Sit4 Genetically Interacts with Vps27 to Regulate Mitochondrial Function and Lifespan in .

The Sit4 protein phosphatase plays a key role in orchestrating various cellular processes essential for maintaining cell viability during aging. We have previously shown that 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 Δ vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0. In addition, exhibited a negative genetic interaction with , as ∆∆ double mutants had a shortened lifespan compared to ∆ and ∆ single mutants. Our results also show that Vps27 did not increase ∆ lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in ∆ cells, as ∆∆ 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.