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The cytokinesis gene KEULE encodes a Sec1 protein that binds the syntaxin KNOLLE.

Assaad FF, Huet Y, Mayer U, Jürgens G - J. Cell Biol. (2001)

Bottom Line: KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes.More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE.Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals.

View Article: PubMed Central - PubMed

Affiliation: Genetics and Microbiology Institute, Ludwig Maximilians University, D-80638 Munich, Germany. fassaad@andrew2.stanford.edu

ABSTRACT
KEULE is required for cytokinesis in Arabidopsis thaliana. We have positionally cloned the KEULE gene and shown that it encodes a Sec1 protein. KEULE is expressed throughout the plant, yet appears enriched in dividing tissues. Cytokinesis-defective mutant sectors were observed in all somatic tissues upon transformation of wild-type plants with a KEULE-green fluorescent protein gene fusion, suggesting that KEULE is required not only during embryogenesis, but at all stages of the plant's life cycle. KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes. More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE. Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals. As a cytokinesis-related Sec1 protein, KEULE appears to represent a novel link between cell cycle progression and the membrane fusion apparatus.

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KEULE and KNOLLE interact. T7-KNOLLE was bacterially overexpressed and bound to α-T7 agarose beads. In the control lanes, bacterially expressed GST-KNOLLE was used in lieu of T7-KNOLLE. The loaded beads were incubated with protein extracts from flowers, leaves, and root lengths. 10% of the bead-bound proteins (T7-KNOLLE, CONTROL) or 1% of the plant extracts (INPUT) are loaded. (A) Coomassie-stained gel of beads incubated with root extract, or of root extract. Arrow points to T7-KNOLLE. GST-KNOLLE fails to bind the α-T7 agarose beads. (B) Westerns were probed with a peptide antibody against the highly conserved KEULE homologue AtSec1a (top) and with the KEULE peptide antibody (bottom). Longer exposures of the upper panel reveal the AtSec1a band in all six lanes, with no differential behavior between the experiment and negative control. The input lanes are loaded with the root extracts used for this experiment. (C) Specificity of the AtSec1a antibody. The AtSec1a peptide antibody recognizes a band at the expected size, 66 kD (arrow), in plant extracts (root). To confirm that this indeed corresponds to AtSec1a, we show that the antibody cross-reacts with a 66-kD protein immunoprecipitated (IP) by an antibody raised against the full length KEULE protein (middle). As expected based on sequence analysis (see Fig. 3), the full length antibody reveals three bands in plant extracts; the lower one presumambly corresponds to AtSec1a (left).
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Figure 7: KEULE and KNOLLE interact. T7-KNOLLE was bacterially overexpressed and bound to α-T7 agarose beads. In the control lanes, bacterially expressed GST-KNOLLE was used in lieu of T7-KNOLLE. The loaded beads were incubated with protein extracts from flowers, leaves, and root lengths. 10% of the bead-bound proteins (T7-KNOLLE, CONTROL) or 1% of the plant extracts (INPUT) are loaded. (A) Coomassie-stained gel of beads incubated with root extract, or of root extract. Arrow points to T7-KNOLLE. GST-KNOLLE fails to bind the α-T7 agarose beads. (B) Westerns were probed with a peptide antibody against the highly conserved KEULE homologue AtSec1a (top) and with the KEULE peptide antibody (bottom). Longer exposures of the upper panel reveal the AtSec1a band in all six lanes, with no differential behavior between the experiment and negative control. The input lanes are loaded with the root extracts used for this experiment. (C) Specificity of the AtSec1a antibody. The AtSec1a peptide antibody recognizes a band at the expected size, 66 kD (arrow), in plant extracts (root). To confirm that this indeed corresponds to AtSec1a, we show that the antibody cross-reacts with a 66-kD protein immunoprecipitated (IP) by an antibody raised against the full length KEULE protein (middle). As expected based on sequence analysis (see Fig. 3), the full length antibody reveals three bands in plant extracts; the lower one presumambly corresponds to AtSec1a (left).

Mentions: To test whether KEULE behaves like other SecI proteins, we examined its ability to bind syntaxins. Candidates for interacting partners are KNOLLE, a cytokinesis-specific syntaxin (Lukowitz et al. 1996; Lauber et al. 1997), as well as its closely related homologues, of which there are eight in the Arabidopsis genome (Sanderfoot et al. 2000). The KNOLLE gene was modified by the addition of a sequence corresponding to the 11–amino acid T7 tag (Novagen). This small epitope has a high affinity for the T7 monoclonal antibody, commercially available in a bead-bound form (α-T7 agarose beads). Recombinant protein produced in E. coli was bound to α-T7 agarose beads, which were incubated with plant extracts from root, flower, and leaf tissues. Bead-bound proteins were subjected to Western analysis. To control for nonspecific binding of proteins from the E.coli and plant extracts to the α-T7 agarose beads, we used a GST-KNOLLE protein fusion in lieu of the T7-KNOLLE fusion. This negative control leaves the active site of the T7 antibody available for non-specific interactions and was therefore chosen over a vector/tag only control. As seen in the Coomassie-stained samples (Fig. 7 A), T7-KNOLLE but not GST-KNOLLE binds the α-T7 agarose beads. The peptide antibody specific to KEULE detects KEULE in the T7-KNOLLE pull down lanes but not in the negative control (Fig. 7 B, bottom). This shows that native KEULE from plant extracts is capable of binding T7-KNOLLE–loaded beads in these assays. Formally, this binding could be indirect, mediated by other proteins from the bacterial or plant extracts. However, as Sec1 proteins are known to bind syntaxins, we are most likely witnessing a direct interaction.


