Ing. For ts-mutants 4? and 4?, but not for cdc33-1 much lower eIF4E-levels were detected when compared to wt (Figure 1D). This was surprising, as these mutants grew quite well at temperatures between 20uC and 30uC and even though eIF4E is an essential protein in yeast [27]. We assume, that at least under these conditions eIF4E is not limiting. To study the properties of mutant proteins, extracts were prepared from the different cell lines grown at 30uC and incubated with 7 mGDP-resin. Only in the case of wild type cells, eIF4E was detected in the 7mGDP eluate (Figure 1D). We also purified extracts from strains 4? and 4? grown at 20uC (they express 1.5- to 2-fold as much eIF4E as when grown at 30uC) with 7mGDP-resin and 117793 web couldn’t detect significant binding (not shown). We conclude that both, the loss of eIF4E-quantity and cap-binding activity of ts mutants are responsible for the loss of adhesion to a solid surface. We extended our studies by producing diploid cell lines which express wt or mutant eIF4E. Diploid temperature-sensitive cell lines completely loose the ability to form pseudohyphae when incubated under limiting nitrogen conditions (see Figure 1B). In order to quantify the observed effects, we used a Flo11-LacZ plasmid construct as a reporter to expression levels. The level of Flo11-expression (LacZ Units; normalized to Flo11 mRNA levels) was considerable lower for ts mutants 4? and 4? and at background level for ts-mutant cdc33-1 when compared to wt cells (see Figure 1C). When determing mRNA-levels by quantitative PCR, we 1313429 reproducibly observe an significantly increased Flo11 mRNA level for mutant cdc33-1 (almost 36higher than wt) which does not compensate for low Flo11-expression (Fig. 1C). All diploid cells (including those expressing wild type eIF4E) showed a very low level of Flo11-expression when grown in minimal SD medium supplemented with essential amino acids which hardly allowed for a proper correlation with pseudohyphenating properties. Only when diploid cells were incubated in SLAD medium with limiting ammonium concentrations (50 mM; about 10006 lower concentration of ammonium salt than normal SD medium), activation of Flo11-LacZ expression could be observed for diploid cells carrying wt eIF4E but not for diploid cells carrying eIF4E ts-mutants (not shown).interact electrostatically with the positive charge of m7G, stabilizing thereby the interaction of capped mRNA with eIF4E [28]. Not surprisingly, this mutant has a strong slow growth and tsphenotype (Figure S1) which is not due to a loss in interaction of eIF4E with its partners p20 and/or eIF4G (see Table S1). The consequences of eIF4E mutation E105Q were evident in both haploid and diploid cells as adhesion and pseudohyphenation were completely lost (Figures 2A and 2B) which for haploid cells was also confirmed by a substantial decrease in Flo11-LacZ expression (Figure 2C). E105Q protein level was somewhat reduced as compared to wild type cells but almost none of mutant E105Q protein was bound to 7mGDP resin (Figure 2D). Less pronounced was the effect of mutant D106N which is next to E105 but less proximal to the positively charged N6-imino group of 7mG (Figure S2). We did not detect notable growth defects for eIF4E mutant D106N (Figure S1) or loss of interaction with eIF4E partners (Table S1), haploid cells showed a pronounced loss of MNS biological activity adhesivity but diploid cells only a mild loss of pseudohyphenation (Figure 2A and B). The loss in adhesivity of haploid mutant D1.Ing. For ts-mutants 4? and 4?, but not for cdc33-1 much lower eIF4E-levels were detected when compared to wt (Figure 1D). This was surprising, as these mutants grew quite well at temperatures between 20uC and 30uC and even though eIF4E is an essential protein in yeast [27]. We assume, that at least under these conditions eIF4E is not limiting. To study the properties of mutant proteins, extracts were prepared from the different cell lines grown at 30uC and incubated with 7 mGDP-resin. Only in the case of wild type cells, eIF4E was detected in the 7mGDP eluate (Figure 1D). We also purified extracts from strains 4? and 4? grown at 20uC (they express 1.5- to 2-fold as much eIF4E as when grown at 30uC) with 7mGDP-resin and couldn’t detect significant binding (not shown). We conclude that both, the loss of eIF4E-quantity and cap-binding activity of ts mutants are responsible for the loss of adhesion to a solid surface. We extended our studies by producing diploid cell lines which express wt or mutant eIF4E. Diploid temperature-sensitive cell lines completely loose the ability to form pseudohyphae when incubated under limiting nitrogen conditions (see Figure 1B). In order to quantify the observed effects, we used a Flo11-LacZ plasmid construct as a reporter to expression levels. The level of Flo11-expression (LacZ Units; normalized to Flo11 mRNA levels) was considerable lower for ts mutants 4? and 4? and at background level for ts-mutant cdc33-1 when compared to wt cells (see Figure 1C). When determing mRNA-levels by quantitative PCR, we 1313429 reproducibly observe an significantly increased Flo11 mRNA level for mutant cdc33-1 (almost 36higher than wt) which does not compensate for low Flo11-expression (Fig. 1C). All diploid cells (including those expressing wild type eIF4E) showed a very low level of Flo11-expression when grown in minimal SD medium supplemented with essential amino acids which hardly allowed for a proper correlation with pseudohyphenating properties. Only when diploid cells were incubated in SLAD medium with limiting ammonium concentrations (50 mM; about 10006 lower concentration of ammonium salt than normal SD medium), activation of Flo11-LacZ expression could be observed for diploid cells carrying wt eIF4E but not for diploid cells carrying eIF4E ts-mutants (not shown).interact electrostatically with the positive charge of m7G, stabilizing thereby the interaction of capped mRNA with eIF4E [28]. Not surprisingly, this mutant has a strong slow growth and tsphenotype (Figure S1) which is not due to a loss in interaction of eIF4E with its partners p20 and/or eIF4G (see Table S1). The consequences of eIF4E mutation E105Q were evident in both haploid and diploid cells as adhesion and pseudohyphenation were completely lost (Figures 2A and 2B) which for haploid cells was also confirmed by a substantial decrease in Flo11-LacZ expression (Figure 2C). E105Q protein level was somewhat reduced as compared to wild type cells but almost none of mutant E105Q protein was bound to 7mGDP resin (Figure 2D). Less pronounced was the effect of mutant D106N which is next to E105 but less proximal to the positively charged N6-imino group of 7mG (Figure S2). We did not detect notable growth defects for eIF4E mutant D106N (Figure S1) or loss of interaction with eIF4E partners (Table S1), haploid cells showed a pronounced loss of adhesivity but diploid cells only a mild loss of pseudohyphenation (Figure 2A and B). The loss in adhesivity of haploid mutant D1.
