---
OA_place: publisher
_id: '17319'
abstract:
- lang: eng
  text: "This thesis comprises two distinct projects, each offering unique insights
    into fundamental\r\ncellular processes. While distinct in their focus, these different
    perspectives have a common\r\ntheme: chemiosmotic theory and utilisation of the
    proton gradient for driving the essential\r\nprocesses like auxin efflux and ATP
    synthesis, effectively bridging the membrane protein\r\nstructure and function
    from the realms of plant biology and cellular bioenergetics.\r\nThe first project
    of this thesis centres on the characterisation of PIN proteins, a class of\r\ntransmembrane
    transporters pivotal in the regulation of auxin transport and distribution in\r\nplants.
    PINs form a conserved and phylogenetically abundant group of transporters present
    in\r\nland plants and certain algae. Despite their great importance, they were
    one of the few elusive\r\nproteins essential for plant development not to be structurally
    and mechanistically\r\ncharacterised since their discovery almost 30 years ago.
    This work aimed to uncover the\r\nstructural and functional dynamics of the PIN
    protein-mediated auxin transport using an array\r\nof experimental techniques,
    including protein purification, biochemical assays and structural\r\nanalysis.
    Through an exhaustive screening process that took several years and included testing\r\ndifferent
    PIN homologues, expression systems, constructs, and purification conditions, we\r\ndeveloped
    a robust protocol for isolating the pure, stable, and monodisperse PIN8 protein.\r\nMoreover,
    utilising biophysical methods and buffer screening, we demonstrated that PIN8\r\nexhibits
    detergent and pH-dependent stability, with mild detergents and lower pH (5.0 and
    6.0)\r\nbeing optimal for the stability of the protein. Using SEC-MALS and crosslinking,
    we\r\ndetermined that PIN8 forms dimers, which was confirmed by our structural
    studies. We\r\nobtained a cryo-EM map of PIN8 at pH 6.0, and, compared to recently
    published structures,\r\nour map implies major pH-dependent conformational changes
    and possibly utilisation of the\r\nproton gradient in the transport mechanism.\r\nThe
    subject of the second project was F1Fo-ATP synthase, an enzyme complex fundamental\r\nto
    cellular energy metabolism. Through an approach integrating biochemical assays
    and\r\nstructural analysis, this research aimed to unveil the molecular mechanism
    of inhibition of ATP\r\nsynthase by yaku´amide, a bioactive compound with potential
    therapeutic implications. Using\r\nsubmitochondrial particles and purified F1Fo-ATP
    synthase, we demonstrated that, contrary to\r\npublished data, yaku´amide inhibits
    both ATP hydrolysis and ATP synthesis reactions.\r\nMoreover, we found that yaku´amide
    inhibitory activity is proton motive force (pmf)\r\ndependent, with lower inhibition
    in a more coupled system. Utilising cryo-EM, we obtained\r\nmaps and models for
    the three main rotational states of murine ATP synthase (State 1 at 3.0 Å,\r\n8\r\nState
    2 at 3.1 Å, and State 3 at 3.2 Å, overall). We observed several new features in
    our maps;\r\nhowever, we cannot definitively determine the exact mechanism of
    yaku amide’s inhibition on\r\nthe protein due to either resolution limits or suboptimal
    binding of the inhibitor."
acknowledged_ssus:
- _id: EM-Fac
- _id: LifeSc
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Kristina
  full_name: Lukic, Kristina
  id: 2B04DB84-F248-11E8-B48F-1D18A9856A87
  last_name: Lukic
  orcid: 0000-0003-1581-881X
citation:
  ama: '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:<a href="https://doi.org/10.15479/at:ista:17319">10.15479/at:ista:17319</a>'
  apa: 'Lukic, K. (2024). <i>Membrane proteins in plant physiology and bioenergetics :
    Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the
    novel inhibitor Yaku’amide B</i>. Institute of Science and Technology Austria.
    <a href="https://doi.org/10.15479/at:ista:17319">https://doi.org/10.15479/at:ista:17319</a>'
  chicago: '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.
    <a href="https://doi.org/10.15479/at:ista:17319">https://doi.org/10.15479/at:ista:17319</a>.'
  ieee: '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.'
  ista: '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.'
  mla: 'Lukic, Kristina. <i>Membrane Proteins in Plant Physiology and Bioenergetics :
    Investigating Auxin Efflux Transporter PIN8 and ATP Synthase Inhibition by the
    Novel Inhibitor Yaku’amide B</i>. Institute of Science and Technology Austria,
    2024, doi:<a href="https://doi.org/10.15479/at:ista:17319">10.15479/at:ista:17319</a>.'
  short: '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.'
corr_author: '1'
date_created: 2024-07-26T09:05:55Z
date_published: 2024-07-26T00:00:00Z
date_updated: 2026-04-07T13:20:44Z
day: '26'
ddc:
- '580'
degree_awarded: PhD
department:
- _id: LeSa
- _id: GradSch
doi: 10.15479/at:ista:17319
file:
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  creator: cchlebak
  date_created: 2024-07-26T13:14:24Z
  date_updated: 2025-01-26T23:30:04Z
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  creator: cchlebak
  date_created: 2024-07-26T13:14:50Z
  date_updated: 2025-01-26T23:30:04Z
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has_accepted_license: '1'
language:
- iso: eng
month: '07'
oa: 1
oa_version: Published Version
page: '224'
publication_identifier:
  issn:
  - 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
status: public
supervisor:
- first_name: Leonid A
  full_name: Sazanov, Leonid A
  id: 338D39FE-F248-11E8-B48F-1D18A9856A87
  last_name: Sazanov
  orcid: 0000-0002-0977-7989
title: 'Membrane proteins in plant physiology and bioenergetics : Investigating auxin
  efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku''amide
  B'
type: dissertation
user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd
year: '2024'
...