytosol where it can be used.
The yeast ADP/ATP carrier AAC2 is the focus of my research. It provides an ideal system for studying structure/function relationships in a membrane transport protein for several reasons. First, it is small ~32,000 Da. Second it has internal sequence homology, being composed of three similar domains of about 100 amino acids each. This greatly simplifies interpretation of site directed mutagenesis experiments and aids in making predictions about structure such as interactions between membrane helices and charge pairs between specific amino acids. Currently 28 sequences of these proteins are known, which helps define the invariant amino acids that are the best candidates for mutagenesis. The best reason for using the yeast AAC2 as a model transporter is the well developed genetic system available in this single celled eucaryote. Specific genes can be deleted precisely from any of the 16 yeast chromosomes. The AAC1 and AAC2 genes have been deleted in this way to make a strain of yeast that serves as a genetic background for mutant AAC2 genes. The loss of this important gene is not fatal to yeast, because they can grow by fermentation without functional mitochondria. This permits introduction of mutant AAC2 genes and evaluation of their function by an in vivo growth test on a non-fermentable carbon source such as glycerol. Mutants that are unable to grow on glycerol are then subjected to selection for regain of function. Sequencing the mutant plasmid AAC2 gene from revertant yeast colonies identifies second site revertant mutations. Frequently, mutations that compensate for each other are quite distant in the amino acid sequence. Over 20 of these pairs have been identified and these have led to detailed structural models of the AAC2 membrane channel. Four charge pairs have been identified in places unimagined from biochemical data. This approach has been very successful and it will be continued.
Other ways of probing structure genetically involve cysteine residues. The AAC2 has four cysteines. All four of these have been changed to Ser without affecting the ability to grow on glycerol. An effort is now underway to make a cys minus protein and use it to test predictions of helix interactions. We are also making a histidine tagged mutant for use in analyzing wild type/mutant heterodimers. These results could answer questions concerning which helices are adjacent in the structure and how the AAC2 dimer is formed. A third project is development of a method to screen human patients for mutations in the ADP/ATP carrier genes. There are three genes for this protein in humans and these are differentially expressed in different tissues. Since humans need a functional copy of this gene, defects caused by mild mutations could result in diseases with muscle weakness (myopathy) or immotile or poorly motile sperm. Such patients would be good candidates to screen by PCR and dissociative gradient gel electrophoresis to find point mutations that could then be sequenced.

CURRENT RESEARCH SUPPORT:
"Analysis of mitochondrial carrier structure and function"; NIH R01 HL54248
$377,000 TDC.

PUBLICATIONS:

