Todd R. Graham, Ph.D.

Professor

tr.graham@vanderbilt.edu
Faculty Appointments
Professor of Biological Sciences Professor of Cell and Developmental BiologyProfessor of Cell and Developmental BiologyProfessor of Cell and Developmental BiologyProfessor of Cell and Developmental BiologyProfessor of Cell and Developmental BiologyProfessor of Cell and Developmental BiologyProfessor of Cell and Developmental BiologyProfessor of Cell and Developmental Biology
Education
Ph.D., Cell & Molecular Biology, Saint Louis University, Saint Louis, MissouriPh.D., Cell & Molecular Biology, Saint Louis University, Saint Louis, MissouriPh.D., Cell & Molecular Biology, Saint Louis University, Saint Louis, MissouriPh.D., Cell & Molecular Biology, Saint Louis University, Saint Louis, MissouriB.S., Maryville College, Maryville, TennesseeB.S., Maryville College, Maryville, TennesseeB.S., Maryville College, Maryville, TennesseeB.S., Maryville College, Maryville, Tennessee
Office Address
Department of Biological Sciences
VU Station B Box 35-1634
Nashville, TN 37235-1634
Research Description
The research goals of the Graham laboratory are to understand the molecular mechanisms underpinning vesicle-mediated protein transport and membrane biogenesis. Most of our effort is focused on determining how type IV P-type ATPases (P4-ATPases) contribute to the establishment of membrane asymmetry and budding of transport vesicles from organelle membranes using the yeast model system.
The P4-ATPases flip specific phospholipid species, such as phosphatidylserine, from the extracellular leaflet of the plasma membrane to the cytosolic leaflet, thus producing an asymmetric membrane structure that is conserved among most eukaryotic cells. This phospholipid asymmetry has a major influence on the localization and activity of many different plasma membrane proteins. Moreover, regulated disruption of membrane asymmetry is a signaling device used in blood clotting reactions and for recognition of apoptotic cells. Humans have 14 P4-ATPases and members of this protein family are implicated in severe liver and neurological disease. Additionally, murine P4-ATPases are implicated in B-cell deficiency, obesity and type 2 diabetes, motor neuron degeneration, defects in bile secretion, and reduced male fertility. A current project in the laboratory is to define the mechanism of substrate recognition and translocation by P4-ATPases using molecular genetic and biochemical approaches. The best characterized P-type ATPases transport small cations across membranes to establish ion gradients and so phospholipid molecules are an unusual substrate for this protein family. Our work is suggesting a novel transport mechanism for the P4-ATPases and is providing insight into how these transporters evolved the ability to transport their "giant substrate".
In addition to establishing membrane asymmetry, we discovered that P4-ATPases play a crucial role in budding protein transport vesicles from Golgi and endosomal membranes. For example, a P4-ATPase called Drs2 translocates phosphatidylserine across the membrane of the trans-Golgi network and this flippase activity is required to bud AP-1/clathrin-coated vesicles that transport proteins from the Golgi to endosomes. Our work has uncovered both positive and negative regulators of Drs2 activity representing proteins and lipids known to have critical roles in vesicular transport. The ATP-powered, unidirectional translocation of phosphatidylserine to cytosolic leaflet should have a dramatic influence on the biophysical properties of the membrane; enhancing the anionic membrane potential of the cytosolic surface as well as inducing curvature through a bilayer couple mechanism. Another current project in the lab is to determine how these P4-ATPase effects on the membrane are coordinated with the vesicle budding machinery to sort and package cargo proteins into newly forming vesicles. The protein trafficking events dependent on P4-ATPase function have a major influence on the protein composition of the plasma membrane and organelles of the secretory and endocytic pathways.

Research Keywords
Protein transport and membrane biogenesis
Publications
Xu P, Baldridge RD, Chi RJ, Burd CG, Graham TR. Phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular transport. J. Cell Biol [print-electronic]. 2013 Sep 9/16/2013; 202(6): 875-86. PMID: 24019533, PMCID: PMC3776346, PII: jcb.201305094, DOI: 10.1083/jcb.201305094, ISSN: 1540-8140.

