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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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Ubiquitous expression of SNP33. (A) Total protein extracts of different organs were separated by SDS-PAGE, transferred to PVDF membranes, and detected with anti-KN serum (second panel; arrowhead, size of KN) or anti-SNP33 serum (third panel; arrowhead, size of SNP33). A Coomassie-stained gel (top) is shown as loading control; protein concentration of stem extract was adjusted to that of cell culture extract based on another Coomassie-stained gel (unpublished data). (B) Expression of GFP under the control of a 2.1-kb genomic fragment containing the SNP33 promotor (Fig. 4, fragment C) was analyzed. Examples of tissues with strong GFP fluorescence are shown. (a and b) Transgenic root (arrowhead) next to a wild-type root shown in bright field (a) and fluorescence using a GFP filter (b). (c) Young leaves emerging between the petioles of the cotyledons (arrowhead). (d) Ovules in an opened silique. (e) Vascular tissue and hydathode (asterisk) of a cotyledon. (f) Abscission (arrowhead) and dehiscence zones (arrows) of a silique. Bar: (a–d) 150 μm; (e) 75 μm; (f) 950 μm.
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fig3: Ubiquitous expression of SNP33. (A) Total protein extracts of different organs were separated by SDS-PAGE, transferred to PVDF membranes, and detected with anti-KN serum (second panel; arrowhead, size of KN) or anti-SNP33 serum (third panel; arrowhead, size of SNP33). A Coomassie-stained gel (top) is shown as loading control; protein concentration of stem extract was adjusted to that of cell culture extract based on another Coomassie-stained gel (unpublished data). (B) Expression of GFP under the control of a 2.1-kb genomic fragment containing the SNP33 promotor (Fig. 4, fragment C) was analyzed. Examples of tissues with strong GFP fluorescence are shown. (a and b) Transgenic root (arrowhead) next to a wild-type root shown in bright field (a) and fluorescence using a GFP filter (b). (c) Young leaves emerging between the petioles of the cotyledons (arrowhead). (d) Ovules in an opened silique. (e) Vascular tissue and hydathode (asterisk) of a cotyledon. (f) Abscission (arrowhead) and dehiscence zones (arrows) of a silique. Bar: (a–d) 150 μm; (e) 75 μm; (f) 950 μm.

Mentions: To compare SNP33 and KN expression, protein blots of extracts from different organs were probed with both antisera (Fig. 3 A). KN expression was restricted to organs containing proliferating tissues as reported previously (Lauber et al., 1997). By contrast, SNP33 seemed to be expressed in all organs analyzed. The expression level in leaves was variable, ranging from barely detectable to high. The reason for this variability of expression, which was observed repeatedly, is not known.


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)

Ubiquitous expression of SNP33. (A) Total protein extracts of different organs were separated by SDS-PAGE, transferred to PVDF membranes, and detected with anti-KN serum (second panel; arrowhead, size of KN) or anti-SNP33 serum (third panel; arrowhead, size of SNP33). A Coomassie-stained gel (top) is shown as loading control; protein concentration of stem extract was adjusted to that of cell culture extract based on another Coomassie-stained gel (unpublished data). (B) Expression of GFP under the control of a 2.1-kb genomic fragment containing the SNP33 promotor (Fig. 4, fragment C) was analyzed. Examples of tissues with strong GFP fluorescence are shown. (a and b) Transgenic root (arrowhead) next to a wild-type root shown in bright field (a) and fluorescence using a GFP filter (b). (c) Young leaves emerging between the petioles of the cotyledons (arrowhead). (d) Ovules in an opened silique. (e) Vascular tissue and hydathode (asterisk) of a cotyledon. (f) Abscission (arrowhead) and dehiscence zones (arrows) of a silique. Bar: (a–d) 150 μm; (e) 75 μm; (f) 950 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198836&req=5

fig3: Ubiquitous expression of SNP33. (A) Total protein extracts of different organs were separated by SDS-PAGE, transferred to PVDF membranes, and detected with anti-KN serum (second panel; arrowhead, size of KN) or anti-SNP33 serum (third panel; arrowhead, size of SNP33). A Coomassie-stained gel (top) is shown as loading control; protein concentration of stem extract was adjusted to that of cell culture extract based on another Coomassie-stained gel (unpublished data). (B) Expression of GFP under the control of a 2.1-kb genomic fragment containing the SNP33 promotor (Fig. 4, fragment C) was analyzed. Examples of tissues with strong GFP fluorescence are shown. (a and b) Transgenic root (arrowhead) next to a wild-type root shown in bright field (a) and fluorescence using a GFP filter (b). (c) Young leaves emerging between the petioles of the cotyledons (arrowhead). (d) Ovules in an opened silique. (e) Vascular tissue and hydathode (asterisk) of a cotyledon. (f) Abscission (arrowhead) and dehiscence zones (arrows) of a silique. Bar: (a–d) 150 μm; (e) 75 μm; (f) 950 μm.
Mentions: To compare SNP33 and KN expression, protein blots of extracts from different organs were probed with both antisera (Fig. 3 A). KN expression was restricted to organs containing proliferating tissues as reported previously (Lauber et al., 1997). By contrast, SNP33 seemed to be expressed in all organs analyzed. The expression level in leaves was variable, ranging from barely detectable to high. The reason for this variability of expression, which was observed repeatedly, is not known.

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