RESUMEN
Although cell size regulation is crucial for cellular functions in a variety of organisms from bacteria to humans, the underlying mechanisms remain elusive. Here, we identify Rim21, a component of the pH-sensing Rim101 pathway, as a positive regulator of cell size through a flow cytometry-based genome-wide screen of Saccharomyces cerevisiae deletion mutants. We found that mutants defective in the Rim101 pathway were consistently smaller than wildtype cells in the log and stationary phases. We show that the expression of the active form of Rim101 increased the size of wildtype cells. Furthermore, the size of wildtype cells increased in response to external alkalization. Microscopic observation revealed that this cell size increase was associated with changes in both vacuolar and cytoplasmic volume. We also found that these volume changes were dependent on Rim21 and Rim101. In addition, a mutant lacking Vph1, a component of V-ATPase that is transcriptionally regulated by Rim101, was also smaller than wildtype cells, with no increase in size in response to alkalization. We demonstrate that the loss of Vph1 suppressed the Rim101-induced increase in cell size under physiological pH conditions. Taken together, our results suggest that the cell size of budding yeast is regulated by the Rim101-V-ATPase axis under physiological conditions as well as in response to alkaline stresses.
Asunto(s)
Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Concentración de Iones de Hidrógeno , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Eliminación de SecuenciaRESUMEN
Copper is essential for the activity and stability of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Loss-of-function mutations in genes required for copper transport to CcO result in fatal human disorders. Despite the fundamental importance of copper in mitochondrial and organismal physiology, systematic identification of genes that regulate mitochondrial copper homeostasis is lacking. To discover these genes, we performed a genome-wide screen using a library of DNA-barcoded yeast deletion mutants grown in copper-supplemented media. Our screen recovered a number of genes known to be involved in cellular copper homeostasis as well as genes previously not linked to mitochondrial copper biology. These newly identified genes include the subunits of the adaptor protein 3 complex (AP-3) and components of the cellular pH-sensing pathway Rim20 and Rim21, both of which are known to affect vacuolar function. We find that AP-3 and Rim mutants exhibit decreased vacuolar acidity, which in turn perturbs mitochondrial copper homeostasis and CcO function. CcO activity of these mutants could be rescued by either restoring vacuolar pH or supplementing growth media with additional copper. Consistent with these genetic data, pharmacological inhibition of the vacuolar proton pump leads to decreased mitochondrial copper content and a concomitant decrease in CcO abundance and activity. Taken together, our study uncovered novel genetic regulators of mitochondrial copper homeostasis and provided a mechanism by which vacuolar pH impacts mitochondrial respiration through copper homeostasis.
Asunto(s)
Cobre/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Medios de Cultivo , Complejo IV de Transporte de Electrones/genética , Genoma Fúngico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homeostasis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
Lager yeast is the most popular yeast strain used for beer production in China. The flocculation of yeast plays an important role in cell separation at the end of fermentation. Therefore, appropriately enhancing the flocculation capability of the lager yeast without affecting its fermentation performance would be desirable for beer industry. Our previous study showed that the defect of gene RIM21 might contribute to the enhanced flocculation capability of a lager yeast G03. To further investigate the role of the RIM21 gene in flocculation of strain G03, this study constructed a RIM21-deleted mutant strain G03-RIM21Δ through homologous recombination. Deletion of RIM21 improved the flocculation capability of strain G03 during wort fermentation at 11 °C without changing its fermentation performance significantly. The expression of FLO5, Lg-FLO1 and some other genes involved in cell wall integrity pathway were up-regulated in strain G03-RIM21Δ. In addition, the disruption of RIM21 enhanced resistance of yeast cells to cell wall inhibitors. These results provide a basis for elucidating the flocculation mechanism of lager yeast under low-temperature fermentation conditions.
Asunto(s)
Floculación , Proteínas de Saccharomyces cerevisiae , Saccharomyces , Cerveza , Fermentación , Receptores de Superficie Celular , Saccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Lager yeast is the most popular yeast strain used for beer production in China. The flocculation of yeast plays an important role in cell separation at the end of fermentation. Therefore, appropriately enhancing the flocculation capability of the lager yeast without affecting its fermentation performance would be desirable for beer industry. Our previous study showed that the defect of gene RIM21 might contribute to the enhanced flocculation capability of a lager yeast G03. To further investigate the role of the RIM21 gene in flocculation of strain G03, this study constructed a RIM21-deleted mutant strain G03-RIM21Δ through homologous recombination. Deletion of RIM21 improved the flocculation capability of strain G03 during wort fermentation at 11 °C without changing its fermentation performance significantly. The expression of FLO5, Lg-FLO1 and some other genes involved in cell wall integrity pathway were up-regulated in strain G03-RIM21Δ. In addition, the disruption of RIM21 enhanced resistance of yeast cells to cell wall inhibitors. These results provide a basis for elucidating the flocculation mechanism of lager yeast under low-temperature fermentation conditions.
Asunto(s)
Cerveza , Fermentación , Floculación , Receptores de Superficie Celular , Saccharomyces/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Yeast responds to alterations in plasma membrane lipid asymmetry and external alkalization via the sensor protein Rim21 in the Rim101 pathway. However, the sensing mechanism used by Rim21 remains unclear. Here, we found that the C-terminal cytosolic domain of Rim21 (Rim21C) fused with GFP was associated with the plasma membrane under normal conditions but dissociated upon alterations in lipid asymmetry or external alkalization. This indicates that Rim21C contains a sensor motif. Rim21C contains multiple clusters of charged residues. Among them, three consecutive Glu residues (EEE motif) were essential for Rim21 function and dissociation of Rim21C from the plasma membrane in response to changes in lipid asymmetry. In contrast, positively charged residues adjacent to the EEE motif were required for Rim21C to associate with the membrane. We therefore propose an "antenna hypothesis," in which Rim21C moves to or from the plasma membrane and functions as the sensing mechanism of Rim21.