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The vacuolar ATPase (V-ATPase) is a large, complex enzyme that pumps protons
across the membrane of most organelles in the cell. It generates acidity
within organelles and produces an electrochemical gradient used by
other transport proteins. Like the F-ATPase, which synthesizes ATP
in mitochondria, the enzyme functions as a rotary motor. One sector
of the enzyme, named V1, protrudes from the membrane and hydrolyzes
ATP. The energy is used to drive the rotation of a set of subunits
embedded in the membrane. The membrane sector, named Vo, forms the
proton-conducting pathway. The rotating part of the enzyme is hypothesized
to pick up protons from the cytosolic surface and release them on the
other side of the membrane. We have found several different types of
natural products that are potent and specific inhibitors of V-ATPases.
Some of these are being investigated for use as therapeutic drugs for
treatment of osteoporosis and some forms of cancer. In research laboratories
the most widely used are the bafilomycins and concanamycins. We have
developed methods to generate mutant strains of Neurospora crassa that
are resistant to these antibiotics. Analysis of the mutants has led
to the discovery that bafilomycin and concanamycin bind to the Vo sector
of the enzyme, specifically to the rotating c subunits. These data
suggest a structure for the c subunit and also point to an intriguing
mechanism to explain how the drugs inhibit the enzyme. |
