Structural characterization and subunit stoichiometry of the peripheral stalk subunits of the Saccharomyces cerevisiae vacuolar ATPase

The vacuolar ATPase, or V-ATPase belongs to the same class of rotary motors as the F-ATP synthase and the archaeal A-ATPase. These ubiquitous rotary molecular motors are found across all three domains of life, localized to many intracellular compartments. The V-ATPase is a proton pump that couples energy from ATP hydrolysis to rotational torque, which, in turn, acts to acidify the lumen of intracellular compartments or extracellular space. Loss-of-function mutations in the subunits of the vacuolar ATPase in humans result in a wide variety of diseases, including distal renal tubular acidosis, osteopetrosis and sensorineural deafness. The V-ATPase can also contribute to osteoporosis and cancer metastasis, making it an ideal drug target for inhibition. Indeed, regulation of the acid pumping activity has proven therapeutically effective for those afflicted with such diseases.; The V-ATPase in Saccharomyces cerevisiae has been shown to reversibly assemble and disassemble in response to the presence or absence of glucose in the extracellular environment. Little is currently known about the downstream signal involved in triggering this dissociation, but certain structural aspects of the V-ATPase appear to he implicated in this "reversible dissociation." It is believed that the structural differences between the V-ATPase and the F-ATP synthase enable this regulatory event. Central to this mechanism are the peripheral stalk (or "stator complex") subunits of the V-ATPase, namely, subunits E and G. Deficiencies in both high and low resolution structural information make it difficult to draw conclusions about their involvement in the mechanism of reversible dissociation. Indeed, until recently, the stoichiometry of subunits E and G has been in dispute. Work from this study resolves twenty years of controversy in the field regarding stator subunit stoichiometry. We present evidence for three copies each of subunits E and G, using two novel mass spectrometric techniques. In doing so, we also developed robust methods for the purification of subunits E and G, which may prove useful in future high-resolution structural analysis. Through the use of a novel mass spectrometric technique where non-covalent complexes can be preserved in the gas phase, we have also determined relative binding affinities of the stator subunits to the rest of the complex.; The archaeal A-ATPase differs from both the F-ATP synthase in stator stoichiometry and assembly. It is possible that the enzyme is regulated by an unconventional or unidentified mechanism. In Thermoplasma acidophilum, there is a potentially novel regulatory mechanism involving a self-splicing proteolytic insert, or "intein" in the catalytic subunit of the A-ATPase (the archaeal analog of the V-ATPase). We discuss a preliminary purification methodology for the Thermoplasma A-ATPase, in which we were able to capture both intein-containing and intein-free variations of A-ATPase assembly.; Finally, a preliminary 3D reconstruction of a V1-ATPase with a GFP tag on the central stalk subunit D was created from ∼2000 single particles. This is of particular interest due to the lack of high-resolution structure and orientation data regarding the central stalk. This preliminary model is presented herein.