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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 is peripherally associated with membranes. (A) KEULE appears to be membrane associated: KEULE is present in both the heavy membrane (P14K) and microsomal (P100) fractions. Longer exposure of the blot shows that KEULE is found in the soluble fraction as well. KEULE is shown to be in the soluble fraction of roots (right). As a control, the syntaxin KNOLLE is shown to be present in the membrane but not the soluble (S100K) fractions (bottom). P, pellet; S, supernatant. Note: not equiloaded, the membrane fractions are fivefold more concentrated than the soluble fractions. The arrow points to the KEULE-specific band which migrates anomalously at 100 kD in this sample (run with <100 mM DTT). It is not clear why this band is absent in the P14K fraction. (B and C) Membrane association is peripheral. KEULE can be released from the microsomes (P100K) if these are incubated with high salt (3 M NaCl), if the pH is increased (0.1 M NaCO3, pH 10.9–11.5), or with 2% SDS. (B) Solubilzation is complete with 2% SDS but only partial with high salt and high pH. (C) KEULE and KNOLLE differ with respect to the nature of their membrane association. In contrast to KEULE, KNOLLE was released from the microsomal fraction by SDS but not by high salt or high pH. (Although it appears that less KEULE protein is released from membranes by SDS than by high salt or high pH, this is most likely an artefact due to incomplete deoxycholate/TCA precipitation in the presence of SDS; see Materials and Methods for sample preparation. As in B, the majority of the KEULE protein remains in the pellets; not shown as grossly overexposed.) sup, supernatant.
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Figure 6: KEULE is peripherally associated with membranes. (A) KEULE appears to be membrane associated: KEULE is present in both the heavy membrane (P14K) and microsomal (P100) fractions. Longer exposure of the blot shows that KEULE is found in the soluble fraction as well. KEULE is shown to be in the soluble fraction of roots (right). As a control, the syntaxin KNOLLE is shown to be present in the membrane but not the soluble (S100K) fractions (bottom). P, pellet; S, supernatant. Note: not equiloaded, the membrane fractions are fivefold more concentrated than the soluble fractions. The arrow points to the KEULE-specific band which migrates anomalously at 100 kD in this sample (run with <100 mM DTT). It is not clear why this band is absent in the P14K fraction. (B and C) Membrane association is peripheral. KEULE can be released from the microsomes (P100K) if these are incubated with high salt (3 M NaCl), if the pH is increased (0.1 M NaCO3, pH 10.9–11.5), or with 2% SDS. (B) Solubilzation is complete with 2% SDS but only partial with high salt and high pH. (C) KEULE and KNOLLE differ with respect to the nature of their membrane association. In contrast to KEULE, KNOLLE was released from the microsomal fraction by SDS but not by high salt or high pH. (Although it appears that less KEULE protein is released from membranes by SDS than by high salt or high pH, this is most likely an artefact due to incomplete deoxycholate/TCA precipitation in the presence of SDS; see Materials and Methods for sample preparation. As in B, the majority of the KEULE protein remains in the pellets; not shown as grossly overexposed.) sup, supernatant.

Mentions: As KEULE is a member of a conserved gene family in Arabidopsis, generating specific antibodies has only been possible by using peptides as antigens. We generated and affinity purified a rabbit antibody against a synthetic peptide specific to KEULE (see Materials and Methods). This antibody recognizes a 73-kD band which is specific to KEULE, as evidenced by the observation that it is present in cytokinesis-defective mutants nonallelic to keule (designated keule-like in Fig. 5 A), but never in keule mutants (Fig. 5 A), even after overexposure of the blots. This KEULE-specific band is of the predicted size based on the coding sequence. However, there are two contaminating lower molecular bands revealed by this peptide antibody (Fig. 5 A, lower panel shows the dominant one). These have precluded the use of the antibody in immunoprecipitation or immunolocalization experiments, but have not hindered Western analysis. As judged by comparing extracts from keule mutants, transgenic plants carrying epitope-tagged KEULE, and wild-type plants, KEULE can migrate anomalously at ∼100 kD or higher in SDS-PAGE gels (an example is given in Fig. 6 A). This is especially pronounced when reductant levels are <100 mM DTT and may be due to intramolecular (or possibly even intermolecular) disulfide bridges.


