Yoko Otsubo

Shugoshin family proteins localize to centromeres and play pivotal roles in chromosome segregation during mitosis and meiosis. In fission yeast, the Shugoshin paralog Sgo2 relocates from centromeres to subtelomeres during interphase, where it contributes to gene repression by establishing a subtelomere-specific condensed chromatin structure known as the knob. However, the mechanisms underlying subtelomere-specific Sgo2 localization and knob formation during interphase remain poorly understood. Here, we identified Nts1, a component of the histone deacetylase complex, as a key regulator of Sgo2 localization through a genetic screen. Deletion of both nts1 + and set2 + (which encodes a histone H3-K36 methyltransferase) resulted in an almost complete loss of Sgo2 localization and knob formation at subtelomeres, indicating that Nts1 and Set2 function redundantly to target Sgo2 to subtelomeres. Notably, Nts1 localizes to subtelomeres during interphase and promotes histone H4 deacetylation, suggesting that histone deacetylation serves as a landmark for subtelomere-specific Sgo2 localization and knob formation.

Extender of chronological lifespan 1 (Ecl1) inhibits target of rapamycin complex 1 (TORC1) and is necessary for appropriate cellular responses to various stressors, such as starvation, in fission yeast. However, little is known about the effect of posttranslational modifications on Ecl1 regulation. Thus, we investigated the phosphorylation levels of Ecl1 extracted from yeast under conditions of sulfur or metal starvation. Mass spectrometry analysis revealed that Ecl1 was phosphorylated at Thr7, and the level was decreased by starvation. The phosphorylation-mimetic mutation of Thr7 significantly reduced the effects of Ecl1-induced cellular responses to starvation, suggesting that Ecl1 function was suppressed by Thr7 phosphorylation. By contrast, regardless of starvation exposure, TORC1 was significantly suppressed, even when Thr7 phosphorylation-mimetic Ecl1 was overexpressed. This indicated that Ecl1 suppressed TORC1 regardless of Thr7 phosphorylation. We newly identified that Ecl1 physically interacted with TORC1 subunit RAPTOR (Mip1). Based on these evidences, we propose that, Ecl1 has dual functional modes: quantity-dependent TORC1 inhibition and Thr7 phosphorylation-dependent control of cellular function.

ABSTRACT Certain proteins assemble into diverse complex states, each having a distinct and unique function in the cell. Target of rapamycin (Tor) complex 1 (TORC1) plays a central role in signalling pathways that allow cells to respond to the environment, including nutritional status signalling. TORC1 is widely recognised for its association with various diseases. The budding yeast Saccharomyces cerevisiae has two types of TORC1, Tor1-containing TORC1 and Tor2-containing TORC1, which comprise different constituent proteins but are considered to have the same function. Here, we computationally modelled the relevant complex structures and then, based on the structures, rationally engineered a Tor2 mutant that could form Tor complex 2 (TORC2) but not TORC1, resulting in a redesign of the complex states. Functional analysis of the Tor2 mutant revealed that the two types of TORC1 induce different phenotypes, with changes observed in rapamycin, caffeine and pH dependencies of cell growth, as well as in replicative and chronological lifespan. These findings uncovered by a general approach with huge potential – model structure-based engineering – are expected to provide further insights into various fields such as molecular evolution and lifespan.