The cytokinesis gene KEULE encodes a Sec1 protein that binds the syntaxin KNOLLE.

Assaad FF, Huet Y, Mayer U, Jürgens G - J. Cell Biol. (2001)

KEULE and KNOLLE interact. T7-KNOLLE was bacterially overexpressed and bound to α-T7 agarose beads. In the control lanes, bacterially expressed GST-KNOLLE was used in lieu of T7-KNOLLE. The loaded beads were incubated with protein extracts from flowers, leaves, and root lengths. 10% of the bead-bound proteins (T7-KNOLLE, CONTROL) or 1% of the plant extracts (INPUT) are loaded. (A) Coomassie-stained gel of beads incubated with root extract, or of root extract. Arrow points to T7-KNOLLE. GST-KNOLLE fails to bind the α-T7 agarose beads. (B) Westerns were probed with a peptide antibody against the highly conserved KEULE homologue AtSec1a (top) and with the KEULE peptide antibody (bottom). Longer exposures of the upper panel reveal the AtSec1a band in all six lanes, with no differential behavior between the experiment and negative control. The input lanes are loaded with the root extracts used for this experiment. (C) Specificity of the AtSec1a antibody. The AtSec1a peptide antibody recognizes a band at the expected size, 66 kD (arrow), in plant extracts (root). To confirm that this indeed corresponds to AtSec1a, we show that the antibody cross-reacts with a 66-kD protein immunoprecipitated (IP) by an antibody raised against the full length KEULE protein (middle). As expected based on sequence analysis (see Fig. 3), the full length antibody reveals three bands in plant extracts; the lower one presumambly corresponds to AtSec1a (left).
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Figure 7: KEULE and KNOLLE interact. T7-KNOLLE was bacterially overexpressed and bound to α-T7 agarose beads. In the control lanes, bacterially expressed GST-KNOLLE was used in lieu of T7-KNOLLE. The loaded beads were incubated with protein extracts from flowers, leaves, and root lengths. 10% of the bead-bound proteins (T7-KNOLLE, CONTROL) or 1% of the plant extracts (INPUT) are loaded. (A) Coomassie-stained gel of beads incubated with root extract, or of root extract. Arrow points to T7-KNOLLE. GST-KNOLLE fails to bind the α-T7 agarose beads. (B) Westerns were probed with a peptide antibody against the highly conserved KEULE homologue AtSec1a (top) and with the KEULE peptide antibody (bottom). Longer exposures of the upper panel reveal the AtSec1a band in all six lanes, with no differential behavior between the experiment and negative control. The input lanes are loaded with the root extracts used for this experiment. (C) Specificity of the AtSec1a antibody. The AtSec1a peptide antibody recognizes a band at the expected size, 66 kD (arrow), in plant extracts (root). To confirm that this indeed corresponds to AtSec1a, we show that the antibody cross-reacts with a 66-kD protein immunoprecipitated (IP) by an antibody raised against the full length KEULE protein (middle). As expected based on sequence analysis (see Fig. 3), the full length antibody reveals three bands in plant extracts; the lower one presumambly corresponds to AtSec1a (left).
Mentions: To test whether KEULE behaves like other SecI proteins, we examined its ability to bind syntaxins. Candidates for interacting partners are KNOLLE, a cytokinesis-specific syntaxin (Lukowitz et al. 1996; Lauber et al. 1997), as well as its closely related homologues, of which there are eight in the Arabidopsis genome (Sanderfoot et al. 2000). The KNOLLE gene was modified by the addition of a sequence corresponding to the 11–amino acid T7 tag (Novagen). This small epitope has a high affinity for the T7 monoclonal antibody, commercially available in a bead-bound form (α-T7 agarose beads). Recombinant protein produced in E. coli was bound to α-T7 agarose beads, which were incubated with plant extracts from root, flower, and leaf tissues. Bead-bound proteins were subjected to Western analysis. To control for nonspecific binding of proteins from the E.coli and plant extracts to the α-T7 agarose beads, we used a GST-KNOLLE protein fusion in lieu of the T7-KNOLLE fusion. This negative control leaves the active site of the T7 antibody available for non-specific interactions and was therefore chosen over a vector/tag only control. As seen in the Coomassie-stained samples (Fig. 7 A), T7-KNOLLE but not GST-KNOLLE binds the α-T7 agarose beads. The peptide antibody specific to KEULE detects KEULE in the T7-KNOLLE pull down lanes but not in the negative control (Fig. 7 B, bottom). This shows that native KEULE from plant extracts is capable of binding T7-KNOLLE–loaded beads in these assays. Formally, this binding could be indirect, mediated by other proteins from the bacterial or plant extracts. However, as Sec1 proteins are known to bind syntaxins, we are most likely witnessing a direct interaction.

Bottom Line: KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes.More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE.Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals.

View Article: PubMed Central - PubMed

Affiliation: Genetics and Microbiology Institute, Ludwig Maximilians University, D-80638 Munich, Germany. fassaad@andrew2.stanford.edu

ABSTRACT
KEULE is required for cytokinesis in Arabidopsis thaliana. We have positionally cloned the KEULE gene and shown that it encodes a Sec1 protein. KEULE is expressed throughout the plant, yet appears enriched in dividing tissues. Cytokinesis-defective mutant sectors were observed in all somatic tissues upon transformation of wild-type plants with a KEULE-green fluorescent protein gene fusion, suggesting that KEULE is required not only during embryogenesis, but at all stages of the plant's life cycle. KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes. More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE. Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals. As a cytokinesis-related Sec1 protein, KEULE appears to represent a novel link between cell cycle progression and the membrane fusion apparatus.

Show MeSH
Related in: MedlinePlus