Evolution of the Vacuolar H+-ATPase Enzyme Complex

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Title: Evolution of the Vacuolar H+-ATPase Enzyme Complex
Author: Finnigan, Gregory Charles, 1983-
Abstract: The vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit enzyme complex responsible for acidification of cellular organelles. The V-ATPase hydrolyzes ATP to pump protons across membranes to create an electrochemical gradient. Acidification of vesicular compartments is critical in numerous biological processes including protein trafficking, endocytosis, and ion homeostasis; defects in V-ATPase function can also lead to human diseases. While the function of the V-ATPase enzyme is highly conserved across eukaryotes, the molecular architecture of this protein complex has undergone unique structural changes through evolutionary time. The goal of this work is to investigate the assembly, transport, and evolution of this critical molecular machine in the model organism <italic>Saccharomyces cerevisiae</italic>. A series of genetic screens was performed in budding yeast to identify factors and pathways that are involved in promoting full V-ATPase function. I utilized several "assembly factor" alleles to serve as sensitized genetic backgrounds to partially reduce enzyme function; this work implicated sphingolipid composition in promoting full vacuolar ATPase enzyme function. I also used ancestral gene reconstruction to analyze the two isoforms of subunit a of the V<sub>0</sub> subdomain (Vph1p and Stv1p) by recreating the most recent common ancestral subunit (Anc.a). Characterization of Anc.a demonstrated that this ancient subunit was able to properly assemble and function within a hybrid V-ATPase complex. While the Vph1p-containing complex localized to the vacuole membrane and the Stv1p-containing complex was present on the Golgi/endosome, incorporation of Anc.a caused the V-ATPase to localize to both types of cellular compartments. Finally, I used ancestral reconstruction to investigate the lineage-specific gene duplication of one of the proteolipid subunits of the V<sub>0</sub> subcomplex that occurred within the fungal clade. I demonstrate that inclusion of a third proteolipid subunit within fungi (as compared to two subunits within metazoans) could have occurred via neutral processes by asymmetric degeneration of subunit-subunit interfaces that "ratcheted" the duplicated subunit with the V<sub>0</sub> ring. These results present a model that describes how macromolecular machines can increase in complexity through evolutionary time. This dissertation includes previously published co-authored material and unpublished co-authored material.
Description: xvii, 167 p. : ill. (some col.)
URI: http://hdl.handle.net/1794/11535
Date: 2011-06


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