Baldridge RD, Xu P, Graham TR. Type IV P-type ATPases distinguish mono- versus diacyl phosphatidylserine using a cytofacial exit gate in the membrane domain. J. Biol. Chem [print-electronic]. 2013 Jul 7/5/2013; 288(27): 19516-27. PMID: 23709217, PMCID: PMC3707653, PII: M113.476911, DOI: 10.1074/jbc.M113.476911, ISSN: 1083-351X.

Graham TR. Arl1 gets into the membrane remodeling business with a flippase and ArfGEF. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2013 Feb 2/19/2013; 110(8): 2691-2. PMID: 23401560, PMCID: PMC3581979, PII: 1300420110, DOI: 10.1073/pnas.1300420110, ISSN: 1091-6490.

Baldridge RD, Graham TR. Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2013 Jan 1/29/2013; 110(5): E358-67. PMID: 23302692, PMCID: PMC3562821, PII: 1216948110, DOI: 10.1073/pnas.1216948110, ISSN: 1091-6490.

Sebastian TT, Baldridge RD, Xu P, Graham TR. Phospholipid flippases: building asymmetric membranes and transport vesicles. Biochim. Biophys. Acta [print-electronic]. 2012 Aug; 1821(8): 1068-77. PMID: 22234261, PMCID: PMC3368091, PII: S1388-1981(11)00272-1, DOI: 10.1016/j.bbalip.2011.12.007, ISSN: 0006-3002.

Baldridge RD, Graham TR. Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2012 Feb 2/7/2012; 109(6): E290-8. PMID: 22308393, PMCID: PMC3277569, PII: 1115725109, DOI: 10.1073/pnas.1115725109, ISSN: 1091-6490.

Graham TR, Burd CG. Coordination of Golgi functions by phosphatidylinositol 4-kinases. Trends Cell Biol [print-electronic]. 2011 Feb; 21(2): 113-21. PMID: 21282087, PMCID: PMC3053015, PII: S0962-8924(10)00214-X, DOI: 10.1016/j.tcb.2010.10.002, ISSN: 1879-3088.

Graham TR, Kozlov MM. Interplay of proteins and lipids in generating membrane curvature. Curr. Opin. Cell Biol [print-electronic]. 2010 Aug; 22(4): 430-6. PMID: 20605711, PMCID: PMC3770468, PII: S0955-0674(10)00065-7, DOI: 10.1016/j.ceb.2010.05.002, ISSN: 1879-0410.

Natarajan P, Liu K, Patil DV, Sciorra VA, Jackson CL, Graham TR. Regulation of a Golgi flippase by phosphoinositides and an ArfGEF. Nat. Cell Biol [print-electronic]. 2009 Dec; 11(12): 1421-6. PMID: 19898464, PMCID: PMC2787759, PII: ncb1989, DOI: 10.1038/ncb1989, ISSN: 1476-4679.

Zhou X, Graham TR. Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2009 Sep 9/29/2009; 106(39): 16586-91. PMID: 19805341, PMCID: PMC2757829, PII: 0904293106, DOI: 10.1073/pnas.0904293106, ISSN: 1091-6490.

Muthusamy BP, Natarajan P, Zhou X, Graham TR. Linking phospholipid flippases to vesicle-mediated protein transport. Biochim. Biophys. Acta [print-electronic]. 2009 Jul; 1791(7): 612-9. PMID: 19286470, PMCID: PMC3770137, PII: S1388-1981(09)00072-9, DOI: 10.1016/j.bbalip.2009.03.004, ISSN: 0006-3002.

Muthusamy BP, Raychaudhuri S, Natarajan P, Abe F, Liu K, Prinz WA, Graham TR. Control of protein and sterol trafficking by antagonistic activities of a type IV P-type ATPase and oxysterol binding protein homologue. Mol. Biol. Cell [print-electronic]. 2009 Jun; 20(12): 2920-31. PMID: 19403696, PMCID: PMC2695799, PII: E08-10-1036, DOI: 10.1091/mbc.E08-10-1036, ISSN: 1939-4586.

