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cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.

Chang F, Drubin D, Nurse P - J. Cell Biol. (1997)

Bottom Line: Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes.Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested.Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

View Article: PubMed Central - PubMed

Affiliation: Imperial Cancer Research Fund, London, United Kingdom. fc99@columbia.edu

ABSTRACT
As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

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cdc12p fragment binds to profilin cdc3p in vitro. (A) Purified proteins used in these studies. (Lane 1) Purified GST-cdc3.  (Lane 2) Purified GST. (Lane 3) Purified His-tagged cdc12p fragment. (B and C) cdc12p binds to GST-cdc3 but not to GST. In each  180-μl reaction, cdc12p (1.6 μM final concentration) and 20-μl 50% slurries of glutathione agarose beads containing indicated concentrations of GST-cdc3 or GST were added to each reaction. Reactions were incubated at room temperature for 1 h, and the beads were  pelleted. (B) Coomassie-stained gel of total proteins before pelleting (lane 1) and pellet fractions (lanes 2–10) at equivalent volumes.  Bands were quantitated by densitometry relative to total protein in lane 1 and plotted in C. (D and E) Poly-l-proline inhibits binding.  Varying amounts of poly-l-proline were added to the binding reactions similar to ones described above except cdc12p was 0.8 μM. (D)  Coomassie-stained gel of the pellet fractions. (E) cdc12p in the pellets was quantitated by densitometry relative to supernatant fractions  (not shown).
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Figure 9: cdc12p fragment binds to profilin cdc3p in vitro. (A) Purified proteins used in these studies. (Lane 1) Purified GST-cdc3. (Lane 2) Purified GST. (Lane 3) Purified His-tagged cdc12p fragment. (B and C) cdc12p binds to GST-cdc3 but not to GST. In each 180-μl reaction, cdc12p (1.6 μM final concentration) and 20-μl 50% slurries of glutathione agarose beads containing indicated concentrations of GST-cdc3 or GST were added to each reaction. Reactions were incubated at room temperature for 1 h, and the beads were pelleted. (B) Coomassie-stained gel of total proteins before pelleting (lane 1) and pellet fractions (lanes 2–10) at equivalent volumes. Bands were quantitated by densitometry relative to total protein in lane 1 and plotted in C. (D and E) Poly-l-proline inhibits binding. Varying amounts of poly-l-proline were added to the binding reactions similar to ones described above except cdc12p was 0.8 μM. (D) Coomassie-stained gel of the pellet fractions. (E) cdc12p in the pellets was quantitated by densitometry relative to supernatant fractions (not shown).

Mentions: Next, we tested whether cdc3p and cdc12p interact directly by an in vitro binding assay. GST–cdc3p fusion protein and GST protein were expressed in E. coli and purified on glutathione-coupled agarose beads (Fig. 9 A, lanes 1 and 2). As a full-length cdc12 fusion protein was not expressed well in E. coli, a smaller His6-tagged fragment of cdc12p that included the central proline-rich FH1 domain was expressed in E. coli and purified using Ni-affinity chromatography (Fig. 9 A, lane 3). The cdc12p protein fragment was mixed with GST-cdc3 beads or GST beads, and binding was assayed by determining the amount of cdc12p that pelleted with the beads. Fig. 9, B and C, show that cdc12p bound to beads containing GST-cdc3 but did not bind to beads with GST or glutathione alone. This cdc12p binding was dependent on the concentration of cdc3p. To test if the proline-rich region of cdc12p might be responsible for profilin binding, we assayed for binding in the presence of poly-l-proline competitor. Binding was inhibited by the addition of PLP peptides (Fig. 9, D and E). A 5:1fold molar excess of the PLP peptide to cdc12 was sufficient for inhibition, while a 1:2 molar deficiency was not, suggesting that this inhibition is specific. Addition of 1% BSA did not abolish binding (data not shown). Further experiments will be necessary to derive an accurate binding constant. These data indicate that the cdc12p fragment is capable of binding to cdc3p profilin in a direct and specific manner and that the interaction is likely to be mediated by the PLP binding site in profilin- and proline-rich sequence (s) present in the FH1 domain of cdc12p.


cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.

