Study On The Regulatory Mechanism That Suppresses Exit From The Quiescent State In Saccharomyces Cerevisiae

In response to nutrient starvation,cells of the budding yeast Saccharomyces cerevisiae form two different types of quiescent states: stationary cells and spores.When conditions become favorable(e.g.,in the presence of glucose),these cells exit from quiescent state and enter into mitosis.The process for spores to exit from quiescent state is called germination.Although many researchers have been conducted studies as to how quiescent cells sense nutrients in the environment and resume growth,the mechanisms that regulate cell exit from quiescent phase are still unclear.Defining the relationship between the regulatory mechanisms and metabolism of quiescent and proliferative transitions remain a major challenge in biology.In this study,we reported for the first time that the presence of a regulatory process which suppresses exit from quiescent state(the negative regulatory process).We demonstrated that exit and maintenance of the quiescent state can be manipulated by modulating the negative regulatory process.Clinically,quiescent cells are problematic because of their stress-resistant property.Therefore,the discovery of this mechanism would provide a theoretical basis for the development of novel drugs and strategies to kill quiescent pathogens and cancer cells.In this paper,biochemical analyses,gene manipulation and other methods to explore,the main results are as follows:(1)Germination of S.cerevisiae spores is induced by heat treatment without nutrients.We found that ascal lysate,which includes extraspore fluid in the ascus,induced morphological changes when the lysate was treated with heat.By incubation with the heated lysate,levels of the mitotic cyclin Clb2 were elevated in spores,showing that the morphological changes were attributable to germination.Further studies showed that direct heating of spores could induce germination without addition of nutrients.(2)Tpi1 is a regulatory molecule that inhibits spore germination.By analysis of heat-induced spore germination,we found that certain levels of glucose were present in the ascus.Nevertheless,spores do not germinate in asci if spores were not subjected to heat treatment,because inhibitory molecules are present in the ascal cytosol and presumably spores.The heat-induced spore germination occurs due to the heat inactivation of the inhibitory molecule.We found that the inhibitory molecule was a glycolytic enzyme triosephosphate isomerase(Tpi1).Genetic and biochemical analyses showed that the catalytic activity was required for suppression of spore germination.(3)Inhibition of spore germination is mediated by Tpi1 and its product GAPTpi1 is an enzyme that converts dihydroxyacetone phosphate(DHAP)to glyceraldehyde triphosphate(GAP).It was found that GAP could inhibit spore germination.It is worth noting that Tpi1 can inhibit germination even if it is present outside of spores.This is because DHAP is released from spores in the presence of glucose.These results suggest that there is a unique mechanism to regulate germination in asci.(4)The negative regulatory pathway is conserved in eukaryotic cellsStationary cells are another quiescent form of S.cerevisiae.Similar to spores,the exit from stationary state can be inhibited by GAP.In the absence of nutrients,the exit from stationary state can also be induced by heating.However,the growth of yeast cells in logarithmic phase was not affected by GAP.Furthermore,we found that GAP suppress proliferation of quiescent cells of a pathogenic yest Candida glabrata and mammalian cells.Thus,the negative regulatory process which mediated by GAP is conserved in eukaryotic cells.