Liu K, Surendhran K, Nothwehr SF, Graham TR. P4-ATPase requirement for AP-1/clathrin function in protein transport from the trans-Golgi network and early endosomes. Mol. Biol. Cell [print-electronic]. 2008 Aug; 19(8): 3526-35. PMID: 18508916, PMCID: PMC2488278, PII: E08-01-0025, DOI: 10.1091/mbc.E08-01-0025, ISSN: 1939-4586.

Liu K, Hua Z, Nepute JA, Graham TR. Yeast P4-ATPases Drs2p and Dnf1p are essential cargos of the NPFXD/Sla1p endocytic pathway. Mol. Biol. Cell [print-electronic]. 2007 Feb; 18(2): 487-500. PMID: 17122361, PMCID: PMC1783782, PII: E06-07-0592, DOI: 10.1091/mbc.E06-07-0592, ISSN: 1059-1524.

Chen S, Wang J, Muthusamy BP, Liu K, Zare S, Andersen RJ, Graham TR. Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane. Traffic [print-electronic]. 2006 Nov; 7(11): 1503-17. PMID: 16956384, PII: TRA485, DOI: 10.1111/j.1600-0854.2006.00485.x, ISSN: 1398-9219.

Parsons AB, Lopez A, Givoni IE, Williams DE, Gray CA, Porter J, Chua G, Sopko R, Brost RL, Ho CH, Wang J, Ketela T, Brenner C, Brill JA, Fernandez GE, Lorenz TC, Payne GS, Ishihara S, Ohya Y, Andrews B, Hughes TR, Frey BJ, Graham TR, Andersen RJ, Boone C. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell. 2006 Aug 8/11/2006; 126(3): 611-25. PMID: 16901791, PII: S0092-8674(06)00906-8, DOI: 10.1016/j.cell.2006.06.040, ISSN: 0092-8674.

Xiao J, Kim LS, Graham TR. Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo. Mol. Biol. Cell [print-electronic]. 2006 Jul; 17(7): 3281-90. PMID: 16687570, PMCID: PMC1483056, PII: E06-02-0106, DOI: 10.1091/mbc.E06-02-0106, ISSN: 1059-1524.

Natarajan P, Graham TR. Measuring translocation of fluorescent lipid derivatives across yeast Golgi membranes. Methods. 2006 Jun; 39(2): 163-8. PMID: 16828307, PII: S1046-2023(06)00080-6, DOI: 10.1016/j.ymeth.2006.05.009, ISSN: 1046-2023.

Graham TR. Flippases and vesicle-mediated protein transport. Trends Cell Biol. 2004 Dec; 14(12): 670-7. PMID: 15564043, PII: S0962-8924(04)00287-9, DOI: 10.1016/j.tcb.2004.10.008, ISSN: 0962-8924.

Natarajan P, Wang J, Hua Z, Graham TR. Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2004 Jul 7/20/2004; 101(29): 10614-9. PMID: 15249668, PMCID: PMC489982, PII: 0404146101, DOI: 10.1073/pnas.0404146101, ISSN: 0027-8424.

Graham TR. Membrane targeting: getting Arl to the Golgi. Curr. Biol. 2004 Jun 6/22/2004; 14(12): R483-5. PMID: 15203023, PII: S096098220400421X, DOI: 10.1016/j.cub.2004.06.017, ISSN: 0960-9822.

Chim N, Gall WE, Xiao J, Harris MP, Graham TR, Krezel AM. Solution structure of the ubiquitin-binding domain in Swa2p from Saccharomyces cerevisiae. Proteins. 2004 Mar 3/1/2004; 54(4): 784-93. PMID: 14997574, DOI: 10.1002/prot.10636, ISSN: 1097-0134.

Chantalat S, Park SK, Hua Z, Liu K, Gobin R, Peyroche A, Rambourg A, Graham TR, Jackson CL. The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae. J. Cell. Sci [print-electronic]. 2004 Feb 2/15/2004; 117(Pt 5): 711-22. PMID: 14734650, PII: jcs.00896, DOI: 10.1242/jcs.00896, ISSN: 0021-9533.

