%0 PDF %T Understanding molecular mechanisms of homeostasis: The role of the Vacuolar H+-ATPase in amino acid sensing %A Stransky, Laura. %D 2017-09-05T11:33:02.844-04:00 %8 2017-09-05 %R http://localhost/files/3b591m861 %X Abstract: Cells continuously sense and respond to environmental conditions to maintain homeostasis. The signaling underling this ability is complex and incompletely understood, particularly with respect to how amino acids are sensed. Vacuolar-type H+-ATPase (V‑ATPase) function is required for amino acid-dependent activation of mTORC1, a critical node of cellular homeostasis and amino acid sensing. Further, the V-ATPase undergoes amino acid-dependent association with the mTORC1 activation machinery. V‑ATPase activity is quickly and locally tuned by changes in the equilibrium between assembled, functional holoenzymes, and disassembled, nonfunctional V1 and V0 domains, a process termed regulated assembly. We hypothesized that amino acids influence V‑ATPase assembly, and therefore activity, coupling free amino acid availability to their production by lysosomal protein turnover and serving as a signal to mTORC1. Indeed, we find that V‑ATPase assembly and activity are increased by amino acid starvation, likely facilitating increased lysosomal protein turnover. This change is independent of PI3K and mTORC1 signaling, which regulate the V‑ATPase in response to other stimuli. However, PKA and AMPK may be important for controlling V‑ATPase activity in response to amino acid starvation. Further, changes in V‑ATPase activity in response to starvation of individual amino acids indicate a complex network for sensing and responding to changes in amino acid levels. We further hypothesized that increased V‑ATPase assembly during amino acid starvation serves as a negative signal to mTORC1. However, changes in V‑ATPase activity or assembly do not correlate with changes in mTORC1 signaling. This demonstrates that changes in V‑ATPase assembly are not a signal to mTORC1. To determine the mechanism by which the V‑ATPase changes mTORC1 activity, we assessed activation of AMPK, which also relies on the V‑ATPase for its activity. Co-inhibition of AMPK and the V‑ATPase rescues mTORC1 activation and localization to lysosomes. Therefore, we conclude that activation of AMPK is at least partially responsible for repression of mTORC1 by V‑ATPase inhibition. This work identifies amino acid availability as an important and novel stimulus for the regulation of the V‑ATPase, and identifies a mechanism by which V‑ATPase inhibition impinges on mTORC1 signaling. Together, this gives new insight into mechanisms of cellular homeostasis.; Thesis (Ph.D.)--Tufts University, 2017.; Submitted to the Dept. of Cellular & Molecular Physiology.; Advisor: Michael Forgac.; Committee: Brent Cochran, Philip Hinds, Peter Juo, and Brendan Manning.; Keywords: Physiology, Cellular biology, and Biochemistry. %[ 2022-10-11 %9 Text %~ Tufts Digital Library %W Institution