---
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:10.15479/at:ista:17319'
apa: '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'
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.
https://doi.org/10.15479/at:ista:17319.'
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. 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.'
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:
- access_level: open_access
checksum: 95517e697ea6a87e267e649cad560989
content_type: application/pdf
creator: cchlebak
date_created: 2024-07-26T13:14:24Z
date_updated: 2025-01-26T23:30:04Z
embargo: 2025-01-26
file_id: '17320'
file_name: Thesis_Kristina_Lukic.pdf
file_size: 24639084
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creator: cchlebak
date_created: 2024-07-26T13:14:50Z
date_updated: 2025-01-26T23:30:04Z
embargo_to: open_access
file_id: '17321'
file_name: Thesis_Kristina_Lukic.docx
file_size: 96334272
relation: source_file
file_date_updated: 2025-01-26T23:30:04Z
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'
...
---
_id: '8993'
abstract:
- lang: eng
text: N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar)
transport of the hormone auxin in plants. For decades, it has been a pivotal tool
in elucidating the unique polar auxin transport-based processes underlying plant
growth and development. Its exact mode of action has long been sought after and
is still being debated, with prevailing mechanistic schemes describing only indirect
connections between NPA and the main transporters responsible for directional
transport, namely PIN auxin exporters. Here we present data supporting a model
in which NPA associates with PINs in a more direct manner than hitherto postulated.
We show that NPA inhibits PIN activity in a heterologous oocyte system and that
expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to
specific saturable NPA binding. We thus propose that PINs are a bona fide NPA
target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent
auxin transport and a logical parsimonious explanation for the known physiological
effects of NPA on plant growth, as well as an alternative hypothesis to interpret
past and future results. We also introduce PIN dimerization and describe an effect
of NPA on this, suggesting that NPA binding could be exploited to gain insights
into structural aspects of PINs related to their transport mechanism.
acknowledgement: "This work was supported by Austrian Science Fund Grant FWF P21533-B20
(to L.A.); German Research Foundation Grant DFG HA3468/6-1 (to U.Z.H.); and European
Research Council Grant 742985 (to J.F.). We thank Herta Steinkellner and Alexandra
Castilho for N. benthamiana plants, Fabian Nagelreiter for statistical advice, Lanassa
Bassukas for help with [ɣ32P]-\r\nATP assays, and Josef Penninger for providing
access to mass spectrometry instruments at the Vienna BioCenter Core Facilities.
We thank PNAS reviewers for the many comments and suggestions that helped to improve
this manuscript."
article_number: e2020857118
article_processing_charge: No
article_type: original
author:
- first_name: Lindy
full_name: Abas, Lindy
last_name: Abas
- first_name: Martina
full_name: Kolb, Martina
last_name: Kolb
- first_name: Johannes
full_name: Stadlmann, Johannes
last_name: Stadlmann
- first_name: Dorina P.
full_name: Janacek, Dorina P.
last_name: Janacek
- first_name: Kristina
full_name: Lukic, Kristina
id: 2B04DB84-F248-11E8-B48F-1D18A9856A87
last_name: Lukic
orcid: 0000-0003-1581-881X
- first_name: Claus
full_name: Schwechheimer, Claus
last_name: Schwechheimer
- first_name: Leonid A
full_name: Sazanov, Leonid A
id: 338D39FE-F248-11E8-B48F-1D18A9856A87
last_name: Sazanov
orcid: 0000-0002-0977-7989
- first_name: Lukas
full_name: Mach, Lukas
last_name: Mach
- first_name: Jiří
full_name: Friml, Jiří
id: 4159519E-F248-11E8-B48F-1D18A9856A87
last_name: Friml
orcid: 0000-0002-8302-7596
- first_name: Ulrich Z.
full_name: Hammes, Ulrich Z.
last_name: Hammes
citation:
ama: Abas L, Kolb M, Stadlmann J, et al. Naphthylphthalamic acid associates with
and inhibits PIN auxin transporters. Proceedings of the National Academy of
Sciences of the United States of America. 2021;118(1). doi:10.1073/pnas.2020857118
apa: Abas, L., Kolb, M., Stadlmann, J., Janacek, D. P., Lukic, K., Schwechheimer,
C., … Hammes, U. Z. (2021). Naphthylphthalamic acid associates with and inhibits
PIN auxin transporters. Proceedings of the National Academy of Sciences of
the United States of America. National Academy of Sciences. https://doi.org/10.1073/pnas.2020857118
chicago: Abas, Lindy, Martina Kolb, Johannes Stadlmann, Dorina P. Janacek, Kristina
Lukic, Claus Schwechheimer, Leonid A Sazanov, Lukas Mach, Jiří Friml, and Ulrich
Z. Hammes. “Naphthylphthalamic Acid Associates with and Inhibits PIN Auxin Transporters.”
Proceedings of the National Academy of Sciences of the United States of America.
National Academy of Sciences, 2021. https://doi.org/10.1073/pnas.2020857118.
ieee: L. Abas et al., “Naphthylphthalamic acid associates with and inhibits
PIN auxin transporters,” Proceedings of the National Academy of Sciences of
the United States of America, vol. 118, no. 1. National Academy of Sciences,
2021.
ista: Abas L, Kolb M, Stadlmann J, Janacek DP, Lukic K, Schwechheimer C, Sazanov
LA, Mach L, Friml J, Hammes UZ. 2021. Naphthylphthalamic acid associates with
and inhibits PIN auxin transporters. Proceedings of the National Academy of Sciences
of the United States of America. 118(1), e2020857118.
mla: Abas, Lindy, et al. “Naphthylphthalamic Acid Associates with and Inhibits PIN
Auxin Transporters.” Proceedings of the National Academy of Sciences of the
United States of America, vol. 118, no. 1, e2020857118, National Academy of
Sciences, 2021, doi:10.1073/pnas.2020857118.
short: L. Abas, M. Kolb, J. Stadlmann, D.P. Janacek, K. Lukic, C. Schwechheimer,
L.A. Sazanov, L. Mach, J. Friml, U.Z. Hammes, Proceedings of the National Academy
of Sciences of the United States of America 118 (2021).
date_created: 2021-01-03T23:01:23Z
date_published: 2021-01-05T00:00:00Z
date_updated: 2026-06-18T19:38:20Z
day: '05'
ddc:
- '580'
department:
- _id: JiFr
- _id: LeSa
doi: 10.1073/pnas.2020857118
ec_funded: 1
external_id:
isi:
- '000607270100073'
pmid:
- '33443187'
intvolume: ' 118'
isi: 1
issue: '1'
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.1073/pnas.2020857118
month: '01'
oa: 1
oa_version: Published Version
pmid: 1
project:
- _id: 261099A6-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '742985'
name: Tracing Evolution of Auxin Transport and Polarity in Plants
publication: Proceedings of the National Academy of Sciences of the United States
of America
publication_identifier:
eissn:
- 1091-6490
issn:
- 0027-8424
publication_status: published
publisher: National Academy of Sciences
quality_controlled: '1'
related_material:
link:
- relation: erratum
url: https://doi.org/10.1073/pnas.2102232118
scopus_import: '1'
status: public
title: Naphthylphthalamic acid associates with and inhibits PIN auxin transporters
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 118
year: '2021'
...