Proton-translocating ATPases synthesize most of the ATP in biological systems, and have a very similar structure and function in bacteria, plants, and animals. The proposed research will investigate the factors controlling the synthesis and assembly of the E.coli ATPase in order to understand how control over gene expression affects assembly and activity. Assembly and function will also be studied for a hybrid ATPase consisting of subunits from both E.coli and the obligate aerobe Bacillus megaterium. The subunits of the E.coli ATPase are coded for by the genes of the unc operon which have been shown to be differentially translated in vitro from a single polycistronic mRNA. In-frame fusions of unc genes to lacZ will be used to measure in vivo expression of chromosomal unc genes and to determine the mechanism of autogenous control over unc gene expression. A system will be developed to induce unc gene-dependent lethal proton permeability, and that system will then be used to study the biochemistry of assembly of the proton channel. Hybrid E.coli-B.megaterium ATPase complexes can be formed when E.coli unc mutants are complemented with cloned B.megaterium ATPase DNA. To further the understanding of the primary structures of ATPase subunits and their interactions in the complex, the remaining B.megaterium ATPase genes will be cloned and sequenced. By constructing plasmids containing various combinations of B.megaterium genes and testing the ability of each plasmid to complement E.coli mutants, the species-specific requirements for assembly of foreign subunits into these hybrid complexes will be determined. Also, since these ATPases can couple proton conduction to ATP synthesis but not hydrolysis, the same experiments will determine the subunits involved in energy coupling. Last, hybrid subunit will be constructed to determine which parts of individual ATPase subunits, and even which amino acid residues, are involved in energy coupling and in subunit-subunit interactions during assembly.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Modified Research Career Development Award (K04)
Project #
5K04AI000882-05
Application #
3070931
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1990-01-01
Project End
1993-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Wayne State University
Department
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Bokhari, Abdullah A B; Solomon, Tsione; Desai, Sanjay A (2008) Two distinct mechanisms of transport through the plasmodial surface anion channel. J Membr Biol 226:27-34
Staines, Henry M; Alkhalil, Abdulnaser; Allen, Richard J et al. (2007) Electrophysiological studies of malaria parasite-infected erythrocytes: current status. Int J Parasitol 37:475-82
Alkhalil, Abdulnaser; Hill, David A; Desai, Sanjay A (2007) Babesia and plasmodia increase host erythrocyte permeability through distinct mechanisms. Cell Microbiol 9:851-60
Lisk, Godfrey; Scott, Seth; Solomon, Tsione et al. (2007) Solute-inhibitor interactions in the plasmodial surface anion channel reveal complexities in the transport process. Mol Pharmacol 71:1241-50
Lisk, Godfrey; Kang, Myungsa; Cohn, Jamieson V et al. (2006) Specific inhibition of the plasmodial surface anion channel by dantrolene. Eukaryot Cell 5:1882-93
Lisk, Godfrey; Desai, Sanjay A (2006) Improved perfusion conditions for patch-clamp recordings on human erythrocytes. Biochem Biophys Res Commun 347:158-65
Desai, Sanjay A (2005) Open and closed states of the plasmodial surface anion channel. Nanomedicine 1:58-66
Kang, Myungsa; Lisk, Godfrey; Hollingworth, Stephen et al. (2005) Malaria parasites are rapidly killed by dantrolene derivatives specific for the plasmodial surface anion channel. Mol Pharmacol 68:34-40
Desai, Sanjay A; Alkhalil, Abdulnaser; Kang, Myungsa et al. (2005) Plasmodial surface anion channel-independent phloridzin resistance in Plasmodium falciparum. J Biol Chem 280:16861-7
Lisk, Godfrey; Desai, Sanjay A (2005) The plasmodial surface anion channel is functionally conserved in divergent malaria parasites. Eukaryot Cell 4:2153-9

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