1. Nelson, D.R. and Robinson, N.C. (1983) Membrane proteins: A summary of known structural information. Methods in Enzymology 97:571-618.
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2. Hanahan, D.J. and Nelson, D.R. (1984) Phospholipids as dynamic participants in biological processes. Journal of Lipid Research 25:1528-1535.
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3. Nelson, D.R. and Hanahan, D.J. (1985) Phospholipid and detergent effects on (Ca2+ + Mg2+) ATPase purified from human erythrocytes. Arch. Biochem. Biophys. 236:720-730.
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4. Nelson, D.R. and Strobel, H.W. (1987) Evolution of Cytochrome P-450 Proteins.
Molecular Biology and Evolution 4:572-593.
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5. Nelson, D.R. and Strobel, H.W. (1988) On the membrane topology of vertebrate cytochrome P450 proteins. Journal of Biological Chemistry 263:6038-6050.
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6. Nelson, D.R. and Strobel, H.W. (1989) Secondary structure prediction of 52 membrane bound cytochromes P450 shows a strong structural similarity to P450cam. Biochemistry 28:656-660.
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7. Strobel, H.W., Nadler, S.N. and Nelson, D.R. (1989) Cytochrome P450: Cytochrome P450 reductase interactions. Drug Metabolism Reviews 20:519- 533.
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8. Nebert, D.W., Nelson, D.R., Coon, M.J., Estabrook, R.W., Fujii-Kuriyama, Y., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., Johnson, E.F., Loper, J.C., Sato, R., Waterman, M.R. and Waxman, D.J. (1991), The P450 superfamily: update on new sequences, gene mapping and recommended nomenclature. DNA and Cell Biology 10:1-14.
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9. Nelson, D.R., Lawson, J.E., Klingenberg, M. and Douglas, M.G. (1992) A genetic approach to studying the structure of membrane transport proteins. Application to the yeast adenine nucleotide translocator. In Molecular Mechanisms of Transport. Quagliariello, E. and Palmieri, F., eds. Elsevier Science Publishers, Amsterdam pp. 197-204.
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10. Nelson,D.R., Kamataki,T., Waxman,D.J., Guengerich,F.P., Estabrook,R.W., Feyereisen,R., Gonzalez,F.J. Coon,M.J., Gunsalus,I.C., Gotoh,O., Okuda,K. and Nebert,D.W.(1993) The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes and nomenclature. DNA and Cell Biol. 12:1- 51.
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11. Nelson, D.R., Lawson, J.E., Klingenberg, M. and Douglas, M.G. (1993) Site directed mutagenesis of the yeast mitochondrial ADP/ATP translocator. Six arginines and one lysine are essential. J. Molec. Biol. 231:1159-1170.
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12. Nelson, D.R. and Douglas, M.G. (1993) Function based mapping of a membrane transport protein by selection for second site revertants. J. Molec. Biol. 230:1171-1182.
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13. Klingenberg,M. and Nelson,D.R. (1994) Structure function relationships of the ADP/ATP carrier. Biochim. Biophys. Acta 1187:241-244.
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14. Nelson,D.R. (1995) Cytochrome P450 Nomenclature and alignment of selected sequences. in: Cytochrome P450: Structure, Mechanism and Biochemistry (second edition), ed. P.R. Ortiz de Montellano, Plenum Press, New York. pp. 575-606.
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15. Klingenberg,M. and Nelson,D. (1995) Structure-function relationships in the mitochondrial carrier family. in Biochemistry of Cell Membranes, eds. Papa,S. and Tager,J.M., Birkhauser Verlag, Basel, Switzerland. pp. 191-219.
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16. Nelson, D.R., Koymans,L., Kamataki, T., Stegeman,J.J, Feyereisen, R., Waxman, D.J., Waterman, M.R., Gotoh, O., Coon, M.J., Estabrook, R.W., Gunsalus, I.C., and Nebert, D.W. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers, and nomenclature. Pharmacogenetics6, 1-42.
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17. Durst, F. and Nelson, D.R. (1995) Diversity and Evolution of plant P450 and P450 reductases. Drug Metabolism and Drug Interactions 12, 189-206.
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18. Nelson,D.R. (1996) The yeast ADP/ATP carrier. Mutagenesis and second site revertants. Biochim. Biophys. Acta 1275, 133-137.
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19. Kawashima, H., Sequeira, D.J., Nelson,D.R. and Strobel, H.W. (1996) Protein expression and catalytic activity toward imipramine N-demethylation of a novel rat brain cytochrome P450 CYP2D18. J. Biol. Chem. submitted.
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20. Mčller, V., Heidkamper, D., Nelson, D.R. and Klingenberg, M. (1996) Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginines on oxidative phosphorylation and AAC expression. Biochemistry submitted.
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21. Heidkamper, D., Muller, V., Nelson, D.R. and Klingenberg, M. (1996) Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations on the transport and the four ADP versus ADP exchange modes. Biochemistry submitted.
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22. Kaplan, R.S., Mayor, J.A., Kakhniashvili, D., Gremse, D., Wood, D.O. and Nelson, D.R. (1996) Deletion of the Nuclear Gene encoding the mitochondrial citrate transport protein from Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. submitted.
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