A freezing robot for cryo-EM

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Project Summary Our laboratory has a long standing interest in understanding the catalytic and regulatory mechanism of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase), a dynamic multisubunit membrane integral rotary motor enzyme found in all eukaryotic cells. The V-ATPase acidifies the lumen of organelles and, in professional acid secreting cells, the extracellular space. Enzyme function is required for fundamental cellular processes such as endocytosis, bone remodeling, protein trafficking, acid-base balance, sperm maturation, and neurotransmitter release. While complete loss of V-ATPase function is embryonic lethal, partial loss or hyperactivity is associated with numerous human diseases such as osteopetrosis, diabetes, male infertility, neurodegeneration, and cancer. Moreover, some viruses such as influenza rely on the acidic environment created by the V-ATPase for infection. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism and regulation of the eukaryotic V-ATPase. In cells, V- ATPase activity is regulated by a unique mechanism referred to as “reversible disassembly”, wherein the complex reversibly dissociates into V1-ATPase and Vo proton channel, with both sub-complexes becoming autoinhibited. Despite its important role in V-ATPase physiology, the molecular mechanism of reversible disassembly is poorly understood. This gap in knowledge is largely due to a lack of both high-resolution structural information and an in vitro model system to study the process under defined conditions, aspects that we are working to address. An interesting, and technically challenging feature of the mammalian V-ATPase is that most of its subunits are expressed as multiple isoforms. However, as such isoforms display differential tissue enrichment, they may provide opportunities for targeted therapeutics. Indeed, several diseases have been linked to malfunction or upregulation of specific isoform containing V-ATPase. However, how different isoform combinations determine tissue localization, and whether these isoform specific complexes have unique biochemical or regulatory properties, is currently unknown. We have started to develop a system to purify wild type and mutant forms of human V-ATPase in an isoform specific fashion for biochemical and structural analyses. Further, we are developing single-domain antibodies (Nanobodies) against specific subunit isoforms to serve as research tools, and to explore isoform specific modulation of V-ATPase activity in disease. Our research program employs the tools of structural biology, cell biology, biochemistry and biophysics to address broad questions of V-ATPase catalytic and regulatory mechanisms. For some fundamental aspects of V- ATPase structure and regulation, we study the enzyme from yeast, a well documented model system for the human V-ATPase. We use human tissue culture for questions that cannot be addressed in yeast, such as structure and biochemical properties of specific isoform containing enzymes. The long term goal of our research is to find ways to modulate the activity of disease causing V-ATPases in an isoform specific way.