Yeast cells, wild-type

Incorporation of a stable isotopic tag into proteins/peptides in metabolically active cells was first described to quantify protein abundance in yeast (43). Wild-type and mutant cell populations were grown in media containing the naturally abundant isotopes of nitrogen and enriched in 15N, respectively, followed by trypsin digestion and LC-ESI-MS/MS analysis to identify and quantify relative phosphopeptide levels in both populations (43). 

PSI] is the prion of Sup35p, a translation termination factor (Ter-Avanesyan et al., 1994 Wickner, 1994). Conversion of wild-type yeast cells to the infected state results in reduction of the termination activity and, consequendy, to a nonsense suppression phenotype. This property can be used to detect [PSI] by genetic selection (Fig. 2). Sup35p is an essential gene whose knockout leads to cell death. Therefore, it appears that the [PSI] condition corresponds to only partial inactivation of Sup35p and enough of the normal protein is left to avert cell death. 

Symbol for the temperature coefficient, a quotient equal to Vt+wIvt, where Vr+io and Vj are the rates of a process (e.g., an enzyme-catalyzed reaction) at two temperatures differing by 10°C. This parameter is usually evaluated at saturating concentrations of substrate(s), so that temperature-dependent changes in Michaelis constant(s) are inconsequential. The <2io value is a characteristic property of a particular enzyme from a specific organism and cell type. For example, one cannot use the Qio value for one hexokinase from yeast to infer the temperature dependence of another hexokinase, say from rat brain. Likewise, the Qio value need not remain the same for a mutant form and a wild-type enzyme. 

As illustrated by the [PS / ] phenomenon the relative abundance of the eRFl/eRF3 complexes is an important determinant for the efficiency of translation termination. Similar effects have been obtained in depletion studies and with eRF3 and eRFl mutants that decrease their cellular concentration (Stansfield et al. 1996 Moskalenko et al. 2003 Chabelskaya et al. 2004 Salas-Marco and Bedwell 2004). Wild type yeast contains approximately 10- to 20-fold fewer termination factors compared to ribosomes (Didichenko et al. 1991 Stansfield et al. 1992). The threshold level of eRFl/eRF3 required to maintain viability is even lower. A 10-fold decrease reduces viability by only about 10% (Valouev et al. 2002) and a decrease in eRF3 of 99% does not affect viability of the cells significantly (Chabelskaya et al. 2004). As eRF3 is associated with polysomes or ribosomes, the mechanism of translation termination depends on efficient recycling (Didichenko et al. 1991 and compare below). 

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