Chang F, Drubin D, Nurse P - J. Cell Biol. (1997)

cdc12p fragment binds to profilin cdc3p in vitro. (A) Purified proteins used in these studies. (Lane 1) Purified GST-cdc3.  (Lane 2) Purified GST. (Lane 3) Purified His-tagged cdc12p fragment. (B and C) cdc12p binds to GST-cdc3 but not to GST. In each  180-μl reaction, cdc12p (1.6 μM final concentration) and 20-μl 50% slurries of glutathione agarose beads containing indicated concentrations of GST-cdc3 or GST were added to each reaction. Reactions were incubated at room temperature for 1 h, and the beads were  pelleted. (B) Coomassie-stained gel of total proteins before pelleting (lane 1) and pellet fractions (lanes 2–10) at equivalent volumes.  Bands were quantitated by densitometry relative to total protein in lane 1 and plotted in C. (D and E) Poly-l-proline inhibits binding.  Varying amounts of poly-l-proline were added to the binding reactions similar to ones described above except cdc12p was 0.8 μM. (D)  Coomassie-stained gel of the pellet fractions. (E) cdc12p in the pellets was quantitated by densitometry relative to supernatant fractions  (not shown).
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Figure 9: cdc12p fragment binds to profilin cdc3p in vitro. (A) Purified proteins used in these studies. (Lane 1) Purified GST-cdc3. (Lane 2) Purified GST. (Lane 3) Purified His-tagged cdc12p fragment. (B and C) cdc12p binds to GST-cdc3 but not to GST. In each 180-μl reaction, cdc12p (1.6 μM final concentration) and 20-μl 50% slurries of glutathione agarose beads containing indicated concentrations of GST-cdc3 or GST were added to each reaction. Reactions were incubated at room temperature for 1 h, and the beads were pelleted. (B) Coomassie-stained gel of total proteins before pelleting (lane 1) and pellet fractions (lanes 2–10) at equivalent volumes. Bands were quantitated by densitometry relative to total protein in lane 1 and plotted in C. (D and E) Poly-l-proline inhibits binding. Varying amounts of poly-l-proline were added to the binding reactions similar to ones described above except cdc12p was 0.8 μM. (D) Coomassie-stained gel of the pellet fractions. (E) cdc12p in the pellets was quantitated by densitometry relative to supernatant fractions (not shown).
Mentions: Next, we tested whether cdc3p and cdc12p interact directly by an in vitro binding assay. GST–cdc3p fusion protein and GST protein were expressed in E. coli and purified on glutathione-coupled agarose beads (Fig. 9 A, lanes 1 and 2). As a full-length cdc12 fusion protein was not expressed well in E. coli, a smaller His6-tagged fragment of cdc12p that included the central proline-rich FH1 domain was expressed in E. coli and purified using Ni-affinity chromatography (Fig. 9 A, lane 3). The cdc12p protein fragment was mixed with GST-cdc3 beads or GST beads, and binding was assayed by determining the amount of cdc12p that pelleted with the beads. Fig. 9, B and C, show that cdc12p bound to beads containing GST-cdc3 but did not bind to beads with GST or glutathione alone. This cdc12p binding was dependent on the concentration of cdc3p. To test if the proline-rich region of cdc12p might be responsible for profilin binding, we assayed for binding in the presence of poly-l-proline competitor. Binding was inhibited by the addition of PLP peptides (Fig. 9, D and E). A 5:1fold molar excess of the PLP peptide to cdc12 was sufficient for inhibition, while a 1:2 molar deficiency was not, suggesting that this inhibition is specific. Addition of 1% BSA did not abolish binding (data not shown). Further experiments will be necessary to derive an accurate binding constant. These data indicate that the cdc12p fragment is capable of binding to cdc3p profilin in a direct and specific manner and that the interaction is likely to be mediated by the PLP binding site in profilin- and proline-rich sequence (s) present in the FH1 domain of cdc12p.

Bottom Line: Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes.Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested.Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

View Article: PubMed Central - PubMed

Affiliation: Imperial Cancer Research Fund, London, United Kingdom. fc99@columbia.edu

ABSTRACT
As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

Show MeSH
Related in: MedlinePlus