Hua Z, Graham TR. Requirement for neo1p in retrograde transport from the Golgi complex to the endoplasmic reticulum. Mol. Biol. Cell [print-electronic]. 2003 Dec; 14(12): 4971-83. PMID: 12960419, PMCID: PMC284799, PII: E03-07-0463, DOI: 10.1091/mbc.E03-07-0463, ISSN: 1059-1524.

Gall WE, Geething NC, Hua Z, Ingram MF, Liu K, Chen SI, Graham TR. Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo. Curr. Biol. 2002 Sep 9/17/2002; 12(18): 1623-7. PMID: 12372257, PII: S096098220201148X, ISSN: 0960-9822.

Hua Z, Fatheddin P, Graham TR. An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system. Mol. Biol. Cell. 2002 Sep; 13(9): 3162-77. PMID: 12221123, PMCID: PMC124150, DOI: 10.1091/mbc.E02-03-0172, ISSN: 1059-1524.

Graham TR. Metabolic labeling and immunoprecipitation of yeast proteins. Curr Protoc Cell Biol. 2001 May; Chapter 7: Unit 7.6. PMID: 18228384, DOI: 10.1002/0471143030.cb0706s06, ISSN: 1934-2616.

Gall WE, Higginbotham MA, Chen C, Ingram MF, Cyr DM, Graham TR. The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast. Curr. Biol. 2000 Nov 11/2/2000; 10(21): 1349-58. PMID: 11084334, PII: S0960-9822(00)00771-5, ISSN: 0960-9822.

Gall WE, MA Higginbotham, C-Y Chen, MF Ingram, DM Cyr, and TR Graham. The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast. 2000.

Hopkins BD, Sato K, Nakano A, Graham TR. Introduction of Kex2 cleavage sites in fusion proteins for monitoring localization and transport in yeast secretory pathway. Meth. Enzymol. 2000; 327: 107-18. PMID: 11044978, PII: S0076-6879(00)27271-6, ISSN: 0076-6879.

Brigance WT, Barlowe C, Graham TR. Organization of the yeast Golgi complex into at least four functionally distinct compartments. Mol. Biol. Cell. 2000 Jan; 11(1): 171-82. PMID: 10637300, PMCID: PMC14766, ISSN: 1059-1524.

Chen CY, Ingram MF, Rosal PH, Graham TR. Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function. J. Cell Biol. 1999 Dec 12/13/1999; 147(6): 1223-36. PMID: 10601336, PMCID: PMC2168089, ISSN: 0021-9525.

Chen, C.-Y., M.F. Ingram, P. Rosal, and T.R. Graham. Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function. J. Cell Biol. 1999; 147: 1223-36.

Reynolds TB, Hopkins BD, Lyons MR, Graham TR. The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast golgi glycosyltransferase. J. Cell Biol. 1998 Nov 11/16/1998; 143(4): 935-46. PMID: 9817752, PMCID: PMC2132948, ISSN: 0021-9525.

Chen CY, Graham TR. An arf1Delta synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport in Saccharomyces cerevisiae. Genetics. 1998 Oct; 150(2): 577-89. PMID: 9755191, PMCID: PMC1460353, ISSN: 0016-6731.

Gaynor EC, Graham TR, Emr SD. COPI in ER/Golgi and intra-Golgi transport: do yeast COPI mutants point the way?. Biochim. Biophys. Acta. 1998 Aug 8/14/1998; 1404(1-2): 33-51. PMID: 9714721, PII: S0167-4889(98)00045-7, ISSN: 0006-3002.

Gaynor EC, Chen CY, Emr SD, Graham TR. ARF is required for maintenance of yeast Golgi and endosome structure and function. Mol. Biol. Cell. 1998 Mar; 9(3): 653-70. PMID: 9487133, PMCID: PMC25294, ISSN: 1059-1524.

Chen, C.-Y., and T. R. Graham. An arf1 synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport. Genetics. 1998; 150: 577-89.

