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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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Characterization of an antiserum raised against SNP33. (A) The anti-SNP33 serum was tested on extracts from bacteria expressing GST-SNP33 (SNP33), an NH2-terminal fragment of SNP33 fused to GST (SNP33(N)) or a GST fusion of the related AtSNAP29 (SNP29). The left panel shows a Coomassie-stained gel to compare the amounts of total protein loaded. BE, bacterial extract without recombinant protein. For the Western blot (right panel), 1:200 dilutions of the extracts were used. (B and C) Total protein extracts from wild-type (WT) and two snp33 (mut-1 and mut-2) mutant callus cultures were separated on SDS-PAGE gels, transferred to PVDF membranes, and detected with anti-SNP33 serum (B) or anti-KN serum (C, control). The arrowheads mark the sizes of the expected proteins. KN expression was the same in all extracts, indicating equal loading. A band of about 33 kD was detected by the anti-SNP33 serum in wild-type but not in snp33 mutant extracts.
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fig2: Characterization of an antiserum raised against SNP33. (A) The anti-SNP33 serum was tested on extracts from bacteria expressing GST-SNP33 (SNP33), an NH2-terminal fragment of SNP33 fused to GST (SNP33(N)) or a GST fusion of the related AtSNAP29 (SNP29). The left panel shows a Coomassie-stained gel to compare the amounts of total protein loaded. BE, bacterial extract without recombinant protein. For the Western blot (right panel), 1:200 dilutions of the extracts were used. (B and C) Total protein extracts from wild-type (WT) and two snp33 (mut-1 and mut-2) mutant callus cultures were separated on SDS-PAGE gels, transferred to PVDF membranes, and detected with anti-SNP33 serum (B) or anti-KN serum (C, control). The arrowheads mark the sizes of the expected proteins. KN expression was the same in all extracts, indicating equal loading. A band of about 33 kD was detected by the anti-SNP33 serum in wild-type but not in snp33 mutant extracts.

Mentions: For interaction to occur in vivo, SNP33 and KN need to be expressed in overlapping domains. To test for SNP33 expression, we generated a polyclonal antiserum against the full-length protein. The antiserum recognized different forms of recombinant SNP33 protein but not the related AtSNAP29 (Fig. 2 A). On Western blots of plant extracts, a band corresponding to the predicted size of SNP33 was detected. This band was only present in wild-type extracts but not in extracts from a snp33 T-DNA insertion mutant (see below), confirming the specificity of the anti-SNP33 antiserum (Fig. 2 B).


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)

Characterization of an antiserum raised against SNP33. (A) The anti-SNP33 serum was tested on extracts from bacteria expressing GST-SNP33 (SNP33), an NH2-terminal fragment of SNP33 fused to GST (SNP33(N)) or a GST fusion of the related AtSNAP29 (SNP29). The left panel shows a Coomassie-stained gel to compare the amounts of total protein loaded. BE, bacterial extract without recombinant protein. For the Western blot (right panel), 1:200 dilutions of the extracts were used. (B and C) Total protein extracts from wild-type (WT) and two snp33 (mut-1 and mut-2) mutant callus cultures were separated on SDS-PAGE gels, transferred to PVDF membranes, and detected with anti-SNP33 serum (B) or anti-KN serum (C, control). The arrowheads mark the sizes of the expected proteins. KN expression was the same in all extracts, indicating equal loading. A band of about 33 kD was detected by the anti-SNP33 serum in wild-type but not in snp33 mutant extracts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198836&req=5

fig2: Characterization of an antiserum raised against SNP33. (A) The anti-SNP33 serum was tested on extracts from bacteria expressing GST-SNP33 (SNP33), an NH2-terminal fragment of SNP33 fused to GST (SNP33(N)) or a GST fusion of the related AtSNAP29 (SNP29). The left panel shows a Coomassie-stained gel to compare the amounts of total protein loaded. BE, bacterial extract without recombinant protein. For the Western blot (right panel), 1:200 dilutions of the extracts were used. (B and C) Total protein extracts from wild-type (WT) and two snp33 (mut-1 and mut-2) mutant callus cultures were separated on SDS-PAGE gels, transferred to PVDF membranes, and detected with anti-SNP33 serum (B) or anti-KN serum (C, control). The arrowheads mark the sizes of the expected proteins. KN expression was the same in all extracts, indicating equal loading. A band of about 33 kD was detected by the anti-SNP33 serum in wild-type but not in snp33 mutant extracts.
Mentions: For interaction to occur in vivo, SNP33 and KN need to be expressed in overlapping domains. To test for SNP33 expression, we generated a polyclonal antiserum against the full-length protein. The antiserum recognized different forms of recombinant SNP33 protein but not the related AtSNAP29 (Fig. 2 A). On Western blots of plant extracts, a band corresponding to the predicted size of SNP33 was detected. This band was only present in wild-type extracts but not in extracts from a snp33 T-DNA insertion mutant (see below), confirming the specificity of the anti-SNP33 antiserum (Fig. 2 B).

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