Membrane proteins in plant physiology and bioenergetics: Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku'amide B
Lukic K. 2024. Membrane proteins in plant physiology and bioenergetics: Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B. Institute of Science and Technology Austria.
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Thesis
| PhD
| Published
| English
Author
Supervisor
Corresponding author has ISTA affiliation
Department
Series Title
ISTA Thesis
Abstract
This thesis comprises two distinct projects, each offering unique insights into fundamental
cellular processes. While distinct in their focus, these different perspectives have a common
theme: chemiosmotic theory and utilisation of the proton gradient for driving the essential
processes like auxin efflux and ATP synthesis, effectively bridging the membrane protein
structure and function from the realms of plant biology and cellular bioenergetics.
The first project of this thesis centres on the characterisation of PIN proteins, a class of
transmembrane transporters pivotal in the regulation of auxin transport and distribution in
plants. PINs form a conserved and phylogenetically abundant group of transporters present in
land plants and certain algae. Despite their great importance, they were one of the few elusive
proteins essential for plant development not to be structurally and mechanistically
characterised since their discovery almost 30 years ago. This work aimed to uncover the
structural and functional dynamics of the PIN protein-mediated auxin transport using an array
of experimental techniques, including protein purification, biochemical assays and structural
analysis. Through an exhaustive screening process that took several years and included testing
different PIN homologues, expression systems, constructs, and purification conditions, we
developed a robust protocol for isolating the pure, stable, and monodisperse PIN8 protein.
Moreover, utilising biophysical methods and buffer screening, we demonstrated that PIN8
exhibits detergent and pH-dependent stability, with mild detergents and lower pH (5.0 and 6.0)
being optimal for the stability of the protein. Using SEC-MALS and crosslinking, we
determined that PIN8 forms dimers, which was confirmed by our structural studies. We
obtained a cryo-EM map of PIN8 at pH 6.0, and, compared to recently published structures,
our map implies major pH-dependent conformational changes and possibly utilisation of the
proton gradient in the transport mechanism.
The subject of the second project was F1Fo-ATP synthase, an enzyme complex fundamental
to cellular energy metabolism. Through an approach integrating biochemical assays and
structural analysis, this research aimed to unveil the molecular mechanism of inhibition of ATP
synthase by yaku´amide, a bioactive compound with potential therapeutic implications. Using
submitochondrial particles and purified F1Fo-ATP synthase, we demonstrated that, contrary to
published data, yaku´amide inhibits both ATP hydrolysis and ATP synthesis reactions.
Moreover, we found that yaku´amide inhibitory activity is proton motive force (pmf)
dependent, with lower inhibition in a more coupled system. Utilising cryo-EM, we obtained
maps and models for the three main rotational states of murine ATP synthase (State 1 at 3.0 Å,
8
State 2 at 3.1 Å, and State 3 at 3.2 Å, overall). We observed several new features in our maps;
however, we cannot definitively determine the exact mechanism of yaku amide’s inhibition on
the protein due to either resolution limits or suboptimal binding of the inhibitor.
Publishing Year
Date Published
2024-07-26
Publisher
Institute of Science and Technology Austria
Acknowledged SSUs
Page
224
ISSN
IST-REx-ID
Cite this
Lukic K. Membrane proteins in plant physiology and bioenergetics: Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B. 2024. doi:10.15479/at:ista:17319
Lukic, K. (2024). Membrane proteins in plant physiology and bioenergetics: Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:17319
Lukic, Kristina. “Membrane Proteins in Plant Physiology and Bioenergetics: Investigating Auxin Efflux Transporter PIN8 and ATP Synthase Inhibition by the Novel Inhibitor Yaku’amide B.” Institute of Science and Technology Austria, 2024. https://doi.org/10.15479/at:ista:17319.
K. Lukic, “Membrane proteins in plant physiology and bioenergetics: Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B,” Institute of Science and Technology Austria, 2024.
Lukic K. 2024. Membrane proteins in plant physiology and bioenergetics: Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku’amide B. Institute of Science and Technology Austria.
Lukic, Kristina. Membrane Proteins in Plant Physiology and Bioenergetics: Investigating Auxin Efflux Transporter PIN8 and ATP Synthase Inhibition by the Novel Inhibitor Yaku’amide B. Institute of Science and Technology Austria, 2024, doi:10.15479/at:ista:17319.
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