Understanding molecular mechanisms of homeostasis: The role of the Vacuolar H+-ATPase in amino acid sensing
Stransky, Laura.
2017
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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 ... read moresensing. 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.read less - ID:
- 3b591m861
- Component ID:
- tufts:22773
- To Cite:
- TARC Citation Guide EndNote
