Research Projects
Structure and Function of the vacuolar ATPase
The vacuolar ATPase (V-ATPase) is a large, complex enzyme that transports protons across membranes. It generates an electrochemical gradient and acidifies the interior of multiple organelles in all eukaryotic cells. We use the filamentous fungus Neurospora crassa to investigate this enzyme. With Neurospora we use the techniques of both biochemistry and molecular genetics to study the structure and function of the V-ATPase.

Fungal cells contain numerous vacuoles, easily seen when the cells are exposed to dyes that accumulate in acidic compartments (Fig. 1). These organelles are acidified by the V-ATPase located on their membranes. Because the enzyme is large and protrudes from the membrane on a stalk, it is visible in the electron microscope. Figure 2 shows a vacuolar vesicle, thickly studded with V-ATPases. At higher magnification the characteristic "ball and stalk" structure of the enzyme appears in the electron micrograph (Fig. 3).

The vacuolar ATPase consists of at least 13 types of subunits, some of which are present in multiple copies. Figure 4 illustrates our current hypothesis of the arrangement of subunits. The A and B subunits form the ATP binding site; the membrane-embedded a, c, c' and c'' subunits form the proton-translocating pathway. Functioning like a tiny molecular motor, the enzyme hydrolyses ATP and causes the cluster of c, c' and c'' subunits to rotate. Protons move across the membrane at the interface between the aand c subunits.

We have generated mutant strains lacking different components of the enzyme. We are using the mutant strains to investigate the function of each of the subunits and to determine if the subunits interact with other proteins in the cell.

Figure 1. Fungal cells contain numerous vacuoles, easily seen when the cells are exposed to dyes that accumulate in acidic compartments.
Figure 2. A vacuolar vesicle, thickly studded with V-ATPases.
Figure 3. At higher magnification the characteristic "ball and stalk" structure of the enzyme appears in the electron micrograph.
Figure 4a. Our current hypothesis of the arrangement of subunits. The A and B subunits form the ATP binding site; the membrane-embedded a, c, c' and c'' subunits form the proton-translocating pathway.

Figure 4b. Functioning like a tiny molecular motor, the enzyme hydrolyses ATP and causes the cluster of c, c' and c'' subunits to rotate. Protons move across the membrane at the interface between the a and c subunits.

Antibiotic compounds that inhibit the vacuolar ATPase
We have identified three classes of compounds that are highly potent and specific inhibitors of vacuolar ATPases. The most widely used are the antibiotics bafilomycin and concanamycin. Pharmaceutical research labs have shown much interest in these drugs because of the possibility of using them to treat osteoporosis. During normal turnover of bone tissue the vacuolar ATPase pumps acid onto the bone surface, causing the solubilization of the calcium matrix. Compounds that inhibit this process could be used to slow the loss of bone tissue.

Using genetics, we are identifying the sites at which bafilomycin and concanamycin bind the vacuolar ATPase. Mutation of residues in the c subunit causes the enzyme to become resistant to these drugs. We believe the drugs block the rotation of the c subunits, acting like "a stone in the gears" (Fig. 5).

In a collaborative effort we have identified two other classes of natural products as potent inhibitors of V-ATPases. The benzolactone enamides, such as salicylihalamide and lobatamide, are particularly interesting because they show great specificity towards vacuolar ATPases from animal cells. In collaboration with Dr. John Porco at Boston University we have been analyzing the inhibitory activity of derivatives of the lobatamides. We are just beginning to characterize the third class of inhibitory natural products.
Figure 5. Mutation of residues in the c subunit causes the enzyme to become resistant to these drugs. We believe the drugs block the rotation of the c subunits, acting like "a stone in the gears."

Calcium transporters in the vacuolar membrane
One important function of the vacuole is to control the level of calcium in the cell. Uptake and release of calcium from the vacuole is hypothesized to be a signal that regulates morphogenesis. Because the vacuolar ATPase generates the electrochemical gradient across the vacuolar membrane, it is essential for concentrating calcium. Mutant strains that lack the vacuolar ATPase are deficient in vacuolar calcium, and they are also morphologically abnormal.

The yeast Saccharomyces cerevisiae has at least two different types of proteins in the vacuolar membrane that directly transport calcium. Whether these transporters have unique functions or overlapping functions is not clear. We have identified three genes encoding putative vacuolar calcium transporters in Neurospora. We are generating strains with mutations in these genes. Analysis of these strains should show us the function of the transporters and the degree to which they interact with each other and the vacuolar ATPase.

The hex gene and Woronin Bodies
A few years ago we discovered, by accident, a gene that encodes the major protein component of the Woronin Body. (See also, Jedd and Chua, 2000, Nat. Cell Biol. 2:226-231.) This small organelle, derived from peroxisomes, has been observed only in a sub-group of the ascomycete fungi. Neurospora is not truly cellular but has compartments separated by septae; each septum has a small pore in the center. Normally, cytoplasm and even large organelles can move freely through these septal pores. However, if the hyphae are damaged, the Woronin bodies move into the septal pores and quickly plug them. To learn more about the formation and function of these intriguing organelles, we have been investigating mutant strains of Neurospora that are defective in forming septal plugs (Fig. 6)

Figure 6. Shown here are mutant strains of Neurospora that are defective in forming septal plugs.