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A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast.

Alberghina L, Rossi RL, Querin L, Wanke V, Vanoni M - J. Cell Biol. (2004)

Bottom Line: Cells grown in glucose are larger than cells grown in ethanol.Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type.A new molecular network accounting for the setting of Ps is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy. lilia.alberghina@unimib.it

ABSTRACT
Saccharomyces cerevisiae must reach a carbon source-modulated critical cell size, protein content per cell at the onset of DNA replication (Ps), in order to enter S phase. Cells grown in glucose are larger than cells grown in ethanol. Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type. Cln3 Delta, far1 Delta, and strains overexpressing Far1 do not delay budding during an ethanol glucose shift-up as wild type does. Together, these findings indicate that Cln3 has to overcome Far1 to trigger Cln-Cdc28 activation, which then turns on SBF- and MBF-dependent transcription. We show that a second threshold is required together with the Cln3/Far1 threshold for carbon source modulation of Ps. A new molecular network accounting for the setting of Ps is proposed.

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Far1-dependent delay of budding in an ethanol to glucose nutritional shift up. (A) Far1 and Cln3 protein levels in wild-type and FAR1 overexpressing strain (FAR1tet): both strains were grown in SCE (E) and SCD (D) and mid log phase cells were collected for protein extraction followed by Western blot analysis with anti-Myc antibodies as detailed in Materials and methods. (B) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells in wild-type (black), far1Δ (red), cln3Δ (green), and FAR1 overexpressing (FAR1tet, blue) strains. (C) Western blots of Far1 and Cln3 proteins from cells overexpressing FAR1 (FAR1tet strain) during an ethanol/glucose shift up. (D) Western blot of Far1 and Cln3 proteins from wild-type cells (W303-CF strain) during an ethanol/glucose shift-up. (E) Cln3 (green) and Far1 (red) protein levels and their ratio (blue) were determined during ethanol/glucose shift-up by quantification of Western blot bands of wild-type cells (W303-CF strain) as detailed in Materials and methods. Percentage of budded cells (black) and Cln3/Far1 mean values ratio (blue) are also plotted as a function of time. Protein levels shown are average ± SD of data derived from independent experiments repeated at least twice. Blots shown are representative of experiments repeated at least twice with similar results.
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fig4: Far1-dependent delay of budding in an ethanol to glucose nutritional shift up. (A) Far1 and Cln3 protein levels in wild-type and FAR1 overexpressing strain (FAR1tet): both strains were grown in SCE (E) and SCD (D) and mid log phase cells were collected for protein extraction followed by Western blot analysis with anti-Myc antibodies as detailed in Materials and methods. (B) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells in wild-type (black), far1Δ (red), cln3Δ (green), and FAR1 overexpressing (FAR1tet, blue) strains. (C) Western blots of Far1 and Cln3 proteins from cells overexpressing FAR1 (FAR1tet strain) during an ethanol/glucose shift up. (D) Western blot of Far1 and Cln3 proteins from wild-type cells (W303-CF strain) during an ethanol/glucose shift-up. (E) Cln3 (green) and Far1 (red) protein levels and their ratio (blue) were determined during ethanol/glucose shift-up by quantification of Western blot bands of wild-type cells (W303-CF strain) as detailed in Materials and methods. Percentage of budded cells (black) and Cln3/Far1 mean values ratio (blue) are also plotted as a function of time. Protein levels shown are average ± SD of data derived from independent experiments repeated at least twice. Blots shown are representative of experiments repeated at least twice with similar results.

Mentions: We showed previously that cells undergoing a nutritional shift-up delay execution of the G1 to S transition as indicated by a transient drop in the fraction of budded cells, while at the same time cell size increases, consistent with a resetting of Ps to a new, higher value that is typical of the new medium (Alberghina et al., 1998). If the above described Cln3/Far1 threshold is acting as a regulatory device during the ethanol glucose shift-up, we expect that the signature drop in budding should be lost in cells where the balance between the two components of the threshold is altered. In fact, the absence of the Far1 inhibitor in far1Δ cells should make cells respond to the change in carbon source as soon as Cln3 increases. Accordingly, in cln3Δ cells the target for inhibition by Far1 is absent, so that no drop in budding should be expected. Finally, because overexpression of Far1 results in a large excess of Far1 over Cln3 (Fig. 4 A, where protein levels can be directly compared because both Far1 and Cln3 are fused to the same 15-myc tag), we expect that this strain should display a phenotype similar to that of cln3Δ cells. Data reported in Fig. 4 B show budding kinetics during a shift-up for wild-type (black), far1Δ (red), cln3Δ (green), and Far1 overexpressing (Far1tet, blue) cells. In keeping with our predictions, the drop in budding is present only in wild-type cells, and is lost in strains where the normal interplay between Cln3 and Far1 is disrupted.


A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast.

