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The septins function in G1 pathways that influence the pattern of cell growth in budding yeast.

Egelhofer TA, Villén J, McCusker D, Gygi SP, Kellogg DR - PLoS ONE (2008)

Bottom Line: However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle.Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1.Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America.

ABSTRACT
The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation.

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Shs1 is required for normal localization of the Cdc11 septin in cln1Δ cln2Δ cells.(A–C) shs1Δ cln1Δ GAL1-CLN2 cells and (D–E) cln1Δ GAL1-CLN2 control cells were grown to log phase in YP media containing galactose and switched to YPD media for 4.5 hours. Cdc11 localization was determined with an anti-Cdc11 antibody. Arrows point to abnormal Cdc11 localization. The arrow head points to normal Cdc11 localization. Bar, 5 µm for all panels. (F) The percentage of budded cells in cln1Δ GAL1-CLN2 and shs1Δ cln1Δ GAL1-CLN2 cells that had any polarized Cdc11 localization in the mother or daughter cell was determined, including cells that had polarized but abnormal localization of Cdc11. The percentage of budded cells that had normal Cdc11 localization at the bud neck was also determined. 200 cells were counted for each strain.
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pone-0002022-g003: Shs1 is required for normal localization of the Cdc11 septin in cln1Δ cln2Δ cells.(A–C) shs1Δ cln1Δ GAL1-CLN2 cells and (D–E) cln1Δ GAL1-CLN2 control cells were grown to log phase in YP media containing galactose and switched to YPD media for 4.5 hours. Cdc11 localization was determined with an anti-Cdc11 antibody. Arrows point to abnormal Cdc11 localization. The arrow head points to normal Cdc11 localization. Bar, 5 µm for all panels. (F) The percentage of budded cells in cln1Δ GAL1-CLN2 and shs1Δ cln1Δ GAL1-CLN2 cells that had any polarized Cdc11 localization in the mother or daughter cell was determined, including cells that had polarized but abnormal localization of Cdc11. The percentage of budded cells that had normal Cdc11 localization at the bud neck was also determined. 200 cells were counted for each strain.

Mentions: Loss of Cdc3, Cdc10, Cdc11 or Cdc12 results in rapid loss of localization of the septins; however, loss of Shs1 has no effect on septin localization [16], [33], [34]. Since Shs1 is essential for viability in cln1Δ cln2Δ cells, we tested whether Shs1 is essential for localization of the other septins in cells that lack Cln1 and Cln2. We shifted shs1Δ cln1Δ GAL1-CLN2 and cln1Δ GAL1-CLN2 control cells into dextrose for 4.5 hours and used immunofluorescence to test for Cdc11 localization. Most shs1Δ cln1Δ GAL1-CLN2 cells showed a complete failure to localize Cdc11 (Figure 3A), although some unbudded cells had diffuse Cdc11 localization over one end of the cell (arrow, Figure 3B). Of the few budded shs1Δ cln1Δ GAL1-CLN2 cells, only 30% had polarized Cdc11 localization in the mother or daughter cell and the Cdc11 localization in the majority of these cells was aberrant (arrow, Figure 3C). Only 7.5% of budded cells had normal Cdc11 localization (Figure 3F). In cln1Δ GAL-CLN2 control cells, 100% of budded cells had polarized Cdc11 localization and 80% had normal Cdc11 localization (arrowhead, Figure 3D and 3F). In addition, the defects in Cdc11 localization that were observed in cells that lack Cln1 and Cln2 were less severe than the defects observed in cells that lack Shs1, Cln1, and Cln2 (arrow, Figure 3E). These results demonstrate that Shs1 is required for the normal localization of Cdc11 in cells that lack Cln1 and Cln2.


The septins function in G1 pathways that influence the pattern of cell growth in budding yeast.

Egelhofer TA, Villén J, McCusker D, Gygi SP, Kellogg DR - PLoS ONE (2008)

Shs1 is required for normal localization of the Cdc11 septin in cln1Δ cln2Δ cells.(A–C) shs1Δ cln1Δ GAL1-CLN2 cells and (D–E) cln1Δ GAL1-CLN2 control cells were grown to log phase in YP media containing galactose and switched to YPD media for 4.5 hours. Cdc11 localization was determined with an anti-Cdc11 antibody. Arrows point to abnormal Cdc11 localization. The arrow head points to normal Cdc11 localization. Bar, 5 µm for all panels. (F) The percentage of budded cells in cln1Δ GAL1-CLN2 and shs1Δ cln1Δ GAL1-CLN2 cells that had any polarized Cdc11 localization in the mother or daughter cell was determined, including cells that had polarized but abnormal localization of Cdc11. The percentage of budded cells that had normal Cdc11 localization at the bud neck was also determined. 200 cells were counted for each strain.
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Related In: Results  -  Collection

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pone-0002022-g003: Shs1 is required for normal localization of the Cdc11 septin in cln1Δ cln2Δ cells.(A–C) shs1Δ cln1Δ GAL1-CLN2 cells and (D–E) cln1Δ GAL1-CLN2 control cells were grown to log phase in YP media containing galactose and switched to YPD media for 4.5 hours. Cdc11 localization was determined with an anti-Cdc11 antibody. Arrows point to abnormal Cdc11 localization. The arrow head points to normal Cdc11 localization. Bar, 5 µm for all panels. (F) The percentage of budded cells in cln1Δ GAL1-CLN2 and shs1Δ cln1Δ GAL1-CLN2 cells that had any polarized Cdc11 localization in the mother or daughter cell was determined, including cells that had polarized but abnormal localization of Cdc11. The percentage of budded cells that had normal Cdc11 localization at the bud neck was also determined. 200 cells were counted for each strain.
Mentions: Loss of Cdc3, Cdc10, Cdc11 or Cdc12 results in rapid loss of localization of the septins; however, loss of Shs1 has no effect on septin localization [16], [33], [34]. Since Shs1 is essential for viability in cln1Δ cln2Δ cells, we tested whether Shs1 is essential for localization of the other septins in cells that lack Cln1 and Cln2. We shifted shs1Δ cln1Δ GAL1-CLN2 and cln1Δ GAL1-CLN2 control cells into dextrose for 4.5 hours and used immunofluorescence to test for Cdc11 localization. Most shs1Δ cln1Δ GAL1-CLN2 cells showed a complete failure to localize Cdc11 (Figure 3A), although some unbudded cells had diffuse Cdc11 localization over one end of the cell (arrow, Figure 3B). Of the few budded shs1Δ cln1Δ GAL1-CLN2 cells, only 30% had polarized Cdc11 localization in the mother or daughter cell and the Cdc11 localization in the majority of these cells was aberrant (arrow, Figure 3C). Only 7.5% of budded cells had normal Cdc11 localization (Figure 3F). In cln1Δ GAL-CLN2 control cells, 100% of budded cells had polarized Cdc11 localization and 80% had normal Cdc11 localization (arrowhead, Figure 3D and 3F). In addition, the defects in Cdc11 localization that were observed in cells that lack Cln1 and Cln2 were less severe than the defects observed in cells that lack Shs1, Cln1, and Cln2 (arrow, Figure 3E). These results demonstrate that Shs1 is required for the normal localization of Cdc11 in cells that lack Cln1 and Cln2.

Bottom Line: However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle.Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1.Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America.

ABSTRACT
The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation.

Show MeSH
Related in: MedlinePlus