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In their Letter to Molecular Biology of the Cell, Schmoller et al. (2022) raise questions about the results and conclusions presented in our published studies (Dorsey et al., 2018; Litsios et al., 2019). Here, we respond to the criticisms of Schmoller et al. and demonstrate how wide-field fluorescence microscopy experiments to determine nuclear Whi5 concentration dynamics can be confounded by uncontrolled effects, which include photobleaching, partial confocal effects, and nuclear-to-cytoplasmic volume scaling. Further, we provide additional experimental evidence demonstrating that nuclear Whi5 concentration is essentially constant as cells grow in G1 phase and that Cln3 and protein synthesis dynamics occur as reported in Litsios et al. (2019). These results suggest that instead of being triggered by dilution of the stable inhibitor Whi5, Start is rather primarily controlled by the increase in protein synthesis rate in G1 and the concomitant production of the unstable activator Cln3. 

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae , is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c , renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6 , which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions. 

In budding yeast, the transcription factors SBF and MBF activate a large program of gene expression in late G1 phase that underlies commitment to cell division, termed Start. SBF/MBF are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the questions of how SBF/MBF are dynamically distributed across the G1/S regulon and how this impacts the Start transition. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits in fixed cells revealed each transcription factor was organized into discrete clusters containing approximately eight copies regardless of cell size and that the total number of clusters increased as cells grew through G1 phase. Stochastic modeling using reasonable biophysical parameters recapitulated growth-dependent SBF/MBF clustering and predicted TF dynamics that were confirmed in live cell PALM experiments. This spatio-temporal organization of SBF/MBF may help coordinate activation of G1/S regulon and the Start transition.