- Introductory Biology
Research in my laboratory addresses the molecular mechanisms by which cells become multidrug resistant. Such resistance to many different chemotherapeutic agents is a major problem in the treatment of cancer, malaria, and various fungal infections. Our work established that yeast cells (a model eukaryote) have several membrane proteins that modulate a basal level of drug resistance. Most of our effort has centered on understanding the regulation and function of the PDR5 gene product. The PDR5 locus encodes a large protein that is a member of the ABC (ATP-binding cassette) transport family. All of these proteins use the energy liberated from ATP hydrolysis to import or expel a variety of compounds depending upon the specificity of the particular transporter. Included in this family are the mammalian MDR1 multidrug-resistance efflux pump and the cystic fibrosis transmembrane regulator. PDR5 is a multidrug transporter that mediates resistance to many different compounds, including the well-known therapeutic agents fluconazole and adriamycin. We demonstrated that loss-of-function mutations in this locus are very drug hypersensitive because they no longer transport substrate out of the cell. A 2-D topological model is shown below:
A central question with all ABC transporters is the coupling of ATP hydrolysis which occurs entirely in the nucleotide binding domains (labeled with a pink circle) with drug transport which takes place in the transmembrane regions (shown in blue). Using a combination of genetics and biochemical assays that measure ATP hydrolysis, drug binding, and transport, we recently began to map the signaling interface responsible for transmitting information between these critical sites (Sauna et al. 2008). Included in this interface are transmembrane helix-2 shown in red and the N-terminal nucleotide-binding domain. We hope to construct a detailed map that will also include the multiple and probably overlapping drug transport sites.
Current research funding is from the National Institutes of Health.
- Golin, J., Ambudkar, S., Gottesman, M., Habib, A., Sczepanski, J., Ziccardi, W., and May, L. (2003). Studies with novel Pdr5p substrates demonstrate a strong size dependence for xenobiotic efflux. J.Biol. Chem. 278: 5963-5969.
- Hanson, L, L. May, P. Tuma, J. Keeven, P. Mehl, M. Ferenz, S.V. Ambudkar and J. Golin (2005). The role of hydrogen bond acceptor groups in the interaction of substrates with Pdr5p, a major yeast drug transporter. Biochemistry 44:9703-9713.
- Pety de Thozee, C., Cronin, S., Gof, A., Golin, J. and Ghislain, M., (2007). Subcellular trafficking of the yeast plasma membrane ABC transporter Pdr5 is impaired by a mutation in the N-terminal nucleotide binding fold. Molecular Microbiology 63: 811-825.
- Golin, J., Ambudkar, S.V., and May, L. (2007). The yeast multidrug transporter: How does it recognize so many substrates? Biochem Biophy. Res. Com. 356: 1-5. (review article)
- Golin, J., Kon, Z. N., Wu, C. P., Martello, J., Hanson, L., Supernavage, S., Ambudkar, S. V., and Sauna, Z. E. (2007). Complete inhibition of the Pdr5p multidrug efflux pump ATPase by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source. Biochemistry 46 (45): 13109-13119.
- Rutledge, R.M., Ghislain, M., Mullins, J.M., Pety de Thozee, C., and Golin, J. (2008) Pdr5-mediated multidrug resistance requires the CPY-vacuolar sorting protein Vps3: are xenobiotic compounds routed from the vacuole to the plasma membrane transporters for efflux? Mol Gen Genet 279: 573-583.
- Sauna, Z., Bohn, S., Rutledge, R. M., Dougherty, M. P., Cronin, S., May, L., Xia, D., Ambudkar, S. V., and Golin, J. (2008) Mutations define cross talk between the N-terminal nucleotide-binding domain and transmembrane helix-2 of the yeast multidrug transporter Pdr5: Possible conservation of a signaling interface coupling ATP hydrolysis to drug transport. J.Biol. Chem. 283: 35010-35022.
- Ananthaswamy, N., Rutledge, R., Sauna, Z.E., Ambudkar, S.V., Dine, E., Nelson, E., Xia, D. and Golin J. (2010) The signaling interface of the yeast multi-drug transporter Pdr5 adopts a cis conformation and there are functional overlap and equivalence of the deviant and canonical Q-loop residues. Biochemistry 49: 4440-4449.
- Downes, M.T., Mehla , J., Ananthaswamy, N., Wakschlag, A., LaMonde, M., Dine, E., and Ambudkar, S.V., and Golin, J. (2013) The transmembrane interface of the Saccharomyces cervevisia multidrug transporter Pdr5: Val-656 located in intracellular-loop 2 plays a major role in drug resistance. Antimicrob. Agents Chemother. 57: 1025-1034.
- Furman, C*.,Mehla, J*., Ananthaswamy, N., Arya, N., Kulesh, B., Kovach, I., Amubdkar, S.V., and Golin, J., (2013) The deviant ATP-binding-site of the multidrug efflux pump Pdr5 plays an active role in the transport cycle. J.Biol Chem. 288: 30420-30431
- Mehla, J., Ernst, R. Moore., Wakschlag, A., Marquis, M.K., Ambudkar S V.,and Golin, J.(2014) Evidence for a molecular diode-based mechanism in a multispecific ATP-binding cassette (ABC) exporter: Ser-1368 as a gatekeeping residue in the yeast multidrug transporter Pdr5. J.Biol Chem. 289:26597-26606.
*co- first authors