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 is peripherally associated with membranes. (A) KEULE appears to be membrane associated: KEULE is present in both the heavy membrane (P14K) and microsomal (P100) fractions. Longer exposure of the blot shows that KEULE is found in the soluble fraction as well. KEULE is shown to be in the soluble fraction of roots (right). As a control, the syntaxin KNOLLE is shown to be present in the membrane but not the soluble (S100K) fractions (bottom). P, pellet; S, supernatant. Note: not equiloaded, the membrane fractions are fivefold more concentrated than the soluble fractions. The arrow points to the KEULE-specific band which migrates anomalously at 100 kD in this sample (run with <100 mM DTT). It is not clear why this band is absent in the P14K fraction. (B and C) Membrane association is peripheral. KEULE can be released from the microsomes (P100K) if these are incubated with high salt (3 M NaCl), if the pH is increased (0.1 M NaCO3, pH 10.9–11.5), or with 2% SDS. (B) Solubilzation is complete with 2% SDS but only partial with high salt and high pH. (C) KEULE and KNOLLE differ with respect to the nature of their membrane association. In contrast to KEULE, KNOLLE was released from the microsomal fraction by SDS but not by high salt or high pH. (Although it appears that less KEULE protein is released from membranes by SDS than by high salt or high pH, this is most likely an artefact due to incomplete deoxycholate/TCA precipitation in the presence of SDS; see Materials and Methods for sample preparation. As in B, the majority of the KEULE protein remains in the pellets; not shown as grossly overexposed.) sup, supernatant.
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Related In: Results  -  Collection

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Figure 6: KEULE is peripherally associated with membranes. (A) KEULE appears to be membrane associated: KEULE is present in both the heavy membrane (P14K) and microsomal (P100) fractions. Longer exposure of the blot shows that KEULE is found in the soluble fraction as well. KEULE is shown to be in the soluble fraction of roots (right). As a control, the syntaxin KNOLLE is shown to be present in the membrane but not the soluble (S100K) fractions (bottom). P, pellet; S, supernatant. Note: not equiloaded, the membrane fractions are fivefold more concentrated than the soluble fractions. The arrow points to the KEULE-specific band which migrates anomalously at 100 kD in this sample (run with <100 mM DTT). It is not clear why this band is absent in the P14K fraction. (B and C) Membrane association is peripheral. KEULE can be released from the microsomes (P100K) if these are incubated with high salt (3 M NaCl), if the pH is increased (0.1 M NaCO3, pH 10.9–11.5), or with 2% SDS. (B) Solubilzation is complete with 2% SDS but only partial with high salt and high pH. (C) KEULE and KNOLLE differ with respect to the nature of their membrane association. In contrast to KEULE, KNOLLE was released from the microsomal fraction by SDS but not by high salt or high pH. (Although it appears that less KEULE protein is released from membranes by SDS than by high salt or high pH, this is most likely an artefact due to incomplete deoxycholate/TCA precipitation in the presence of SDS; see Materials and Methods for sample preparation. As in B, the majority of the KEULE protein remains in the pellets; not shown as grossly overexposed.) sup, supernatant.
Mentions: As KEULE is a member of a conserved gene family in Arabidopsis, generating specific antibodies has only been possible by using peptides as antigens. We generated and affinity purified a rabbit antibody against a synthetic peptide specific to KEULE (see Materials and Methods). This antibody recognizes a 73-kD band which is specific to KEULE, as evidenced by the observation that it is present in cytokinesis-defective mutants nonallelic to keule (designated keule-like in Fig. 5 A), but never in keule mutants (Fig. 5 A), even after overexposure of the blots. This KEULE-specific band is of the predicted size based on the coding sequence. However, there are two contaminating lower molecular bands revealed by this peptide antibody (Fig. 5 A, lower panel shows the dominant one). These have precluded the use of the antibody in immunoprecipitation or immunolocalization experiments, but have not hindered Western analysis. As judged by comparing extracts from keule mutants, transgenic plants carrying epitope-tagged KEULE, and wild-type plants, KEULE can migrate anomalously at ∼100 kD or higher in SDS-PAGE gels (an example is given in Fig. 6 A). This is especially pronounced when reductant levels are <100 mM DTT and may be due to intramolecular (or possibly even intermolecular) disulfide bridges.

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