Research Interests

The research interest of this laboratory encompasses two distinct topics: protein structure and function as related to the mitochondrial ATP synthase and understanding the biochemical basis of Batten disease. Yeast Saccharomyces cerevisiae is used as a model organism because the enzyme is highly conserved from yeast to mammals and because of the powerful tools afforded by yeast.

The ATP synthase is a multimeric enzyme that is responsible for the synthesis of ATP via oxidative phosphorylation. The catalytic site is in a water-soluble portion of the enzyme, the F1, which is bound to the membrane by a membrane bound portion, the Fo. The crystal structure of the water-soluble bovine F1 portion was determined in the laboratory of Dr. John Walker at the Laboratory of Molecular Biology, Cambridge, U.K., making it one of the largest nonsymmetrical protein structures solved to date. In collaboration with Drs. John Walker and Andrew Leslie in the MRC in Cambridge, U.K., we now have the 2.8A map of the yeast F1-ATPase. This is a major advance as it now allows us to investigate the structure/function relationship of the ATP synthase by a combination of genetic, biochemical, and x-ray crystallographic methods. Some of the aims that are being pursued are:

1. How does the gamma subunit cause the energy transduction step?
2. How does the binding of nucleotides affect the conformation of the active site?
3. What are the active and inhibited conformations of the enzyme?
4. What is the reaction mechanism of the enzyme?
5. What is the crystal structure of the F1F0 ATP synthase?

These questions are being addressed using a combination of genetic, biochemical and x-ray crystallographic methods. The yeast system is currently the only system available that can use all of these techniques to answer these questions. We are also involved in a collaborative project with Dr. Richard Berry from Oxford. Dr. Berry is studying the ATPase by single molecular methods thereby measuring the torque of the rotary mechanism. Dr. Berry, in part, will be using the yeast ATPase to address specific questions relevant to the mechanism of ATP synthesis.

Batten Disease

The second project is aimed at understanding the biochemical basis of Batten disease. Batten disease is the most prominent childhood neurodegenerative disease. The mutations responsible for the disease have been mapped to the human Cln3 gene. Unfortunately,the Cln3 gene product is an orphan protein, i.e., a protein in search of a function. There is a yeast homologue of Cln3, referred to as YHC3 or BTN1. We are using yeast as a model to understand the role of BTN1 protein in yeast. Initial studies in our laboratory have indicated that the protein is localized in the yeast lysosomal vacuole. Some current studies being done include the dynamic movement of BTN1 protein in the cell and the regulation of the expression of BTN1.
The figure below shows the localization of Batten gene product in yeast. The protein is visualized by adding GFP at the N-terminus of the protein thereby providing a fluorescent tag of the protein. The Batten gene product is clearly a membrane bound protein localized in the vacuole. The vacuole is the lysosome of the yeast cell.