This project will explore the mechanisms that govern the assembly, sorting and transport of the vacuolar- type proton-translocating ATPase (V-ATPase) in the simple model eukaryote, the yeast Saccharomyces cerevisiae. The V-ATPase is a molecular machine that couples the hydrolysis of ATP with the translocation of protons into the lumen of organelles, and the V-ATPase is highly conserved across fungi, plants and animals. Our genetic analysis in yeast has identified a group of genes in yeast encoding proteins that function in the endoplasmic reticulum (ER) in assembly of the membrane sector of the V-ATPase. These V-ATPase assembly factors will be characterized by genetic and biochemical approaches, to characterize the assembly pathway for the V-ATPase. Whereas it has been known for years that the V-ATPase is highly conserved, it has only very recently become clear that the V-ATPase assembly factors that we have identified in yeast are conserved in humans.
Three specific aims are proposed.
The first aim focuses on the important question of how each of the V- ATPase assembly factors functions in the highly orchestrated assembly of the 6-subunit integral membrane domain of the V-ATPase in the ER.
This aim i s also focused on investigating the mechanism of ER exit of the V-ATPase, and the function of the proteins identified as required for ER exit of this protein complex.
The second aim centers on characterizing the human V-ATPase assembly factors in yeast. Our collaboration with Professor Dirk Lefeber has provided us with the cDNAs of four human V-ATPase assembly factors (hVma12, hVma21, hVma22 and Ac45), as well as unpublished information on mutant alleles of these genes that cause human disease. These proteins will be characterized for V-ATPase assembly function in the much simpler yeast model system. Finally, the third aim addresses the sorting and retention of the Golgi-endosomal form of the yeast V-ATPase that contains the second isoform of subunit """"""""a"""""""", the Stv1 protein. We will use the knowledge of the Stv1 sorting/retention signal that we have recently identified to investigate and characterize the proteins involved in the sorting and retention of the Stv1-containing V-ATPase complex. The knowledge gained from these studies will provide new insights into the assembly, transport and sorting of this complex protein machine, and will provide insights into the mechanistic basis for the growing number of V-ATPase associated human diseases.

Public Health Relevance

Cells contain membrane-enclosed organelles, and the internal pH of these organelles is controlled by the vacuolar-type ATPase (V-ATPase). The V-ATPase is essentially the same in the simple model organism, the yeast Saccharomyces cerevisiae, and in humans. The V-ATPase is composed of 14 different protein subunits, and the proposed studies investigate the assembly of the very large protein machine. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of organelle acidification in all eukaryotic cells because of the remarkable similarity in mechanisms and proteins that regulate these processes from yeast to humans. These basic studies in yeast are providing important insights into our understanding of many diseases in humans related to defects in organelle acidification. Understanding V-ATPase function should provide important insights into diseases of the kidney (renal tubular acidosis), bone diseases (osteopetrosis), and in tumor metastasis, and new drug targets could arise from our studies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Ainsztein, Alexandra M
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University of Oregon
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Jansen, Eric J R; Timal, Sharita; Ryan, Margret et al. (2016) ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation. Nat Commun 7:11600
Coonrod, Emily M; Graham, Laurie A; Carpp, Lindsay N et al. (2013) Homotypic vacuole fusion in yeast requires organelle acidification and not the V-ATPase membrane domain. Dev Cell 27:462-8
Finnigan, Gregory C; Cronan, Glen E; Park, Hae J et al. (2012) Sorting of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase): identification of a necessary and sufficient Golgi/endosomal retention signal in Stv1p. J Biol Chem 287:19487-500
Finnigan, Gregory C; Hanson-Smith, Victor; Stevens, Tom H et al. (2012) Evolution of increased complexity in a molecular machine. Nature 481:360-4
Finnigan, Gregory C; Ryan, Margret; Stevens, Tom H (2011) A genome-wide enhancer screen implicates sphingolipid composition in vacuolar ATPase function in Saccharomyces cerevisiae. Genetics 187:771-83
Finnigan, Gregory C; Hanson-Smith, Victor; Houser, Benjamin D et al. (2011) The reconstructed ancestral subunit a functions as both V-ATPase isoforms Vph1p and Stv1p in Saccharomyces cerevisiae. Mol Biol Cell 22:3176-91
Flannery, Andrew R; Stevens, Tom H (2008) Functional characterization of the N-terminal domain of subunit H (Vma13p) of the yeast vacuolar ATPase. J Biol Chem 283:29099-108
Neubert, Christoph; Graham, Laurie A; Black-Maier, Eric W et al. (2008) Arabidopsis has two functional orthologs of the yeast V-ATPase assembly factor Vma21p. Traffic 9:1618-28
Ryan, Margret; Graham, Laurie A; Stevens, Tom H (2008) Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum. Mol Biol Cell 19:5131-42
Compton, Mark A; Graham, Laurie A; Stevens, Tom H (2006) Vma9p (subunit e) is an integral membrane V0 subunit of the yeast V-ATPase. J Biol Chem 281:15312-9

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