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cellular processes essential for maintaining cell viability during aging. We have previously shown that SIT4 deletion promotes vacuolar acidification, mitochondrial derepression, and oxidative stress resistance, increasing yeast chronological lifespan. In this study, we performed a proteomic analysis of isolated vacuoles and yeast genetic interaction analysis to unravel how Sit4 influences vacuolar and mitochondrial function. By employing high-resolution mass spectrometry, we show that sit4\u0394 vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0. In addition, SIT4 exhibited a negative genetic interaction with VPS27, as sit4\u2206vps27\u2206 double mutants had a shortened lifespan compared to sit4\u2206 and vps27\u2206 single mutants. Our results also show that Vps27 did not increase sit4\u2206 lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in sit4\u2206 cells, as sit4\u2206vps27\u2206 double mutants exhibited high iron levels and impaired mitochondrial respiration. These findings show, for the first time, cross-talk between Sit4 and Vps27, providing new insights into the mechanisms governing chronological lifespan.<\/jats:p>","DOI":"10.3390\/cells13080655","type":"journal-article","created":{"date-parts":[[2024,4,9]],"date-time":"2024-04-09T04:37:27Z","timestamp":1712637447000},"page":"655","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Sit4 Genetically Interacts with Vps27 to Regulate Mitochondrial Function and Lifespan in Saccharomyces cerevisiae"],"prefix":"10.3390","volume":"13","author":[{"given":"Telma S.","family":"Martins","sequence":"first","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sau\u0301de, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"ICBAS\u2014Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal"}]},{"given":"Miguel","family":"Correia","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sau\u0301de, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"}]},{"given":"Denise","family":"Pinheiro","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sau\u0301de, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"}]},{"given":"Carolina","family":"Lemos","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sau\u0301de, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"ICBAS\u2014Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6105-522X","authenticated-orcid":false,"given":"Marta Vaz","family":"Mendes","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sau\u0301de, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8167-6912","authenticated-orcid":false,"given":"Clara","family":"Pereira","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sau\u0301de, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7868-4663","authenticated-orcid":false,"given":"V\u00edtor","family":"Costa","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sau\u0301de, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"ICBAS\u2014Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,4,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.arr.2016.04.009","article-title":"The Crucial Impact of Lysosomes in Aging and Longevity","volume":"32","author":"Hughes","year":"2016","journal-title":"Ageing Res. 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