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Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis.

Heese M, Gansel X, Sticher L, Wick P, Grebe M, Granier F, Jurgens G - J. Cell Biol. (2001)

Bottom Line: AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate.A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering.Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekularbiologie der Pflanzen, Universität Tübingen, D-72076 Tübingen, Germany.

ABSTRACT
Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

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Interaction of KN with different Arabidopsis SNAP25 homologues. Using the two-hybrid system, the cytoplasmic domain of KN was tested for interaction with full-length constructs of the Arabidopsis SNAP25 homologues. GNOM (GN) bait and empty prey vector (pJG4-5) were used as negative controls. Two independent clones are shown when KN was used as the bait. Interaction was tested in the color assay on X-gal containing plates (top, X-Gal) and in the growth assay on medium lacking leucine (Leu−, bottom). Interaction was only observed upon induction of the prey constructs with galactose (left, GAL) and not on glucose-containing plates (right, GLC).
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fig10: Interaction of KN with different Arabidopsis SNAP25 homologues. Using the two-hybrid system, the cytoplasmic domain of KN was tested for interaction with full-length constructs of the Arabidopsis SNAP25 homologues. GNOM (GN) bait and empty prey vector (pJG4-5) were used as negative controls. Two independent clones are shown when KN was used as the bait. Interaction was tested in the color assay on X-gal containing plates (top, X-Gal) and in the growth assay on medium lacking leucine (Leu−, bottom). Interaction was only observed upon induction of the prey constructs with galactose (left, GAL) and not on glucose-containing plates (right, GLC).

Mentions: No SNP33 protein was detected in snp33 mutant tissue (Fig. 2) even after overexposure of the Western blots, indicating that the mutant represents a complete loss of function. Thus, residual gene function does not account for the rather weak cytokinetic phenotype of snp33 mutants. Sequencing of the Arabidopsis genome identified two additional SNAP25 homologous genes, AtSNAP29 (AGI-ID: At5g07880) and AtSNAP30 (AGI-ID: At1g13890). The deduced proteins show ∼62% identity to AtSNAP33 and 52% identity among each other (Fig. 9), which raised the issue of functional redundancy. Therefore, we tested their ability to interact with the KN syntaxin in the yeast two-hybrid assay (Fig. 10). All three SNAP25 homologues of Arabidopsis interacted with the KN cytoplasmic region, suggesting that cytokinetic defects of snp33 single mutants may be weakened by activities of the other SNAP25 homologues.


Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis.

Heese M, Gansel X, Sticher L, Wick P, Grebe M, Granier F, Jurgens G - J. Cell Biol. (2001)

Interaction of KN with different Arabidopsis SNAP25 homologues. Using the two-hybrid system, the cytoplasmic domain of KN was tested for interaction with full-length constructs of the Arabidopsis SNAP25 homologues. GNOM (GN) bait and empty prey vector (pJG4-5) were used as negative controls. Two independent clones are shown when KN was used as the bait. Interaction was tested in the color assay on X-gal containing plates (top, X-Gal) and in the growth assay on medium lacking leucine (Leu−, bottom). Interaction was only observed upon induction of the prey constructs with galactose (left, GAL) and not on glucose-containing plates (right, GLC).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198836&req=5

fig10: Interaction of KN with different Arabidopsis SNAP25 homologues. Using the two-hybrid system, the cytoplasmic domain of KN was tested for interaction with full-length constructs of the Arabidopsis SNAP25 homologues. GNOM (GN) bait and empty prey vector (pJG4-5) were used as negative controls. Two independent clones are shown when KN was used as the bait. Interaction was tested in the color assay on X-gal containing plates (top, X-Gal) and in the growth assay on medium lacking leucine (Leu−, bottom). Interaction was only observed upon induction of the prey constructs with galactose (left, GAL) and not on glucose-containing plates (right, GLC).
Mentions: No SNP33 protein was detected in snp33 mutant tissue (Fig. 2) even after overexposure of the Western blots, indicating that the mutant represents a complete loss of function. Thus, residual gene function does not account for the rather weak cytokinetic phenotype of snp33 mutants. Sequencing of the Arabidopsis genome identified two additional SNAP25 homologous genes, AtSNAP29 (AGI-ID: At5g07880) and AtSNAP30 (AGI-ID: At1g13890). The deduced proteins show ∼62% identity to AtSNAP33 and 52% identity among each other (Fig. 9), which raised the issue of functional redundancy. Therefore, we tested their ability to interact with the KN syntaxin in the yeast two-hybrid assay (Fig. 10). All three SNAP25 homologues of Arabidopsis interacted with the KN cytoplasmic region, suggesting that cytokinetic defects of snp33 single mutants may be weakened by activities of the other SNAP25 homologues.

Bottom Line: AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate.A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering.Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekularbiologie der Pflanzen, Universität Tübingen, D-72076 Tübingen, Germany.

ABSTRACT
Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

Show MeSH
Related in: MedlinePlus