[{"year":"2024","ddc":["580"],"user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","date_published":"2024-07-26T00:00:00Z","OA_place":"publisher","type":"dissertation","abstract":[{"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.","lang":"eng"}],"_id":"17319","language":[{"iso":"eng"}],"has_accepted_license":"1","day":"26","citation":{"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>","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>.","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.","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.","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>.","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>","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."},"page":"224","status":"public","file_date_updated":"2025-01-26T23:30:04Z","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"}],"date_updated":"2026-04-07T13:20:44Z","publication_identifier":{"issn":["2663-337X"]},"oa_version":"Published Version","alternative_title":["ISTA Thesis"],"title":"Membrane proteins in plant physiology and bioenergetics : Investigating auxin efflux transporter PIN8 and ATP synthase inhibition by the novel inhibitor Yaku'amide B","date_created":"2024-07-26T09:05:55Z","publication_status":"published","supervisor":[{"first_name":"Leonid A","id":"338D39FE-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-0977-7989","full_name":"Sazanov, Leonid A","last_name":"Sazanov"}],"file":[{"access_level":"open_access","date_created":"2024-07-26T13:14:24Z","file_size":24639084,"content_type":"application/pdf","embargo":"2025-01-26","file_name":"Thesis_Kristina_Lukic.pdf","checksum":"95517e697ea6a87e267e649cad560989","creator":"cchlebak","relation":"main_file","date_updated":"2025-01-26T23:30:04Z","file_id":"17320"},{"embargo_to":"open_access","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","date_created":"2024-07-26T13:14:50Z","file_size":96334272,"access_level":"closed","date_updated":"2025-01-26T23:30:04Z","creator":"cchlebak","relation":"source_file","file_id":"17321","checksum":"74325746a9a05078fb9935dbf2aef752","file_name":"Thesis_Kristina_Lukic.docx"}],"publisher":"Institute of Science and Technology Austria","corr_author":"1","article_processing_charge":"No","degree_awarded":"PhD","doi":"10.15479/at:ista:17319","oa":1,"month":"07","department":[{"_id":"LeSa"},{"_id":"GradSch"}],"author":[{"orcid":"0000-0003-1581-881X","first_name":"Kristina","id":"2B04DB84-F248-11E8-B48F-1D18A9856A87","full_name":"Lukic, Kristina","last_name":"Lukic"}]},{"article_processing_charge":"No","publisher":"National Academy of Sciences","related_material":{"link":[{"relation":"erratum","url":"https://doi.org/10.1073/pnas.2102232118"}]},"pmid":1,"oa":1,"doi":"10.1073/pnas.2020857118","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.","author":[{"first_name":"Lindy","last_name":"Abas","full_name":"Abas, Lindy"},{"first_name":"Martina","full_name":"Kolb, Martina","last_name":"Kolb"},{"full_name":"Stadlmann, Johannes","last_name":"Stadlmann","first_name":"Johannes"},{"full_name":"Janacek, Dorina P.","last_name":"Janacek","first_name":"Dorina P."},{"last_name":"Lukic","full_name":"Lukic, Kristina","orcid":"0000-0003-1581-881X","id":"2B04DB84-F248-11E8-B48F-1D18A9856A87","first_name":"Kristina"},{"first_name":"Claus","full_name":"Schwechheimer, Claus","last_name":"Schwechheimer"},{"id":"338D39FE-F248-11E8-B48F-1D18A9856A87","first_name":"Leonid A","orcid":"0000-0002-0977-7989","last_name":"Sazanov","full_name":"Sazanov, Leonid A"},{"full_name":"Mach, Lukas","last_name":"Mach","first_name":"Lukas"},{"id":"4159519E-F248-11E8-B48F-1D18A9856A87","first_name":"Jiří","orcid":"0000-0002-8302-7596","full_name":"Friml, Jiří","last_name":"Friml"},{"last_name":"Hammes","full_name":"Hammes, Ulrich Z.","first_name":"Ulrich Z."}],"volume":118,"month":"01","department":[{"_id":"JiFr"},{"_id":"LeSa"}],"article_type":"original","project":[{"name":"Tracing Evolution of Auxin Transport and Polarity in Plants","_id":"261099A6-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","grant_number":"742985"}],"publication_identifier":{"issn":["0027-8424"],"eissn":["1091-6490"]},"title":"Naphthylphthalamic acid associates with and inhibits PIN auxin transporters","date_created":"2021-01-03T23:01:23Z","oa_version":"Published Version","publication_status":"published","status":"public","issue":"1","ec_funded":1,"scopus_import":"1","publication":"Proceedings of the National Academy of Sciences of the United States of America","date_updated":"2025-05-14T10:58:54Z","isi":1,"article_number":"e2020857118","intvolume":"       118","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","year":"2021","date_published":"2021-01-05T00:00:00Z","main_file_link":[{"url":"https://doi.org/10.1073/pnas.2020857118","open_access":"1"}],"external_id":{"pmid":["33443187"],"isi":["000607270100073"]},"language":[{"iso":"eng"}],"_id":"8993","abstract":[{"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.","lang":"eng"}],"quality_controlled":"1","type":"journal_article","day":"05","citation":{"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. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences. <a href=\"https://doi.org/10.1073/pnas.2020857118\">https://doi.org/10.1073/pnas.2020857118</a>","ieee":"L. Abas <i>et al.</i>, “Naphthylphthalamic acid associates with and inhibits PIN auxin transporters,” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 118, no. 1. National Academy of Sciences, 2021.","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).","mla":"Abas, Lindy, et al. “Naphthylphthalamic Acid Associates with and Inhibits PIN Auxin Transporters.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 118, no. 1, e2020857118, National Academy of Sciences, 2021, doi:<a href=\"https://doi.org/10.1073/pnas.2020857118\">10.1073/pnas.2020857118</a>.","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.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences, 2021. <a href=\"https://doi.org/10.1073/pnas.2020857118\">https://doi.org/10.1073/pnas.2020857118</a>.","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.","ama":"Abas L, Kolb M, Stadlmann J, et al. Naphthylphthalamic acid associates with and inhibits PIN auxin transporters. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. 2021;118(1). doi:<a href=\"https://doi.org/10.1073/pnas.2020857118\">10.1073/pnas.2020857118</a>"}}]