Alberghina L, Rossi RL, Querin L, Wanke V, Vanoni M - J. Cell Biol. (2004)

Far1-dependent delay of budding in an ethanol to glucose nutritional shift up. (A) Far1 and Cln3 protein levels in wild-type and FAR1 overexpressing strain (FAR1tet): both strains were grown in SCE (E) and SCD (D) and mid log phase cells were collected for protein extraction followed by Western blot analysis with anti-Myc antibodies as detailed in Materials and methods. (B) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells in wild-type (black), far1Δ (red), cln3Δ (green), and FAR1 overexpressing (FAR1tet, blue) strains. (C) Western blots of Far1 and Cln3 proteins from cells overexpressing FAR1 (FAR1tet strain) during an ethanol/glucose shift up. (D) Western blot of Far1 and Cln3 proteins from wild-type cells (W303-CF strain) during an ethanol/glucose shift-up. (E) Cln3 (green) and Far1 (red) protein levels and their ratio (blue) were determined during ethanol/glucose shift-up by quantification of Western blot bands of wild-type cells (W303-CF strain) as detailed in Materials and methods. Percentage of budded cells (black) and Cln3/Far1 mean values ratio (blue) are also plotted as a function of time. Protein levels shown are average ± SD of data derived from independent experiments repeated at least twice. Blots shown are representative of experiments repeated at least twice with similar results.
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Related In: Results  -  Collection

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fig4: Far1-dependent delay of budding in an ethanol to glucose nutritional shift up. (A) Far1 and Cln3 protein levels in wild-type and FAR1 overexpressing strain (FAR1tet): both strains were grown in SCE (E) and SCD (D) and mid log phase cells were collected for protein extraction followed by Western blot analysis with anti-Myc antibodies as detailed in Materials and methods. (B) At time 0 2% glucose was added to cells exponentially growing in SCE medium and samples were collected to evaluate the percentage of budded cells in wild-type (black), far1Δ (red), cln3Δ (green), and FAR1 overexpressing (FAR1tet, blue) strains. (C) Western blots of Far1 and Cln3 proteins from cells overexpressing FAR1 (FAR1tet strain) during an ethanol/glucose shift up. (D) Western blot of Far1 and Cln3 proteins from wild-type cells (W303-CF strain) during an ethanol/glucose shift-up. (E) Cln3 (green) and Far1 (red) protein levels and their ratio (blue) were determined during ethanol/glucose shift-up by quantification of Western blot bands of wild-type cells (W303-CF strain) as detailed in Materials and methods. Percentage of budded cells (black) and Cln3/Far1 mean values ratio (blue) are also plotted as a function of time. Protein levels shown are average ± SD of data derived from independent experiments repeated at least twice. Blots shown are representative of experiments repeated at least twice with similar results.
Mentions: We showed previously that cells undergoing a nutritional shift-up delay execution of the G1 to S transition as indicated by a transient drop in the fraction of budded cells, while at the same time cell size increases, consistent with a resetting of Ps to a new, higher value that is typical of the new medium (Alberghina et al., 1998). If the above described Cln3/Far1 threshold is acting as a regulatory device during the ethanol glucose shift-up, we expect that the signature drop in budding should be lost in cells where the balance between the two components of the threshold is altered. In fact, the absence of the Far1 inhibitor in far1Δ cells should make cells respond to the change in carbon source as soon as Cln3 increases. Accordingly, in cln3Δ cells the target for inhibition by Far1 is absent, so that no drop in budding should be expected. Finally, because overexpression of Far1 results in a large excess of Far1 over Cln3 (Fig. 4 A, where protein levels can be directly compared because both Far1 and Cln3 are fused to the same 15-myc tag), we expect that this strain should display a phenotype similar to that of cln3Δ cells. Data reported in Fig. 4 B show budding kinetics during a shift-up for wild-type (black), far1Δ (red), cln3Δ (green), and Far1 overexpressing (Far1tet, blue) cells. In keeping with our predictions, the drop in budding is present only in wild-type cells, and is lost in strains where the normal interplay between Cln3 and Far1 is disrupted.

Bottom Line: Cells grown in glucose are larger than cells grown in ethanol.Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type.A new molecular network accounting for the setting of Ps is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy. lilia.alberghina@unimib.it

ABSTRACT
Saccharomyces cerevisiae must reach a carbon source-modulated critical cell size, protein content per cell at the onset of DNA replication (Ps), in order to enter S phase. Cells grown in glucose are larger than cells grown in ethanol. Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type. Cln3 Delta, far1 Delta, and strains overexpressing Far1 do not delay budding during an ethanol glucose shift-up as wild type does. Together, these findings indicate that Cln3 has to overcome Far1 to trigger Cln-Cdc28 activation, which then turns on SBF- and MBF-dependent transcription. We show that a second threshold is required together with the Cln3/Far1 threshold for carbon source modulation of Ps. A new molecular network accounting for the setting of Ps is proposed.

Show MeSH
Related in: MedlinePlus