Gaynor, E.C., T.R. Graham and S.D. Emr. COPI in ER/Golgi transport and intra-Golgi transport: do yeast COPs point the way?. Bioch. Biophys. Acta. 1998; 1404: 33-51.

Gaynor, E. C., C.-Y. Chen, S. D.Emr, and T. R.Graham. ARF is required for maintenance of yeast Golgi and endosome structure and function. Mol. Biol. Cell. 1998; 9: 653-70.

Reynolds, T.B., B.D. Hopkins, M.R. Lyons and T.R. Graham. The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast Golgi glycosyltransferase. J. Cell Biol. 1998; 143: 935-46.

Graham TR, Krasnov VA. Sorting of yeast alpha 1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein. Mol. Biol. Cell. 1995 Jul; 6(7): 809-24. PMID: 7579696, PMCID: PMC301242, ISSN: 1059-1524.

Graham, T. R., and V. A. Krasnov. Sorting of yeast alpha1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein. 1995.

Krasnov, V. and Graham, T.R. The Golgi complex of Saccharomyces cerevisiae. Can J Botany. 1995; 73: S343-S346.

Graham TR, Seeger M, Payne GS, MacKay VL, Emr SD. Clathrin-dependent localization of alpha 1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae. J. Cell Biol. 1994 Nov; 127(3): 667-78. PMID: 7962051, PMCID: PMC2120240, ISSN: 0021-9525.

Graham, T.R., Seegar, M., MacKay, V., Payne, G.S., and Emr, S.D. Clathrin-dependent localization of a, 3-mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae. J Cell Biology. 1994; 127: 667-78.

Gaynor, E.C., te Heesen, S., Graham, T.R., Aebi, M., and Emr, S.D. Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J Cell Biology. 1994; 127: 653-65.

Graham, T.R. and S.D. Emr. SEC18. In: Guidebook to the Secretory Pathway (J. Rothblatt, P. Novick, and T. Stevens, eds.) Oxford Univ. Press, NY. 1994; 132-3.

Graham, T.R., Scott, P., and Emr, Scott D. Brefeldin A reversibly blocks early but not late protein transport steps in the yeast secretory pathway. EMBO J. 1993; 12: 869-77.

Horazdovsky, B.F., Graham, T.R., and Emr, S.D. ┬┐Vacuolar protein sorting in yeast". Protein Synthesis and Targeting in Yeast (M.F. Tuite, J.E.G. McCarthy, A.J. Brown and F. Sherman, eds.) Springer Verlag, Berlin. 1992.

Lacoste, H.C., Graham, T.R., and Kaplan, A. A sequence in b-hexosaminadase from Dictyostelium discoideum required for sorting of proteins to a compartment involved in developmentally induced secretion. J Biol Chem. 1992; 267: 5942-8.

Graham, T.R. and Emr, S.D. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biology. 1991; 114: 207-18.

Vida, T.A., Herman, P.K., Emr, S.D., and Graham, T.R. Compartmentalized transport, modification and sorting of yeast vacuolar hydrolases. Biomed. Biochem. Acta. 1991; 50: 413-20.

Robinson, J.S., Graham, T.R.,and Emr, S.D. A putative zinc finger protein, Saccaromyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient a factor prohormone maturation. Mol Cell Biol. 1991; 12: 5813-24.

Available Postdoctoral Position Details
Posted: 9/12/2013
Applications are sought for a full time postdoctoral position to study the mechanism of substrate recognition and transport by P4-ATPases. Experience in molecular biology techniques such as cloning, mutagenesis, PCR, constructing gene fusions is essential. In addition, there is a preference for candidates familiar with protein expression and purification, microscopy, and bacterial and/or yeast microbial genetic techniques. Candidates must work well within a team environment and be willing to train graduate and undergraduate students.

Applicants should send a cover letter and curriculum vitae to Professor Todd Graham (tr.graham@vanderbilt.edu) in the Department of Biological Sciences at Vanderbilt University. Please include the names and addresses of three references who are willing to provide letters of recommendation. The position is open starting Sept 1, 2013 and will remain open until filled.