Ghent University Academic Bibliography

Advanced

Identification and characterization of substrates and regulators of the Anaphase Promoting Complex/Cyclosome involved in cell cycle exit in Arabidopsis thaliana

Jefri Heyman UGent (2012)
abstract
Control of cell division and well timed cell cycle exit is crucial for maintaining the balance between the generation of new cells and cell expansion coupled to endoreduplication onset. This balance between cell division and cell differentiation is an important factor controlling the size of meristems, and eventually plant growth and organ size. A premature onset of cell differentiation will result in a decrease in number of dividing cells, whereas a delayed cell cycle exit leads to an increased pool of proliferating cells. The Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase is known to play an important role in determining the timing of cell cycle exit and coupled endoreduplication onset. By ubiquitinating and hence targeting key cell cycle proteins for destruction, the APC/C controls the switch between the mitotic cell cycle and the endocycle. However, whereas the key APC/C targets and regulators have been well described in yeasts, frogs, fruit flies and humans, only little is known in plants. In order to find putative regulators and substrates of the Arabidopsis thaliana APC/C involved in controlling cell cycle exit, tandem affinity purifications (TAP) were performed using different APC/C subunits as bait. Since putative targets and regulators are most likely to co-purify with the CCS52A1 and CCS52A2 subunits, that confer substrate specificity and activation of the APC/C, we focused on interactors of these proteins. CCS52A co-purifying proteins that contain specific degrons, needed for CCS52A interaction, such as a D-box, KEN-box or GxEN-box, are interesting candidates. By screening knock-out mutants of these candidates for altered traits in which APC/CCCS52A activity is known to be involved, such as DNA ploidy levels or meristem size, putative targets or regulators can be identified. Using this approach, we attempted to identify and further characterize novel or plant specific regulators and substrates of the APC/C involved in cell cycle exit. In the first part of this work, we focus on the identified regulators and putative substrates using this approach. In the second part of this work, alternative approaches to identify APC/C targets were initiated. In order to find APC/CCCS52A2 specific substrates, ccs52a2 mutant seeds were mutagenized and a screen to identify suppressor mutants was initiated. Additionally, an alternative TAP-based approach was set up to identify APC/CCCS52A targets on a proteomewide scale. Here, a TAP-tagged ubiquitin construct using an adapted TAP-tag, allowing purification under fully denaturing conditions, was generated that would allow tandem purification of ubiquitin-tagged protein conjugates. By introducing this construct into a Scope 40 ccs52a mutant background and using a two-step purification and differential identification approach of mutant versus wild type plants, we hoped to reveal specific APC/CCCS52A targets.
Please use this url to cite or link to this publication:
author
promoter
UGent
organization
year
type
dissertation (monograph)
subject
pages
243 + annexe pages
publisher
Ghent University. Faculty of Sciences
place of publication
Ghent, Belgium
defense location
Zwijnaarde : Technologiepark (FSVM building)
defense date
2012-10-12 16:00
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
3007231
handle
http://hdl.handle.net/1854/LU-3007231
date created
2012-10-05 10:35:37
date last changed
2012-10-08 11:38:53
@phdthesis{3007231,
  abstract     = {Control of cell division and well timed cell cycle exit is crucial for maintaining the balance between the generation of new cells and cell expansion coupled to endoreduplication onset. This balance between cell division and cell differentiation is an important factor controlling the size of meristems, and eventually plant growth and organ size. A premature onset of cell differentiation will result in a decrease in number of dividing cells, whereas a delayed cell cycle exit leads to an increased pool of proliferating cells. The Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase is known to play an important role in determining the timing of cell cycle exit and coupled endoreduplication onset. By ubiquitinating and hence targeting key cell cycle proteins for destruction, the APC/C controls the switch between the mitotic cell cycle and the endocycle. However, whereas the key APC/C targets and regulators have been well described in yeasts, frogs, fruit flies and humans, only little is known in plants. In order to find putative regulators and substrates of the Arabidopsis thaliana APC/C involved in controlling cell cycle exit, tandem affinity purifications (TAP) were performed using different APC/C subunits as bait. Since putative targets and regulators are most likely to co-purify with the CCS52A1 and CCS52A2 subunits, that confer substrate specificity and activation of the APC/C, we focused on interactors of these proteins. CCS52A co-purifying proteins that contain specific degrons, needed for CCS52A interaction, such as a D-box, KEN-box or GxEN-box, are interesting candidates. By screening knock-out mutants of these candidates for altered traits in which APC/CCCS52A activity is known to be involved, such as DNA ploidy levels or meristem size, putative targets or regulators can be identified. Using this approach, we attempted to identify and further characterize novel or plant specific regulators and substrates of the APC/C involved in cell cycle exit. In the first part of this work, we focus on the identified regulators and putative substrates using this approach. In the second part of this work, alternative approaches to identify APC/C targets were initiated. In order to find APC/CCCS52A2 specific substrates, ccs52a2 mutant seeds were mutagenized and a screen to identify suppressor mutants was initiated. Additionally, an alternative TAP-based approach was set up to identify APC/CCCS52A targets on a proteomewide scale. Here, a TAP-tagged ubiquitin construct using an adapted TAP-tag, allowing purification under fully denaturing conditions, was generated that would allow tandem purification of ubiquitin-tagged protein conjugates. By introducing this construct into a Scope 40 ccs52a mutant background and using a two-step purification and differential identification approach of mutant versus wild type plants, we hoped to reveal specific APC/CCCS52A targets.},
  author       = {Heyman, Jefri},
  language     = {eng},
  pages        = {243 + annexe},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {Identification and characterization of substrates and regulators of the Anaphase Promoting Complex/Cyclosome involved in cell cycle exit in Arabidopsis thaliana},
  year         = {2012},
}

Chicago
Heyman, Jefri. 2012. “Identification and Characterization of Substrates and Regulators of the Anaphase Promoting Complex/Cyclosome Involved in Cell Cycle Exit in Arabidopsis Thaliana”. Ghent, Belgium: Ghent University. Faculty of Sciences.
APA
Heyman, J. (2012). Identification and characterization of substrates and regulators of the Anaphase Promoting Complex/Cyclosome involved in cell cycle exit in Arabidopsis thaliana. Ghent University. Faculty of Sciences, Ghent, Belgium.
Vancouver
1.
Heyman J. Identification and characterization of substrates and regulators of the Anaphase Promoting Complex/Cyclosome involved in cell cycle exit in Arabidopsis thaliana. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2012.
MLA
Heyman, Jefri. “Identification and Characterization of Substrates and Regulators of the Anaphase Promoting Complex/Cyclosome Involved in Cell Cycle Exit in Arabidopsis Thaliana.” 2012 : n. pag. Print.