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The Quiescent State
[Greg Petsko] |
Projects Page
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People working on this project: |
Yeast Genetics Luda Berenfeld DJ-1/ThiJ Mark Wilson |
Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a station ary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Stationary phase, also called the G0 state of the cell cycle, is a remarkable state. Yeast cells in stationary phase can survive for months without added nutrients; indeed, the cells can survive prolonged incubation in distilled water! Most of the cells in a yeast colony on a plate are in stationary phase. Most of the cells of most microorganisms are in stationary phase most of the time in the wild; the proliferating cell, which has been the object of most studies, is actually a rare state. The same is true for many of the cell types in the human body. For example, neurons are in stationary phase from the time of their terminal differentiation. Diseases that require proliferation, such as neuronal cancer, must overcome the blocks to growth that stationary phase set s up; this is just one reason why understanding the biology of stationary phase is important.
Recent characterization of mutant yeast cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis (remodelling?), and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. Determining the factors responsible for entry into stationary phase, survival of cells in G0, and exit to proliferation is the goal of this project. For our model system we choose the budding yeast Saccharomyces cerevisiae. We have found a substance, azetidine carboxylic acid (AZC), that induces a G0-like state in yeast by flooding the cell with misfolded proteins. We have obtained mutants that are resistant to AZC, and others that are supersensitive to the drug. We are characterizing the genes responsible. We have also used the cDNA microarray technology to determine which genes are turned off and on when cells are exposed to AZC. We find about 300 genes whose mRNA is up- or down-regulated by more then 5-fold; many are not homologous to any known protein and we are in the process of cloning and characterizing them as well. One of the most interesting effects that AZC produces is an immediate global reduction in the mRNA coding for all of the ribosomal subunits. We are searching for the factors that regulate this translational shutdown.
In addition, we are screening for genes that control membrane remodelling during entry into and exit from stationary phase, as well as genes that regulate the turnover of proteins during G0. We expect that some of the genes we uncover by these experiments will yield proteins whose three-dimensional structure will need to be determined by X-ray crystallography and whose activity will need to be characterized enzymatically, and those experiments we will do.
Trotter EW, Kao CM, Berenfeld L, Botstein D, Petsko GA, Gray JV Misfolded proteins are competent to mediate a subset of the responses to heat shock in Saccharomyces cerevisiae. J Biol Chem. (2002) 277(47):44817-25.
Trotter EW, Berenfeld L, Krause SA, Petsko GA, Gray JV Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomycescerevisiae. Proc Natl Acad Sci (2001) 98(13):7313-8.