Prof. Barry Bowman - UCSC Biomedical Research Faculty

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The Biochemistry and Cell Biology of Membrane Proteins

Barry Bowman, MCD Biology

One third of the genes in all organisms encode membrane proteins, most of which transport molecules from one compartment to another. We use Neurospora crassa as our model organism. The complete genome has been sequenced for this filamentous fungus. It has 10,000 genes, twice the number in yeast, and a complete collection of knockout mutants is being generated. (Keywords: cell biology, biochemistry, ATP, membrane proteins)

The Vacuolar ATPase

In the past, the focus of our research has been on one of the major ion pumps in the cell, the vacuolar ATPase. Found in all components of the endomembrane system within cells, it is a large, complex enzyme that generates an electrochemical gradient for protons. This enzyme is composed of the products of at least 14 genes. It has an amazing mechanism, coupling the movement of protons to the function of a rotary motor that spins at 10,000 rpm. Some of the questions we have addressed are:

What are the functions of the subunits in the vacuolar ATPase? Do some of these subunits mediate the regulation of the enzyme and its interaction with other proteins in the cell?

What are the roles of the vacuolar ATPase in the cell? We found that disruption of genes that encode subunits of the vacuolar ATPase causes severe morphological changes. Analysis of these mutants may allow us to determine how the enzyme is involved in phenomena such as calcium homeostasis.

What is the mechanism of action of drugs that inhibit the vacuolar ATPase? We have discovered that several different kinds of antibiotics are potent inhibitors of the vacuolar ATPase. Other labs are trying to develop these drugs for use in treating diseases such as osteoporosis. By generating mutant forms of the vacuolar ATPase we have determined the binding site of one class of these antibiotics. The data have allowed us to make a high resolution model for the structure of the proton-binding polypeptides in the enzyme, as shown below.

Calcium Transport Proteins

Where is calcium in the cell and how does it get there? Calcium is an important signalling molecule. Release and uptake of calcium by organelles generates changes in concentration in local areas of the cytosol. Surprisingly little is known about the mechanism by which calcium is sequestered in different organelles or transported in and out of the cell.

We have identified 11 genes that appear to encode calcium transport proteins. Some of these are pumps, using the energy of ATP to move calcium. Other proteins are carriers that may couple the transport of calcium with the movement of Na+ or H+. By using knockout strains, or by tagging these proteins with GFP, we hope to identify the location and role of each one in the cell. The putative location of the cax carrier protein and four Ca-pumping ATPases are shown below.

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